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https://openalex.org/W2542496409
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https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-016-3186-0
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English
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Dwarfism with joint laxity in Friesian horses is associated with a splice site mutation in B4GALT7
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BMC genomics
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cc-by
| 8,518
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* Correspondence: p.a.j.leegwater@uu.nl
1Department of Clinical Sciences of Companion Animals, Faculty of
Veterinary Medicine, Utrecht University, PO Box 80154NL-3508 TD Utrecht,
The Netherlands
Full list of author information is available at the end of the article Leegwater et al. BMC Genomics (2016) 17:839
DOI 10.1186/s12864-016-3186-0 Leegwater et al. BMC Genomics (2016) 17:839
DOI 10.1186/s12864-016-3186-0 © The Author(s). 2016 Open Access 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. Dwarfism with joint laxity in Friesian horses
is associated with a splice site mutation in
B4GALT7 Peter A. Leegwater1*
, Manon Vos-Loohuis1, Bart J. Ducro2, Iris J. Boegheim1,3, Frank G. van Steenbeek1,
Isaac J. Nijman4, Glen R. Monroe4, John W. M. Bastiaansen2, Bert W. Dibbits2, Leanne H. van de Goor5, Ids Hellinga6,
Willem Back3,7 and Anouk Schurink2 Abstract Background: Inbreeding and population bottlenecks in the ancestry of Friesian horses has led to health issues such
as dwarfism. The limbs of dwarfs are short and the ribs are protruding inwards at the costochondral junction, while
the head and back appear normal. A striking feature of the condition is the flexor tendon laxity that leads to
hyperextension of the fetlock joints. The growth plates of dwarfs display disorganized and thickened chondrocyte
columns. The aim of this study was to identify the gene defect that causes the recessively inherited trait in Friesian
horses to understand the disease process at the molecular level. Results: We have localized the genetic cause of the dwarfism phenotype by a genome wide approach to a 3 Mb
region on the p-arm of equine chromosome 14. The DNA of two dwarfs and one control Friesian horse was
sequenced completely and we identified the missense mutation ECA14:g.4535550C > T that cosegregated with the
phenotype in all Friesians analyzed. The mutation leads to the amino acid substitution p.(Arg17Lys) of xylosylprotein
beta 1,4-galactosyltransferase 7 encoded by B4GALT7. The protein is one of the enzymes that synthesize the
tetrasaccharide linker between protein and glycosaminoglycan moieties of proteoglycans of the extracellular matrix. The mutation not only affects a conserved arginine codon but also the last nucleotide of the first exon of the gene
and we show that it impedes splicing of the primary transcript in cultured fibroblasts from a heterozygous horse. As a
result, the level of B4GALT7 mRNA in fibroblasts from a dwarf is only 2 % compared to normal levels. Mutations in
B4GALT7 in humans are associated with Ehlers-Danlos syndrome progeroid type 1 and Larsen of Reunion Island
syndrome. Growth retardation and ligamentous laxity are common manifestations of these syndromes. Conclusions: We suggest that the identified mutation of equine B4GALT7 leads to the typical dwarfism phenotype in
Friesian horses due to deficient splicing of transcripts of the gene. The mutated gene implicates the extracellular matrix
in the regular organization of chrondrocyte columns of the growth plate. Conservation of individual amino acids may
not be necessary at the protein level but instead may reflect underlying conservation of nucleotide sequence that are
required for efficient splicing. Keywords: Proteoglycan, Growth retardation, Hypermobile joints, Galactosyltransferase I, Linkeropathy, Genome, Equus
caballus, Extracellular matrix © The Author(s). * Correspondence: p.a.j.leegwater@uu.nl Background gene mutation. The identification of the gene enables
the comparison of the phenotype across species and
enhances the understanding of the processes of growth
and development. g
A dwarfism trait has been segregating in the Friesian
horse breed for decades [1], (OMIA 000299-9796 [2]). Characteristic for the trait is the physeal growth retard-
ation of limbs and ribs, resulting in a disproportionate
form of dwarfism. The affected horses exhibit hyper-
extension of the fetlock joints of all limbs with varying
severity. Flexor tendon laxity, which is often seen in
newborn foals of all breeds, fails to recover in dwarf
foals and instead tends to increase further during aging. As a consequence, these dwarf Friesians develop an ab-
normal gait in which the limbs undergo extreme out-
ward rotation at the level of carpus and hocks. The
ribcage is abnormal in most cases with thickened and
S-shaped costochondral junctions, leading to an inward
protrusion of the chest at the level of Th10-16 (Fig. 1b,c). Mature dwarfs have a head of the same size as un-
affected horses, a broader chest with narrowing at the
costochondral junction, a disproportionally long back
and abnormally short limbs. The abdomen has a weak
and rounded appearance, and the musculature over the
body is poorly developed. Involvement of the hypothalamic-
pituitary growth axis in the pathogenesis of the condition
has been excluded [3]. Abstract 2016 Open Access 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. Leegwater et al. BMC Genomics (2016) 17:839 Page 2 of 9 Page 2 of 9 Gene mapping pp
g
To substantiate the localization of the dwarfism gene on
chromosome ECA14 we performed a genome wide com-
parison of a group of dwarfs with a group of controls
from the Friesian horse breed. The allelic association
reached genome wide significance in the telomeric re-
gion of the p-arm of ECA14 with a Bonferroni corrected
Pgenome = 2.90 × 10−19 for BIEC2-239391 at position
3776009, the SNP that was most significantly associated
with dwarfism (Fig. 2a). In total 35 SNPs passed the
Bonferroni corrected significance level (1.68 × 10−6) and
all were located on ECA14 between positions 1 and
9510581. Inspection of the genotypes of the individual
horses in the region showed that only the dwarfs shared
a haplotype of 3 Mb homozygously, confirming that the
phenotype originated from a single founder (Fig. 2b). The genotypes clearly pointed to recombination events
that were evident in several cases and that placed the
critical region between positions 3151847 and 6229282
on ECA14. According to the annotation release 101 of
the NCBI of the equine reference genome the critical
region contained 66 genes [5]. A monogenic recessive mode of inheritance is most
likely and, considering the breed structure with a small
number of founders and narrow population bottlenecks,
it is expected that the dwarfs are homozygous for the
responsible gene mutation [4]. A genome wide associ-
ation study of 10 cases and 10 control Friesian horses
has been reported earlier [4]. The dwarfism locus was
assigned to the telomeric region of the p-arm of
chromosome 14 (ECA14), although genome wide signifi-
cance was not reached. The aim of the present study
was to confirm and further define the critical chromo-
some region of the locus and to identify the responsible DNA sequence analysis q
y
Full genome DNA sequence data was generated of four
dwarfs and three control Friesian horses by Next Gener-
ation Sequencing. The DNA sequence of the critical
region of ECA14 of the dwarfs was compared with those
of the controls, the reference genome, and the Quarter
Horse that is publicly available [6]. As dwarfism has not
been reported in the Quarter Horse breed we assumed
that the causative mutation is not present in this popula-
tion and that the horse was homozygous for the refer-
ence allele. The variations of the dwarfs as compared
with the reference genome were filtered by snpSift [7]
for possible effects on amino acid sequence or splicing
(Additional file 1). Using the Integrative Genomics
Viewer [8] we then searched for the variations that were
absent in the Quarter Horse and not homozygously
present in the control Friesian horses. Only one
nonsynonymous mutation fulfilled these criteria. The
mutation
was
ECA14:g.4535550C > T
in
B4GALT7
and corresponds to XM_014730464.1:c.50G > A and
XP_014585950.1:p.(Arg17Lys). Arginine
and
lysine
are both basic amino acids that are interchangeably
seen in many conserved protein domains. In this
case, however, the arginine residue at position 17 of
the equine B4GALT7 encoded protein xylosylprotein Fig. 1 Dwarfism in the Friesian horse breed. a A female dwarf next
to two normal female Friesian horses. The dwarf has a height at the
withers of 1.12 m; the horse in the middle has a height of 1.54 m,
which is close to the minimum allowed by the breed standard (1.53 m);
the horse on the right has a height of 1.66 m. b and c Photographic
illustrations of the typical pectus excavatum phenotype in the Friesian
dwarf of the right b and the left side c Fig. 1 Dwarfism in the Friesian horse breed. a A female dwarf next
to two normal female Friesian horses. The dwarf has a height at the
withers of 1.12 m; the horse in the middle has a height of 1.54 m,
which is close to the minimum allowed by the breed standard (1.53 m);
the horse on the right has a height of 1.66 m. b and c Photographic
illustrations of the typical pectus excavatum phenotype in the Friesian
dwarf of the right b and the left side c Leegwater et al. BMC Genomics (2016) 17:839 Page 3 of 9 cases
controls
3.2 M
6.2 M
B4GALT7
a
b
Fig. DNA sequence analysis The results of the telomeric 134 SNPs of the p-arm that passed quality control are shown. Red: homozygous genotype
of major allele in the dwarfs; green: homozygous genotype of minor allele; yellow: heterozygous genotype. The region of homozygosity in the dwarfs
is bordered by the SNPs BIEC2-239119 and BIEC2-240544 Fig. 2 Localization of the dwarfism locus in Friesian horses. a Manhattan plot of the genome wide association study. The DNA of 19 dwarfs and 65
controls of the Friesian horse breed was typed with the Illumina EquineSNP50 array. The allele frequency differences between the groups were
assessed with GenABEL software and plotted as the –log10p value from Chi-square tests. The red line indicates the Bonferroni corrected significance
level. b Homozygosity mapping. Individual genotypes of informative SNPs from the telomeric region (0 - 10 Mb) of ECA14 of cases and controls were
extracted from the array data. The results of the telomeric 134 SNPs of the p-arm that passed quality control are shown. Red: homozygous genotype
of major allele in the dwarfs; green: homozygous genotype of minor allele; yellow: heterozygous genotype. The region of homozygosity in the dwarfs
is bordered by the SNPs BIEC2-239119 and BIEC2-240544 Friesian horses 22 were carrier of the mutation and 155
were homozygous for the reference allele (line 1). beta 1,4-galactosyltransferase, polypeptide 7 (galacto-
syltransferase I) is strictly conserved in all verte-
brates analyzed (Fig. 3a). Nonetheless, the mutation
was considered moderate by the snpSift analysis and
benign by PolyPhen-2 [9]. RNA analysis The mutated guanosine nucleotide is the last residue of
exon 1 of B4GALT7 and the position of this first splice
donor relative to the start codon of the gene is con-
served in vertebrates (Fig. 3b). The nucleotide is second
in the triplet coding for arginine and since this amino
acid is conserved, the guanosine is conserved with it. The association of the mutation with the dwarfism
phenotype was evaluated by Sanger DNA sequencing. All 29 dwarfs of which DNA was available were homozy-
gous for the mutation (Fig. 4c, line 2). The 8 obligate
carriers were heterozygous (line 3) and of a group of 177 a
b
Fig. 3 Conservation of B4GALT7. a Alignment of the N-terminal amino acid sequence of xylosylprotein beta 1,4-galactosyltransferase, polypeptide
7 of horse with that of several mammals, an amphibian and a fish. The protein sequences are derived from the reference cDNAs listed below. b
Alignment of the splice donor site of exon 1 of B4GALT7 of horse with that of other vertebrate species. The exon DNA sequence is in upper case
and that of the intron is in lower case. The residues in a and b that are mutated in Friesian horse dwarfs are underlined. Residues that are identical to
those in horse are indicated by a dot; gaps that are introduced to optimize the alignment are indicated by a dash. The exon 1/intron 1 splice junctions
are derived from the respective reference genomes and based on the following cDNA reference sequences. Horse (Equus caballus): XM_014730464;
human: NM_007255; cow (Bos taurus): NM_001075321; pig (Sus scrofa): NM_001168422; rhinoceros (Ceratotherium simum): XM_010145472; rat (Rattus
norvegicus): NM_001031661; bat (Eptesicus fuscus): XM_008143179; Dolphin (Tursiops truncatus): XM_004313659; frog [Xenopus (Silurana) tropicalis]:
(NM_001126545); zebrafish (Danio rerio): NM_001003417 Fig. 3 Conservation of B4GALT7. a Alignment of the N-terminal amino acid sequence of xylosylprotein beta 1,4-galactosyltransferase, polypeptide
7 of horse with that of several mammals, an amphibian and a fish. The protein sequences are derived from the reference cDNAs listed below. b
Alignment of the splice donor site of exon 1 of B4GALT7 of horse with that of other vertebrate species. The exon DNA sequence is in upper case
and that of the intron is in lower case. The residues in a and b that are mutated in Friesian horse dwarfs are underlined. DNA sequence analysis 2 Localization of the dwarfism locus in Friesian horses. a Manhattan plot of the genome wide association study. The DNA of 19 dwarfs and 65
controls of the Friesian horse breed was typed with the Illumina EquineSNP50 array. The allele frequency differences between the groups were
assessed with GenABEL software and plotted as the –log10p value from Chi-square tests. The red line indicates the Bonferroni corrected significance
level. b Homozygosity mapping. Individual genotypes of informative SNPs from the telomeric region (0 - 10 Mb) of ECA14 of cases and controls were
extracted from the array data. The results of the telomeric 134 SNPs of the p-arm that passed quality control are shown. Red: homozygous genotype
of major allele in the dwarfs; green: homozygous genotype of minor allele; yellow: heterozygous genotype. The region of homozygosity in the dwarfs
is bordered by the SNPs BIEC2-239119 and BIEC2-240544 a cases
controls
3.2 M
6.2 M
B4GALT7
b b a Fig. 2 Localization of the dwarfism locus in Friesian horses. a Manhattan plot of the genome wide association study. The DNA of 19 dwarfs and 65
controls of the Friesian horse breed was typed with the Illumina EquineSNP50 array. The allele frequency differences between the groups were
assessed with GenABEL software and plotted as the –log10p value from Chi-square tests. The red line indicates the Bonferroni corrected significance
level. b Homozygosity mapping. Individual genotypes of informative SNPs from the telomeric region (0 - 10 Mb) of ECA14 of cases and controls were
extracted from the array data. The results of the telomeric 134 SNPs of the p-arm that passed quality control are shown. Red: homozygous genotype
of major allele in the dwarfs; green: homozygous genotype of minor allele; yellow: heterozygous genotype. The region of homozygosity in the dwarfs
is bordered by the SNPs BIEC2-239119 and BIEC2-240544 Fig. 2 Localization of the dwarfism locus in Friesian horses. a Manhattan plot of the genome wide association study. The DNA of 19 dwarfs and 65
controls of the Friesian horse breed was typed with the Illumina EquineSNP50 array. The allele frequency differences between the groups were
assessed with GenABEL software and plotted as the –log10p value from Chi-square tests. The red line indicates the Bonferroni corrected significance
level. b Homozygosity mapping. Individual genotypes of informative SNPs from the telomeric region (0 - 10 Mb) of ECA14 of cases and controls were
extracted from the array data. RNA analysis Residues that are identical to
those in horse are indicated by a dot; gaps that are introduced to optimize the alignment are indicated by a dash. The exon 1/intron 1 splice junctions
are derived from the respective reference genomes and based on the following cDNA reference sequences. Horse (Equus caballus): XM_014730464;
human: NM_007255; cow (Bos taurus): NM_001075321; pig (Sus scrofa): NM_001168422; rhinoceros (Ceratotherium simum): XM_010145472; rat (Rattus
norvegicus): NM_001031661; bat (Eptesicus fuscus): XM_008143179; Dolphin (Tursiops truncatus): XM_004313659; frog [Xenopus (Silurana) tropicalis]:
(NM_001126545); zebrafish (Danio rerio): NM_001003417 Leegwater et al. BMC Genomics (2016) 17:839 Page 4 of 9 a
b
c b b b c Fig. 4 Mutation of the terminal nucleotide of exon 1 of B4GALT7 affects proper RNA splicing of intron 1. a Map of the exon 1- exon 2 region of
the equine B4GALT7 gene. The positions of the exons 1 and 2 are indicated by numbered boxes; α: alternative exon; γ: cryptic exon. The position
of the used PCR primers are indicated by > and < signs with Roman numerals. I>: EX1F; <II: IN1R; <III: EX2R. b cDNA fragments derived from a
Friesian horse homozygous for the reference allele (lanes 2, 5 and 9), a heterozygous carrier (lanes 3. 6 and 10) and a Friesian horse dwarf (lanes
4, 7 and 11). Fibroblasts were grown from skin biopsies from the horses and RNA was isolated. cDNA was synthesized with reverse transcriptase
followed by PCR with equine B4GALT7 specific primers. Lanes 1 and 8: 100 bp size standard ladder (2 = 200 bp fragment, 4 = 400 bp fragment);
lanes 2-4: primers I and II; lanes 5-7: primers I and III; lanes 9-11: primers I, II and III. c Genomic DNA and RNA sequence analysis of B4GALT7
fragments from a horse homozygous for the reference allele (G/G), a dwarf (A/A) and a heterozygous carrier (G/A). The arrow indicates the
position of the mutation. Sub: substrate genomic DNA (DNA) from the horses representing the three genotypes (gen) or cDNA synthesized with
RNA from fibroblasts of the heterozygous horse (RNA). Pri: The PCR fragments were generated with primer I and either primer II or III as indicated. RNA analysis The mutant allele A is only observed in the unspliced RNA fragment (line 4) while the properly spliced product from the same heterozygous
horse only shows the reference allele G (line 5) We wondered whether the selection pressure could have
worked the other way around; that is, that the nucleotide
itself was conserved not due to its amino acid coding
properties but due to its splicing function. The mutation
of guanosine to adenosine might affect splicing of the
primary RNA transcript of the gene and splicing require-
ments may block its propagation. According to the
splice site predictor NNSPLICE 0.9 [10] the exon 1/in-
tron 1 junction of the equine reference gene had a splice
donor score of 0.96 on a scale of 0 to 1. The mutated
nucleotide sequence of the dwarfs had a moderate score
of 0.58, suggesting that the mutation could indeed inter-
fere with splicing. was less pronounced in the cDNA from the dwarf (lane
4). The primer set that included the intron 1 reverse pri-
mer produced a cDNA band derived from unspliced
RNA of approximately 220 bp. This band was detected
with the samples from the heterozygous horse and from
the dwarf (lanes 6 and 7), but also, be it weakly, with the
sample from the wildtype horse (lane 5). Combination of
the 3 primers in a semi quantitative PCR showed spliced
and unspliced products with similar intensities in the
cDNA from the dwarf (lane 11). The wild type horse and
the heterozygous horse do not display the unspliced prod-
uct of 220 bp with the 3 primer PCR (lanes 9 and 10). When
we
analyzed
the
cDNA
sequence
of
the
unspliced product from the heterozygous horse (lane 6)
it was derived from the mutant allele only (Fig. 4c, line
4). The cDNA sequence of the properly spliced product
from the same horse (lane 3) indicated that it was derived
from the normal allele (Fig. 4c, line 5). The B4GALT7
cDNA analysis confirms that the mutation r.50 g > a leads
to a splicing deficiency of the primary transcript. To investigate the effect of the mutation on splicing of
the primary transcript we isolated RNA from cultured
skin fibroblasts of a Friesian horse dwarf, of a heterozy-
gous carrier of the B4GALT7 mutation and of a Friesian
horse that was homozygous for the reference allele. RNA analysis Syn-
thesis of cDNA was followed by PCR with a forward
primer derived from B4GALT7 exon 1 and a reverse
primer from intron 1 or exon 2 (Fig. 4a). The RNA of
the wildtype horse and the heterozygous horse yielded a
splicing product with the exonic primers of the expected
length of 401 bp (Fig. 4b, lanes 2 and 3). This product The exon 1/exon 2 primer set produced minor frag-
ments that were larger than the expected length (Fig. 4b,
lane 4). DNA sequence analysis of the fragments derived
from the dwarf showed that the fragment of approximately Leegwater et al. BMC Genomics (2016) 17:839 Page 5 of 9 Page 5 of 9 In the homozygous state, the mutation leads to low
mRNA levels and the expression of the gene is strongly
reduced as measured by qPCR. The improperly spliced
transcripts could be prone to nonsense mediated decay. 560 bp contained an alternative exon of 162 nucleotides be-
tween exon 1 and exon 2 sequences that were spliced at
the proper positions. This alternative exon is annotated as
such in Genbank. It is situated from position 4533657 to
4533818 of chromosome 14 of the reference genome
EquCab 2.0. It should be noted that the gene is situated in
the reverse orientation on the chromosome. The alternative
exon has an open reading frame that is in frame with the
remainder of the coding sequence of the gene and the
amino acid sequence encoded by the exon is conserved in
mammals. The nucleotide sequence AGgt of the exon 1 splice
junction of B4GALT7 and its position with regard to the
start codon are highly conserved (Fig. 3b). One could
argue that the last nucleotide of the exon is expected to
be conserved if the encoded arginine residue would be
essential for the function of the protein. This G residue
is the second nucleotide of the codon and all six triplets
that code for arginine have a G residue at the second
position. Thus, if the arginine is conserved, the guano-
sine is conserved with it. The first position of the codon
under consideration is a conserved A residue, while 4 of
the 6 possible arginine codons start with a C. A func-
tional restriction on the encoded arginine residue would
therefore not necessarily lead to conservation of the A
residue of the AGgt splice junction. RNA analysis The hypothetical
mutation of the A residue to a C would only lead to a
moderate drop of the splice donor score from 0.96 to
0.89. According to this prediction a mutation of the sec-
ond to last A residue to a C would be allowed while in
fact it is highly conserved. Recently, a mutation of the A
residue of a splice donor site in IBA57 with the same
AGgt junction sequence as exon 1 of B4GALT7 was
shown to impede proper splicing, causing a severe
leukoencephalopathy [11]. This mutation did not alter
the encoded amino acid and it stresses the importance
of the exonic terminal nucleotide sequences for splicing
at particular junctions. An in vitro splicing assay may
resolve the importance of the second to last A residue of
the exon 1 of B4GALT7 for proper splicing. Concurrent
with our results, the NNSPLICE program assigns a
much lower splice donor score of 0.58 for the mutation
found in the Friesian dwarfs. Considering all our results,
we conclude that the conservation of the exon 1
terminal sequence in vertebrates reflects a restriction on
a splicing requirement rather than on a functional re-
quirement of the encoded amino acid. Characterization
of naturally occurring mutations that are uncovered
because of an association with disease can render im-
portant insights in splicing requirements [12]. I
h
i
i
B4GALT7
h
Ehl Another minor cDNA fragment of approximately
730 bp contained the same alternative exon sequence to-
gether with a cryptic exon of 169 nucleotides located
between exon 1 and the alternative exon. The genomic
location of the cryptic exon was from 4534375 to
4534543 on chromosome 14 and it contained multiple
stopcodons in frame with the start codon on exon 1. The aberrant splicing from the exon 1 donor due to
the variant r.50 g > a was associated with a severe reduc-
tion of expression of B4GALT7 at the mRNA level. Quantitative PCR measurement of cDNA fragments
indicated that the concentration of transcripts from the
gene in the fibroblasts from the dwarf was only 2 % of
that in fibroblasts from a Friesian horse that did not
carry the mutation (Additional file 2). Discussion Disproportionate dwarfism in Friesian horses is associ-
ated with a mutation in B4GALT7. The mutation
changes a conserved arginine codon to a lysine codon. Both amino acid residues are basic and the effect of the
mutation is considered moderate by the snpSift predic-
tion. The mutation also affects the last nucleotide of
exon 1 of the gene. Unspliced cDNA fragments spanning
the exon 1/intron 1 junction can be detected regardless
of the genotype of the horses. However, the cDNA
sequences from a heterozygous horse clearly show that
RNA derived from the mutant allele is hardly spliced, in
contrast to the RNA from the normal allele (Fig. 4c,
lines 4 and 5). When an exonic and an intronic reverse primer are
allowed to compete in a 3 primer PCR, only the cDNA
from the dwarf displays the spliced and unspliced prod-
ucts in comparable amounts (Fig. 4b, lane 11). The nor-
mally spliced product is seen prominently in the wild
type horse and the horse heterozygous for the mutation,
but the unspliced product cannot be discerned among
the products from these horses (lanes 9 and 10). This
semi quantitative PCR and the cDNA sequence analysis
of the products of the heterozygous horse confirms that
the B4GALT7 mutation strongly reduces the splicing
capacity of the exon 1/intron 1 junction. In humans, mutations in B4GALT7 cause the Ehlers-
Danlos syndrome, progeroid type 1 (EDSP1, OMIM130070)
and Larsen of Reunion Island syndrome (LRS). Only 7 mu-
tations have been described in relation to the recessively
inherited syndromes [13–18]. Most patients were normal
with respect to length and weight at birth but soon were
presented with growth retardation, osteopenia, facial
dysmorphology, loose joints, bone dysplasias, loose
skin and in most cases mild forms of mental retard-
ation. Pectus carinatum was reported for a number of
patients [16, 17]. The human phenotype is highly Leegwater et al. BMC Genomics (2016) 17:839 Page 6 of 9 Page 6 of 9 variable, even in patients sharing the same mutation
homozygously [17]. extracellular matrix. Mutations in any of the enzymes
that build the saccharide linker cause a variety of rare
syndromes with overlapping features, which are called
linkeropathies (reviewed in [21]). Dwarfism in Friesian
horses could therefore be considered as a new presenta-
tion of a linkeropathy. Discussion A founder effect in a closed population of Reunion has
led to at least 22 cases of LRS that were genetically con-
firmed. LRS was described as a subtype of Larsen syn-
drome [19]. The same mutation that causes LRS was
observed homozygously in two siblings from another
population
diagnosed
with
EDSP1. The
progeroid
aspect was not observed in any of the genetically
confirmed cases of EDSP1 nor LRS and it has been
suggested to remove this term from the name of the
EDSP1 syndrome [16, 18]. Remarkably, this is the second gene with a role in
protein glycosylation in which we found a pathogenic
mutation in Friesian horses. Earlier we found a nonsense
mutation in B3GALNT2 involved in muscular dystrophy
with hydrocephalus in stillborn foals [22]. The encoded
beta-1,3-N-acetylgalactosaminyltransferase is involved in
glycosylation of alpha-dystroglycan, which is part of the
complex that connects the cytoskeleton with the extra-
cellular matrix. Clear similarities between the conditions in man and
horse are growth retardation and hypermobile joints. Rib
deformities have been observed in human as well as
equine cases [1, 16, 17]. Pectus carinatum, reported in
human cases, refers to the pectus in which the ribcar-
tilage has been overdeveloped outward, leading to a
‘chicken chest’. In the Friesian horse cases on the
other hand, the ribcartilage has been overdeveloped
inward, leading to pectus excavatum or ‘shoemaker
chest’ in humans. Conclusions We
provide
evidence
indicating
that
dwarfism
in
Friesian horses could be caused by a splicing deficiency
of B4GALT7 that severely reduces expression of the
gene. The
conservation
of
the
affected
nucleotide
reflects a splicing requirement rather than a functional
requirement of the encoded amino acid. The clinical pic-
ture of the Friesian horse dwarfs adds to the phenotypic
variability observed in human patients with B4GALT7
mutations. Crosses between carriers can be prevented by
screening breeding horses for the B4GALT7 mutation
and the dwarfism trait could thus be eliminated from
the breed. The dwarfism in the horse is described as a dispropor-
tionate growth disturbance because all limbs are short,
while the head and back appear rather normal. In con-
trast, almost all confirmed human patients with LRS and
EDSP1 display facial dysmorphism and disproportional
growth was not noted [16, 17]. Cognitive functions do
not seem to be impaired in dwarf horses. Another clear
difference between the phenotype in man and horse is
that loose skin has never been observed in Friesian
dwarfs. Atrophic scarring and/or delayed wound healing
has been reported for a number of human patients but
is never seen in Friesian horse dwarfs. The fibroblasts
from one human patient displayed reduced proliferation
rates [20], while the fibroblasts of the Friesian dwarf pro-
liferated at least as fast as the fibroblasts from normal
Friesians. The differences in the clinical presentations
between human patients and Friesian dwarfs may be due
to the nature of the mutation in horses, which we think
has predominantly an effect on the expression level of a
normally functioning protein. On the other hand, the
protein may have rate limiting key roles in processes that
are different in the two species, such that loss of activity
becomes manifest in different ways. Methods Phenotypes, genotypes and genome-wide association study
Friesian horses (n = 29) were diagnosed as being dwarfs
by local equine veterinarians in consultation with the
Equine Clinic of Utrecht University, usually via a digital
in vivo picture for confirmation of the phenotype. Thirteen of the horses were male, 11 female and the sex
of 5 dwarfs was unknown. The group of unaffected con-
trols (n = 65) were Friesian horses without the character-
istic
appearance
of
dwarfism
[1]. In
addition,
we
obtained blood samples for DNA isolation from 8
parents of dwarfs and DNA samples from 177 Friesian
horses that were part of a DNA bank maintained at the
Dr. Van Haeringen Laboratory B.V.. g
y
Blood samples were taken and DNA was isolated as
described by Orr et al. [4]. Genotypes of 19 dwarfs
and 65 controls were obtained using the Illumina®
EquineSNP50 Genotyping BeadChip containing 54,602
SNPs. Quality
control
was
performed
using
the
check.marker function in the GenABEL package in R
[23]. SNPs with MAF <5 % and call-rate <90 % were
discarded, leaving 29,840 SNPs (54.7 % of all SNPs)
for the analysis. The B4GALT7 gene is highly expressed in the prolifer-
ative zone of the growth plate in rat [16]. Deficiency of
the encoded xylosylprotein beta-1,4-galactosyltransferase
7 apparently induces the irregularities of the chondro-
cyte columns seen in the growth plate of Friesian dwarfs
[1]. The enzyme adds the second of four saccharides that
form the linker between the protein core and the glycos-
aminoglycan moiety of proteoglycans. Proteoglycans are
major
components
of
molecular
networks
of
the Leegwater et al. BMC Genomics (2016) 17:839 Page 7 of 9 Page 7 of 9 The ccfast function in GenABEL package in R [23]
was used to determine the significance of allelic differ-
ences between dwarfs and unaffected horses with a
χ2-test (1df). The Bonferroni corrected significance level
applied was 1.68 × 10−6. Homozygosity mapping in the
significantly associated region was performed by eye
to identify overlapping regions of homozygosity be-
tween dwarfs. were performed using BigDye v3.1 (Thermo Fisher
Scientific, Waltham, MA) according to the manufac-
turer’s protocol. The products were analysed on a
3130XL Genetic Analyzer (Applied Biosystems, Foster
City, CA) and the data was analysed with Lasergene
(version 11 DNASTAR). Homologous DNA and protein sequences from differ-
ent species were retrieved from GenBank and aligned
one by one by eye. RNA analysis Fibroblasts were grown from 6 mm punch biobsies from
the skin of a dwarf, a carrier of the mutation of interest
and a Friesian horse homozygous for the reference allele. The biopsies were washed in Euroflush (IMV technolo-
gies, L’Aigle, France) containing 5000 IU/ml heparin, cut
with scissors and incubated in petri dishes with DMEM/
M199 (1:1) medium containing pen/strep (10,000 U/ml
(all from Thermo Fisher Scientific, Waltham, MA),
2.5 ng/ml basic-FGF5 (Peprotech, Rocky Hill, NJ) and
20 % FCS at 38.5 oC with 5 % CO2 and 5 % O2. Prolifer-
ating fibroblasts were harvested and passaged in culture
flasks using standard procedures. RNA was isolated from
cultured fibroblasts with the RNeasy kit with an on-
column DNase digestion according to the instructions of
the manufacturer (Qiagen, Hilden, Germany). The RIN
value of the RNA was meausured with an Agilent 2100
Bioanalyzer (Santa Clara, CA, USA) and was found to be
9.5 or higher. cDNA was synthesized with the iScript kit
(Bio-Rad Laboratories, Hercules, CA) using 500 ng of
RNA in 20 μl reactions. Splicing products were PCR
amplified from 0.5 μl cDNA product with B4GALT7
exonic
primers
5’-CTGGGAGCTCGAGCTCCATG-3’
(EX1F) and 5’-CTCAGGAAGCGGTGCATGTG-3’ (EX2R)
as described above. Unspliced products were amplified with
primer EX1F and the intronic primer IN1R described above. In a semi quantitative experiment, the 3 primers were com-
bined in a single PCR using the same component concen-
trations and cycling conditions as above. The fragments
were visualized by electrophoresis on a 1 % agarose gel in
0.5x TBE with 0.5 μg/ml ethidium bromide followed by UV
irradiation of the gel. The Illumina data was processed with our inhouse de-
veloped pipeline v 1.2.1 [24] including GATK v3.2.2 [25]
according to the best practices guidelines [26]. Briefly,
we mapped the pairs with BWA-MEM v0.7.5a [27],
marked duplicates, merged lanes, realigned indels. Base
recalibration did not improve our results, so this step
was skipped. Next, GATK Haplotypecaller was used to
call SNPs and indels. Variants are flagged as PASS only if
they do not meet the following criteria: QD < 2.0, MQ <
40.0,FS > 60.0, HaplotypeScore > 13.0, MQRankSum < -12.5,
ReadPosRankSum < -8.0, snpclusters > =3 in 35 bp. For
indels: QD < 2.0, FS >200.0, ReadPosRankSum < -20.0. Detecting recessive candidate variants was done with
snpSift [28] fitting the model of reference or potential
carrier status in the control and homozygous state in the
cases. Genome sequencing Four dwarf cases and three unrelated control were
paired-end sequenced with 150 nucleotide reads for the
full genome on Illumina NextSeq500 to an average
coverage of 4-9x according to the manufacturers proto-
cols. To increase the power to detect causal candidate
variants as a fully homozygous variant, we merged the
data for the four cases yielding an mean coverage of 36x. The Illumina data was processed with our inhouse de-
veloped pipeline v 1.2.1 [24] including GATK v3.2.2 [25]
according to the best practices guidelines [26]. Briefly,
we mapped the pairs with BWA-MEM v0.7.5a [27],
marked duplicates, merged lanes, realigned indels. Base
recalibration did not improve our results, so this step
was skipped. Next, GATK Haplotypecaller was used to
call SNPs and indels. Variants are flagged as PASS only if
they do not meet the following criteria: QD < 2.0, MQ <
40.0,FS > 60.0, HaplotypeScore > 13.0, MQRankSum < -12.5,
ReadPosRankSum < -8.0, snpclusters > =3 in 35 bp. For
indels: QD < 2.0, FS >200.0, ReadPosRankSum < -20.0. Four dwarf cases and three unrelated control were
paired-end sequenced with 150 nucleotide reads for the
full genome on Illumina NextSeq500 to an average
coverage of 4-9x according to the manufacturers proto-
cols. To increase the power to detect causal candidate
variants as a fully homozygous variant, we merged the
data for the four cases yielding an mean coverage of 36x. Methods Identities and differences were indi-
cated by using a word processor. The species were
selected arbitrary to represent close and distant mem-
bers of the animal kingdom. Competing interests 13. Almeida R, Levery SB, Mandel U, Kresse H, Schwientek T, Bennett EP,
Clausen H. Cloning and expression of a proteoglycan UDP-galactose:beta-
xylose beta-1,4-galactosyltransferase I: a seventh member of the human
beta4-galactosyltransferase gene family. J Biol Chem. 1999;274:26165–71. LHvdG is employed by Dr. Van Haeringen Laboratorium B.V. that performs
DNA diagnostic tests on a commercial basis. IH is director of the studbook
office of the KFPS (Koninklijke Vereniging “Het Friesch Paarden-Stamboek” i.e. Royal Friesian Horse Studbook). 14. Okajima T, Fukumoto S, Furukawa K, Urano T, Furukawa K. Molecular basis
for the progeroid variant of Ehlers-Danlos syndrome: identification and
characterization of two mutations in galactosyltransferase I gene. J Biol
Chem. 1999;274:28841–4. Author details
1 1Department of Clinical Sciences of Companion Animals, Faculty of
Veterinary Medicine, Utrecht University, PO Box 80154NL-3508 TD Utrecht,
The Netherlands. 2Animal Breeding and Genomics Centre, Wageningen
University, PO Box 338NL-6700 AH Wageningen, The Netherlands. 3Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht
University, Yalelaan 112-114, NL-3584 CM Utrecht, The Netherlands. 4Department of Medical Genetics, University Medical Center Utrecht, PO Box
85090NL-3508 AB Utrecht, The Netherlands. 5Dr. van Haeringen Laboratorium
B.V., PO Box 408NL-6700 AK Wageningen, The Netherlands. 6Koninklijke
Vereniging “het Friesch Paarden-Stamboek”, PO Box 624NL-9200 AP
Drachten, The Netherlands. 7Department of Surgery and Anaesthesiology of
Domestic Animals, Faculty of Veterinary Medicine, Ghent University,
Salisburylaan 133, B-9820 Merelbeke, Belgium. Funding
Thi
d This study was funded in part by the KFPS. This organization also identified
Friesian horses of interest to the study and facilitated the collection of
samples. 8. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G,
Mesirov JP. Integrative Genomics Viewer. Nat Biotechnol. 2011;29:24–6. 8. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G,
Mesirov JP. Integrative Genomics Viewer. Nat Biotechnol. 2011;29:24–6. 9. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P,
Kondrashov AS, Sunyaev SR. A method and server for predicting damaging
missense mutations. Nat Methods. 2010;7:248–9. 9. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P,
Kondrashov AS, Sunyaev SR. A method and server for predicting damaging
missense mutations. Nat Methods. 2010;7:248–9. Received: 14 May 2016 Accepted: 22 October 2016 Received: 14 May 2016 Accepted: 22 October 2016 Additional file 1: Variants with predicted moderate or high effect on
gene function in the critical chromosome region of Friesian dwarf horses. Output of snpSift analysis of Next Generation Sequence data. (XLSX 15 kb)
Additional file 2: Expression of B4GALT7 in relation to genotype. qPCR
data of B4GALT7 mRNA from fibroblasts. (XLSX 13 kb) Additional file 1: Variants with predicted moderate or high effect on
gene function in the critical chromosome region of Friesian dwarf horses. Output of snpSift analysis of Next Generation Sequence data. (XLSX 15 kb)
Addi i
l fil
2 E
i
f B GALT i
l i
PCR Availability of data and materials The whole genome sequence data is available at the European Nucleotide
Archive (ENA) [30] under study accession number PRJEB13863. 10. Reese MG, Eeckman FH, Kulp D, Haussler D. Improved Splice Site Detection
in Genie. J Comp Biol. 1997;4:311–23. 10. Reese MG, Eeckman FH, Kulp D, Haussler D. Improved Splice Site Detection
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(i.e. Royal Friesian Horse Studbook); LRS: Larsen of Reunion Island syndrome. 4. Orr N, Back W, Gu J, Leegwater P, Govindarajan P, Conroy J, Ducro B, Van
Arendonk JAM, MacHugh DE, Ennis S, Hill EW, Brama PA. Genome-wide SNP
association-based localization of a dwarfism gene in Friesian dwarf horses. Anim Genet. 2010;41 Suppl 2:2–7. 4. Orr N, Back W, Gu J, Leegwater P, Govindarajan P, Conroy J, Ducro B, Van
Arendonk JAM, MacHugh DE, Ennis S, Hill EW, Brama PA. Genome-wide SNP
association-based localization of a dwarfism gene in Friesian dwarf horses. Anim Genet. 2010;41 Suppl 2:2–7. Consent for publication
Not applicable. 15. Faiyaz-Ul-Haque M, Zaidi SHE, Al-Ali M, Al-Mureikhi MS, Kennedy S, Al-Thani
G, Tsui L-C, Teebi AS. A novel missense mutation in the
galactosyltransferase-I (B4GALT7) gene in a family exhibiting facioskeletal
anomalies and Ehlers-Danlos syndrome resembling the progeroid type. Am
J Med Genet A. 2004;128A:39–45. 15. Faiyaz-Ul-Haque M, Zaidi SHE, Al-Ali M, Al-Mureikhi MS, Kennedy S, Al-Thani
G, Tsui L-C, Teebi AS. A novel missense mutation in the
galactosyltransferase-I (B4GALT7) gene in a family exhibiting facioskeletal
anomalies and Ehlers-Danlos syndrome resembling the progeroid type. Am
J Med Genet A. 2004;128A:39–45. References 1. Back W, Van der Lugt JJ, Nikkels PGJ, Van den Belt AJM, Van der Kolk JH,
Stout TAE. Phenotypic diagnosis of dwarfism in six Friesian horses. Equine
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data of B4GALT7 mRNA from fibroblasts. (XLSX 13 kb) 2. University of Sydney: OMIA - Online Mendelian Inheritance in Animals. http://omia.angis.org.au/home/. Accessed 7 Dec 2015. 3. De Graaf-Roelfsema E, Back W, Keizer HA, Stout TA, van der Kolk JH. Normal
function of the hypothalamic-pituitary growth axis in three dwarf Friesian
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qPCR reactions contained 1x IQ SYBRGreen SuperMix
(Bio-Rad, Laboratories, Hercules, CA), 400 nM of each
primer and 1 μl cDNA, obtained as described above, in a
total volume of 15 μl. The reference genes were RPS19
and RPL13A, which were analyzed as described [29]. The
reactions were performed in a MyiQ2 thermal cycler and
the data was analyzed with IQ5 software (both from Bio-
Rad, Laboratories, Hercules, CA). Acknowledgements The photos of Fig. 1 were provided by the Multimedia Department, Faculty
of Veterinary Medicine, Utrecht University. We gratefully acknowledge the
assistence of Mrs. Yteke Elte of the Department of Equine Sciences of
Utrecht University with taking the skin biopsies. We are very grateful to the
owners of the horses that were part of this study. 5. NCBI: map viewer. http://www.ncbi.nlm.nih.gov/projects/mapview/map_
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d PAL, BJD, IH, WB and AS conceived and designed the experiments. MV-L, IJB,
FGvS, GRM, JWMB, BWD and LHvdG performed the experiments. PAL, BJD,
IJN and AS analyzed the data. IH and WB contributed materials. PAL and AS
wrote the manuscript. All authors read and approved the manuscript. 12. Voorbij AM, Van Steenbeek FG, Vos-Loohuis M, Martens EE, Hanson-Nilsson
JM, Van Oost BA, Kooistra HS, Leegwater PA. A contracted DNA repeat in
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and effect impact ‘HIGH’ or ‘MODERATE’ was required. Additional evaluation of the variant of interest was per-
formed with PolyPhen-2 [9]. y
The observed possibly detrimental DNA variant of
B4GALT7 was confirmed and evaluated in the complete
cohort by Sanger sequencing of PCR fragments. The
PCR primer sequences were 5’- AGTTTCTCGGAG
TGTAGAG-3’ (UP1F) and 5’-AGAGACATAGACCCTC
AGAG-3’ (IN1R). The PCR was performed with 50 ng
genomic DNA, 3 U Platinum Taq DNA polymerase
(Thermo
Fisher
Scientific,
Waltham,
MA),
2
mM
MgCl2, 0.2 mM each dNTP, 0.5 μM each primer, 1 M
betaine and 1× Platinum buffer. Temperature cycling
conditions were 5 min at 95 °C, 35 cycles of 30 s at 95 °
C, 30 s at 55 °C, 30 s at 72 °C, and a final elongation step
at 72 °C for 10 min. All amplifications were performed
on an ABI 9700 Thermal Cycler (Applied Biosystems,
Foster City, CA). The PRC primers were degraded by
addition of 1 U Exonuclease I (Thermo Fisher Scientific,
Waltham, MA) and incubation for 15 min. at 37 oC and
15 min. at 85 oC. DNA sequencing tercycle reactions For DNA sequence analysis and confirmation of the
origin of the products, the bands were cut from the gel
and the DNA was isolated with QIAquick gel extraction
kit (Qiagen, Hilden, Germany). The DNA sequencing
procedure was as described above. Quantitative PCR for measurement of B4GALT7
transcripts was performed with forward primer 5’- G
ACGGCAGGTCCAGGTTG-3’
and
reverse
primer
5’- ACAGGCAACGAAGAGGTGG-3’ at an annealing Page 8 of 9 Leegwater et al. BMC Genomics (2016) 17:839 Page 8 of 9 Page 8 of 9 temperature of 55 oC. The forward primer bridges exons 1
and 2, while the reverse primer is situated in exon 2. The
qPCR reactions contained 1x IQ SYBRGreen SuperMix
(Bio-Rad, Laboratories, Hercules, CA), 400 nM of each
primer and 1 μl cDNA, obtained as described above, in a
total volume of 15 μl. The reference genes were RPS19
and RPL13A, which were analyzed as described [29]. The
reactions were performed in a MyiQ2 thermal cycler and
the data was analyzed with IQ5 software (both from Bio-
Rad, Laboratories, Hercules, CA). temperature of 55 oC. The forward primer bridges exons 1
and 2, while the reverse primer is situated in exon 2. Leegwater et al. BMC Genomics (2016) 17:839 Leegwater et al. BMC Genomics (2016) 17:839 Fast and accurate short read alignment with Burrows-
Wheeler Transform. Bioinformatics. 2009;25:1754–60. 27. Li H, Durbin R. Fast and accurate short read alignment 27. Li H, Durbin R. Fast and accurate short read alignment with
Wheeler Transform. Bioinformatics. 2009;25:1754–60. Wheeler Transform. Bioinformatics. 2009;25:1754–60. 28. Cingolani P, Platts A, le Wang L, Coon M, Nguyen T, Wang L, Land SJ, Lu X,
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4 May 2016. 30. EMBL-EBI: European Nucleotide Archive. http://www.ebi.ac.uk/ena. Accessed
4 May 2016. • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
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Submit your manuscript at
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Submit your next manuscript to BioMed Central
and we will help you at every step: Ethics approval The horses that were included in the genome wide mapping, Next
Generation Sequencing and validation experiments were privately owned
and blood samples for DNA isolation were taken with informed consent
from the owners. The skin biopsies were taken from University Utrecht
owned Friesian teaching horses. Blood and skin sampling was done with
approval of the Animal Ethics Committee of the Utrecht University (DEC file
2013.III.01.012). 16. Guo MH, Stoler J, Lui J, Nilsson O, Bianchi DW, Hirschhorn JN, Dauber A. Redefining the progeroid form of Ehlers-Danlos syndrome: report of the
fourth patient with B4GALT7 deficiency and review of the literature. Am J
Med Genet A. 2013;161A:2519–27. 16. Guo MH, Stoler J, Lui J, Nilsson O, Bianchi DW, Hirschhorn JN, Dauber A. Redefining the progeroid form of Ehlers-Danlos syndrome: report of the
fourth patient with B4GALT7 deficiency and review of the literature. Am J
Med Genet A. 2013;161A:2519–27. Page 9 of 9 Leegwater et al. BMC Genomics (2016) 17:839 Leegwater et al. BMC Genomics (2016) 17:839 17. Cartault F, Munier P, Jacquemont ML, Vellayoudom J, Doray B, Payet C,
Randrianaivo H, Laville JM, Munnich A, Cormier-Daire V. Expanding the
clinical spectrum of B4GALT7 deficiency: homozygous p.R270C mutation
with founder effect causes Larsen of Reunion Island syndrome. Eur J Hum
Genet. 2015;23:49–53. 18. Salter CG, Davies JH, Moon RJ, Fairhurst J, Bunyan D; DDD Study, Foulds N. Further defining the phenotypic spectrum of B4GALT7 mutations. Am J
Med Genet A. 2016;doi:10.1002/ajmg.a.37604. 19. Laville JM, Lakermance P, Limouzy F. Larsen’s syndrome: review of the
literature and analysis of thirty-eight cases. J Pediatr Orthop. 1994;14:63–73. 20. Seidler DG, Faiyaz-Ul-Haque M, Hansen U, Yip GW, Zaidi SH, Teebi AS, Kiesel
L, Götte M. Defective glycosylation of decorin and biglycan, altered collagen
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Danlos syndrome patient carrying the novel Arg270Cys substitution in
galactosyltransferase I (beta4GalT-7). J Mol Med (Berl). 2006;84:583–94. 21. Jones KL, Schwarze U, Adam MP, Byers PH, Mefford HC. A homozygous B3GAT3
mutation causes a severe syndrome with multiple fractures, expanding the
phenotype of linkeropathy syndromes. Am J Med Genet A. 2015;167A:2691–6. 22. Ducro BJ, Schurink A, Bastiaansen JWM, Boegheim IJ, van Steenbeek FG,
Vos-Loohuis M, Nijman IJ, Monroe GR, Hellinga I, Dibbits BW, Back W,
Leegwater PA. A nonsense mutation in B3GALNT2 is concordant with
hydrocephalus in Friesian horses. BMC Genomics. 2015;16:761. 23. Aulchenko YS, Ripke S, Isaacs A, van Duijn CM. GenABEL: an R library for
genome-wide association analysis. Bioinformatics. 2007;23:1294–6. 24. University Medical Center Utrecht: Illumina analysis pipeline. https://github. com/CuppenResearch/IAP. Accessed 7 Dec 2015 25. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A,
Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. The Genome
Analysis Toolkit: a MapReduce framework for analyzing next-generation
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Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella K,
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Calls: The Genome Analysis Toolkit Best Practices Pipeline. Curr Protoc
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Calls: The Genome Analysis Toolkit Best Practices Pipeline. Curr Protoc
Bioinformatics. 2013;11:11.10.1–11.10.33. 27. Li H, Durbin R. Submit your next manuscript to BioMed Central
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Параллельный алгоритм метода циклической редукции для периодической краевой задачи
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Лилия Владимировна Логанова, старший преподаватель, каф. технической кибернетики.
Димитрий Львович Головашкин (д.ф.-м.н., с. н. с.), старший научный сотрудник, лаб. ди-
фракционной оптики. Ольга Сергеевна Сягайло, студент. Общероссийский математический портал Л. В. Логанова, Д. Л. Головашкин, О. С. Сягайло, Параллельный алгоритм
метода циклической редукции для периодической краевой задачи, Вестн. Сам. гос. техн. ун-та. Сер. Физ.-мат. науки, 2010, выпуск 1(), 197–204 DOI: 10.14498/vsgtu740 DOI: 10.14498/vsgtu740 Использование Общероссийского математического портала Math-Net.Ru подразумевает, что вы прочитали и
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24 октября 2024 г., 08:50:29 Вестн. Сам. гос. техн. ун-та. Сер. Физ.-мат. науки. — 2010. — №1 (20). — С. 197–204 Л. В. Логанова1, Д. Л. Головашкин2, О. С. Сягайло1 1 Самарский государственный аэрокосмический университет им. ак. С. П. Корол¨eва,
443086, Самара, Московское ш., 34. 2 Институт систем обработки изображений РАН,
443001, Самара, ул. Молодогвардейская, 151. E-mails: tk@smr.ru, dimitriy@smr.ru E-mails: tk@smr.ru, dimitriy@smr.ru Работа посвящена построению параллельного алгоритма, основанного на мето-
де циклической редукции для периодической краевой задачи. Произведено сравне-
ние с известным алгоритмом, приведены результаты численных эксперимен-
тов по исследованию ускорения алгоритма. Показана высокая эффективность
его применения. Ключевые слова: метод циклической редукции, параллельные алгоритмы. Введение. Математическое моделирование получает все большее распро-
странение в различных областях науки и техники, например, вычислительной
теплопередаче [1]. Развитие вычислительных средств приводит к необходимо-
сти интеграции методов математического моделирования и современных ком-
пьютерных технологий. Моделирование физических процессов зачастую сво-
дится к необходимости решения систем линейных алгебраических уравнений
(СЛАУ) вида Ax = b, где матрица A имеет ленточную структуру. Решение
технических задач в таком случае сопровождается существенным объёмом
вычислений. Именно этот фактор определяет растущую потребность в при-
менении параллельных вычислительных систем при решении таких СЛАУ,
и, как следствие, необходимость в синтезе алгоритмов, определяемых такой
архитектурой. Для создания параллельных алгоритмов решения СЛАУ с ленточной мат-
рицей традиционно используются следующие прямые методы: прогонки, цик-
лической редукции, декомпозиции области. Метод прогонки является эконо-
мичным, но плохо масштабируемым. Так, применение метода правой про-
гонки из работ [2,3] непосредственно для одномерной (1D) сеточной области
невозможно. Метод встречных прогонок из [4,5] не может быть применим для
решения разностных уравнений с циклическими граничными условиями. Ал-
горитм, основанный на методе циклической редукции [6,7], пользуется особой
популярностью для решения трёхдиагональных систем как для параллель-
ных, так и для векторных компьютеров, являясь привлекательным в силу 197 Л о г а н о в а Л. В., Г о л о в а ш к и н Д. Л., С я г а й л о О. С. присущей ему масштабируемости и небольшой вычислительной сложности. Данные соображения определили выбор авторов в пользу применения метода
циклической редукции. В современной литературе недостаточное внимание уделяется примене-
нию указанного метода при решении периодических одномерных задач по-
средством разностного подхода. Так, в [6] приведён случай непериодической
задачи, а в [7] рассмотрена двумерная задача. Настоящая работа призвана
восполнить этот пробел. Развивая подход, применяемый в непериодическом случае, авторы обра-
щаются к синтезу алгоритма для одномерной периодической задачи, его па-
раллельного варианта и подтверждают эффективность результатами вычис-
лительных экспериментов. 1. Циклическая редукция для случая трёхдиагональных систем. Предва-
рим представление параллельного алгоритма описанием метода циклической
редукции, приведенного в [6,8], для случая трёхдиагональных систем вида
dx1+ fx2
= b1,
fx1+dx2+ fx3
= b2,
fx2+dx3+fx4
= b3,
. . . {работаем с последним уравнением} else if j = 2(k−p−1)
c = b(p)
(2j−1)r −f (p)x(2j−2)r;
{ else if j
2
c = b(p)
(2j−1)r −f (p)x(2j−2)r;
{ ( j
)
{работаем с остальными уравнениями} ( j
)
{работаем с остальными уравнениями} else c = b(p)
(2j−1)r −f (p)(x2jr + x(2j−2)r);
nd end
Решить d(p)x(2j−1)r = c относительно x(2j−1)r
end
end end Представленный алгоритм записан в нотации Дж. Голуба [10], при этом
верно упомянутое ограничение на размерность системы и d(0) = d, f (0) = f,
b(0) = b. 2. Циклическая редукция для периодической краевой задачи. В практике
вычислительного эксперимента нередко встречаются периодические задачи
на круговых областях, бесконечных решётках и т. п. Для них система (1)
будет модифицирована в следующую:
dx1+ fx2
+
fxn
= b1,
fx1+dx2+ fx3
= b2,
fx2+dx3+fx4
= b3,
. . . ,
fx1
+
fxn−1+dxn
= bn. В отличие от (1), здесь n = 2k, где k ∈N. E-mails: tk@smr.ru, dimitriy@smr.ru ,
fxn−1+dxn
= bn,
(1)
dx1+ fx2
= b1,
fx1+dx2+ fx3
= b2,
fx2+dx3+fx4
= b3,
. . . ,
fxn−1+dxn
= bn,
(1) (1) где d, f, bi ∈R. Отметим существенное ограничение на размерность системы
n = 2k −1, где k ∈N. Основная идея метода циклической редукции [7, 8, 10] заключается в ис-
ключении неизвестных с номерами не кратными 2p на каждом шаге p редук-
ции. Алгоритм можно обобщить и сформулировать следующим образом: {прямой ход редукции} {прямой ход редукции} {прямой ход редукции} { р
д р ду ц
}
{цикл от начальной системы до последней редуцированной системы}
k { р
д р ду ц
}
{цикл от начальной системы до последней редуцированной системы}
for p = 1 : k −1 {цикл от начальной системы до последней редуцированной системы}
for p = 1 : k −1
{поиск коэффициентов новой редуцированной системы}
d(p) = 2[f (p−1)]2 −[d(p−1)]2,
f (p) = [f (p−1)]2
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
r = 2p for p = 1 : k −1
{поиск коэффициентов новой редуцированной системы}
d(p) = 2[f (p−1)]2 −[d(p−1)]2,
( )
(
1) 2 f
[f
]
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
r = 2p r
2
{поиск правых частей новой редуцированной системы}
(
) r
2
{поиск правых частей новой редуцированной системы} р
р
у
р
for j = 1 : 2(k−p) −1
b(p)
jr = f (p−1)(b(p−1)
jr−r/2 + b(p−1)
jr+r/2) −d(p−1)b(p−1)
jr end end {обратный ход редукции} р
р
у
Решить d(k−1)x2k−1 = b(k−1) 2
2
{цикл по редуцированным системам до начальной системы}
for p = k −2 : 0 : −1 p
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
r
2p p
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
r
2p r
2
{цикл по элементам редуцированной системы} r
2
{цикл по элементам редуцированной системы} j
{работаем с первым уравнением} f j
1
c = b(p)
(2j−1)r −f (p)x2jr; 198 Параллельный алгоритм метода циклической редукции {работаем с последним уравнением}
(
) {работаем с последним уравнением} Используя подход [7], запишем
алгоритм циклической редукции, учитывая ограничение на размерность си-
стемы: {прямой ход редукции}
{цикл от начальной системы до предпоследней редуцированной системы}
f
1
k
2 {прямой ход редукции}
{цикл от начальной сис {прямой ход редукции}
{цикл от начальной сис {прямой ход редукции}
{цикл от начальной системы до предпоследней редуцированной системы}
for p = 1 : k −2 { р
д р ду ц
}
{цикл от начальной системы до предпоследней редуцированной системы}
for p = 1 : k −2 for p = 1 : k −2
{поиск коэффициентов новой редуцированной системы}
d(p) = 2[f (p−1)]2 −[d(p−1)]2,
f (p) = [f (p−1)]2
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
r = 2p for p = 1 : k −2
{поиск коэффициентов новой редуцированной системы}
d(p) = 2[f (p−1)]2 −[d(p−1)]2,
f (p) = [f (p−1)]2
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
r = 2p f ( ) = [f (
)]
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
2p r
2
{поиск правых частей новой редуцированной системы}
(k
) r
2
{поиск правых частей новой редуцированной системы}
(k
p) for j = 1 : 2(k−p) −1
b(p)
jr = f (p−1)(b(p−1)
jr−r/2 + b(p−1)
jr+r/2) −d(p−1)b(p−1)
jr
end end end end
{поиск коэффициентов последней редуцированной системы}
k
1 end
{поиск коэффициентов последней редуцированной системы}
p
k
1 p = k −1
d(p) = 2[f (p−1)]2 −[d(p−1)]2,
f (p) = 2[f (p−1)]2
{ p = k −1
d(p) = 2[f (p−1)]2 −[d(p−1)]2,
f (p) = 2[f (p−1)]2 f
= 2[f
]
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
r
2p f
2[f
]
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
2p r = 2p
{поиск правых частей последней редуцированной системы}
(k
) r = 2p
{поиск правых частей последней редуцированной системы}
(
) у
for j = 0 : 2(k−p) −1
if j=0
b(p)
jr = f (p−1)(b(p−1)
2k−r/2 + b(p−1)
jr+r/2) −d(p−1)b(p−1)
jr
else 199 Л о г а н о в а Л. В., Г о л о в а ш к и н Д. Л., С я г а й л о О. С. {работаем с последним уравнением} b(p)
jr = f (p−1)(b(p−1)
jr−r/2 + b(p−1)
jr+r/2) −d(p−1)b(p−1)
jr
d {обратный ход редукции} {обратный ход редукции}
Решить систему относительно x0, x2k−1 Решить систему относительно x
d(k−1)x0 + f (k−1)x2k−1 = b(ik−1)
0
f (k−1)x0 + d(k−1)x2k−1 = b(k−1)
(2k−1) (
{цикл по редуцированным системам до начальной системы}
for p = k −2 : 0 : −1 (
{цикл по редуцированным системам до начальной системы}
for p = k −2 : 0 : −1
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
r = 2p for p
k
2 : 0 :
1
{r – расстояние между двумя соседними уравнениями новой системы
в старой системе}
r = 2p r
2
{цикл по элементам редуцированной системы} r
2
{цикл по элементам редуцированной системы}
(k
p
1) р
у
р
for j = 1 : 2(k−p−1) for j = 1 : 2
{работаем с первым уравнением} for j
1 : 2
{работаем с первым уравнением} if j = 1
c = b(p)
(2j−1)r −f (p)x2jr −f (p)x0
работаем с последним уравнением} if j = 1
c = b(p)
(2j−1)r −f (p)x2jr −f (p)x0
{работаем с последним уравнением} р
ур
else if j = 2(k−p−1)
c = b(p)
(2j−1)r −f (p)x(2j−2)r −f (p)x0; ( j
)
{работаем с остальными уравнениями}
else else
c = b(p)
(2j−1)r −f (p)(x2jr + x(2j−2)r);
nd Решить d(p)x(2j−1)r = c относительно x(2j−1)r
end end Здесь также верно упомянутое ограничение на размерность системы и
d(0) = d, f (0) = f, b(0) = b. , f
f,
3. Параллельный алгоритм метода циклической редукции. Заметим, что
операции, ведущие к новым уравнениям на каждом шаге циклической ре-
дукции, могут выполняться параллельно. Рассмотрим случай, когда есть два
процессора (L = 2). р
р
(
)
Произведём разбиение на равные части вектора правых частей b: b1 =
= b(0 : n
2 −1) и b2 = b(n
2 : n −1) и вектора неизвестных x: x1 = x(0 : n
2 −1)
и x2 = x(n
2 : n −1), где верхний индекс — номер процессора, выполняющего
действия по алгоритму с данной частью вектора. Вычисления по алгоритму включают нижеследующие этапы. 1. В течение k−2 шагов каждым процессором осуществляется поиск коэф-
фициентов и правых частей новой редуцированной системы. По завершении
производится обмен коэффициентами последнего уравнения, пересчитанного
на данном шаге (прямой ход редукции). 1. В течение k−2 шагов каждым процессором осуществляется поиск коэф-
фициентов и правых частей новой редуцированной системы. По завершении
производится обмен коэффициентами последнего уравнения, пересчитанного
на данном шаге (прямой ход редукции). (
)
2. На последнем шаге прямого хода циклической редукции после вычисле-
ния коэффициентов редуцированной системы и правых частей выполняется
пересылка коэффициентов последнего уравнения от второго процессора пер-
вому. у
3. Получив данные, первый процессор производит вычисления x0, x n
2 и пе-
ресылает результат второму процессору (обратный ход редукции). 4. Каждый процессор выполняет обратный ход редукции для хранящейся
у него половины исходной системы без коммуникаций. у
у
Схема работы алгоритма представлена на рис. 1. При прямом ходе (рис. 1, а) 200 Параллельный алгоритм метода циклической редукции Рис. 1. r
2
{цикл по элементам редуцированной системы} Схема работы алгоритма для двух процессоров: а — прямой,
д — обратный ходы редукции, выполняемые всеми процессорами; б, в,
г — пересылки данных Рис. 1. Схема работы алгоритма для двух процессоров: а — прямой,
д — обратный ходы редукции, выполняемые всеми процессорами; б, в,
г — пересылки данных в конце каждого шага редукции все процессоры будут содержать коэффици-
енты в точности половины уравнений новой редуцированной системы. До
начала следующего шага процессорам необходимо обменяться коэффициен-
тами одного уравнения (рис. 1, б). На последнем шаге прямого хода редукции
только второй процессор пересылает коэффициент дополнительного уравне-
ния (рис. 1, в). После получения первым процессором этого коэффициента
начинается обратный ход редукции. Первый шаг обратного хода выполняет-
ся первым процессором в одиночку, результат посылается второму процессо-
ру (рис. 1, г). Далее каждый процессор выполняет обратный ход редукции
(рис. 1, д) без коммуникаций. (р
,
)
у
Рассмотрим теперь четырёхпроцессорный вариант алгоритма (L = 4). Как и ранее, процессоры могут параллельно выполнять операции, порож-
дающие редуцированные системы. Произведём разбиение на равные части
вектора правых частей b: b1, b2, b3, b4 и вектора неизвестных x: x1, x2, x3, x4,
где индекс — номер процессора, выполняющего действия по алгоритму с дан-
ной частью вектора. Вычисления по алгоритму, как и предыдущем случае,
можно разделить на 4 этапа. При прямом ходе в конце каждого шага редукции каждый процессор бу-
дет содержать коэффициенты в точности четверти от общего числа уравне-
ний новой редуцированной системы. До начала следующего шага процессо-
рам необходимо будет переслать друг другу коэффициенты последнего урав-
нения, пересчитанные на данном шаге редукции. На предпоследнем шаге пря-
мого хода редукции второй, третий и четвёртый процессоры пересылают пер-
вому процессору коэффициенты одного уравнения. Последние два шага ре- 201 Л о г а н о в а Л. В., Г о л о в а ш к и н Д. Л., С я г а й л о О. С. дукции реализуются первым процессором. После этого начинается обратный
ход редукции. Первые два шага обратного хода выполняются первым про-
цессором в одиночку, результаты посылаются остальным процессорам. Далее
каждый процессор выполняет обратный ход редукции для хранящейся у него
части уравнений исходной системы без коммуникаций. Обобщим данный подход на произвольное число задач L. Произведем раз-
биение на L равных частей вектора правых частей b: b1, b2, . . . , bL и вектора
неизвестных x: x1, x2, . . . , xL, где индекс — номер процессора, выполняющего
действия по алгоритму с данной частью вектора. r
2
{цикл по элементам редуцированной системы} Вычисления по алгоритму
включают нижеследующие этапы. 1. В течение k−2 шагов каждым процессором осуществляется поиск коэф-
фициентов и правых частей новой редуцированной системы. По завершении
производится обмен коэффициентами последнего уравнения, пересчитанного
на данном шаге (прямой ход редукции). 2. На предпоследнем шаге прямого хода циклической редукции после вы-
числения коэффициентов и правых частей редуцированной системы выпол-
няется пересылка коэффициентов последнего уравнения первому процессору
от процессоров 2, 3, . . . , n. 3. Получив данные, первый процессор реализует последние шаги редук-
ции. 4. В течении log2 L шагов обратного хода первый процессор производит
вычисление искомых значений в одиночку и рассылает остальным процессо-
рам. р
5. Каждый процессор выполняет обратный ход редукции для хранящейся
у него части исходной системы без коммуникаций. 4. Исследование ускорения. Проведём сравнение представленного парал-
лельного алгоритма циклической редукции для периодической краевой за-
дачи и параллельного алгоритма метода встречных циклических прогонок
для одной и той же задачи [9]. Несомненным достоинством представленного
алгоритма является его масштабируемость. Данное свойство позволяет полу-
чить преимущество в скорости вычислений, несмотря на их больший объём
(O(N log2 N) и O(N) — алгоритм из [9]), а также наличие простоев. ( (
2
)
(
)
[ ])
Результаты вычислительных экспериментов подтверждают это. Макси-
мальное ускорение алгоритма из [9] для одномерной сеточной области дости-
гает величины 1,9, в то время как ускорение предложенного алгоритма для
4-х процессоров соответствует 2,5. Недостатком синтезированного алгоритма
является привязка к степени двойки, что сужает область его применения. Вычислительные эксперименты проводились на кластере, состоящем из
четырёх ЭВМ AMD Sempron™Processor 3000+ (1000 MHz) и ОЗУ 1 Гб, свя-
занных локальной сетью Ethernet 100 MB, и работающем под операционной
системой GNU Linux (ядро 2.6.17). На рис. 2 представлены результаты исследования ускорения параллель-
ного алгоритма метода циклической редукции для периодической задачи в
случае двух и четырех процессов. Двухзадачный алгоритм характеризуется максимальным значением уско-
рения в 1,85 раза для (N = 67·106), а четырёхзадачный — в 2,5 раза. В случае
двухзадачного алгоритма ускорение по значению близко к максимально воз-
можному по закону Амдала. 202 Параллельный алгоритм метода циклической редукции Рис. 2. Графики зависимости ускорений от размерности задачи
(n = 2k): сплошная линия — для двухмерной, штриховая — для четы-
рёхмерных задач Рис. 2. Графики зависимости ускорений от размерности задачи
(n = 2k): сплошная линия — для двухмерной, штриховая — для четы-
рёхмерных задач Сложность процесса взаимодействия процессоров между собой привела
к потерям во времени работы четырёхпроцессорного алгоритма. r
2
{цикл по элементам редуцированной системы} Для него
использовалась топология коммуникаций в виде кольца, хотя на самом деле
все пересылки осуществлялись через сервер. Заключение. Применение параллельного алгоритма, основанного на ме-
тоде циклической редукции, при исследовании периодических краевых задач
разностным методом характеризуется высоким ускорением и эффективно-
стью. Представляется целесообразным исследование зависимости ускорения
от схемы коммуникаций и развитие данного подхода для случая двумерной
сеточной области. Работа выполнена при поддержке российско-американской программы «Фундаменталь-
ные исследования и высшее образование» (BRHE), гранта Президента РФ поддержки ве-
дущих научных школ (код проекта НШ–3086.2008.9) и гранта РФФИ (код проекта 07–07–
00210–а). Liliya V. Loganova, Lecturer, Dept. of Technical Cybernetics. Dimitrii L. Golovashkin (Dr.
Sci. (Phys. & Math.)), Leading Research Scientist, Lab. of Diffractive Optics.
Ol’ga S. Syagailo, Student. БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. Самарский А. А., Вабищевич П. Н. Вычислительная теплопередача. — М.: Едиториал
УРСС, 2003. — 784 с. 2. Миренков Н. Н. Параллельные алгоритмы для решения задач на однородных вычисли-
тельных системах // Вычислительные системы, 1973. — №57. — C. 3–32. 3. Бирюкова Л. Н., Четверушкин Б. Н. О возможности реализации квазигидродинамиче-
ской модели полупроводниковой плазмы на многопроцессорных вычислительных си-
стемах // Матем. моделирование, 1991. — Т. 3, №6. — C. 61–71. //
4. Милюкова О. Ю. Параллельный вариант обобщенного попеременно-треугольного ме-
тода для решения эллиптических уравнений // Ж. вычисл. матем. и матем. физ.,
1998. — Т. 38, №12. — C. 2002–2012;
англ. пер.: Milyukova O. Yu. A parallel variant of
the generalized alternating triangular method for elliptic equations // Comput. Math. Math. Phys., 1998. — Vol. 38, No. 12. — P. 1922–1932. 5. Головашкин Д. Л. Параллельные алгоритмы решения сеточных уравнений трехдиаго-
нального вида, основанного на методе встречных прогонок // Матем. моделирование,
2005. — Т. 17, №11. — C. 118–128. ,
6. Ярмушкин С. В., Головашкин Д. Л. Исследование параллельных алгоритмов решения
трёхдиагональных систем линейных алгебраических уравнений // Вестн. Сам. гос. техн
ун-та. Сер. Физ.-мат. науки, 2004. — №26. — C. 78–82. у
р
у
,
7. Самарский А. А., Николаев Е. С. Методы решения сеточных уравнений. — М.: Наука,
1978. — 561 с. 8. Ortega J. M. Introduction to Parallel and Vector Solution of Linear Systems. — New York:
Plenum Press, 1988. — 318 p.;
русск. пер.: Ортега Дж. Введение в параллельные и
векторные методы решения линейных систем. — М.: Мир, 1991. — 367 с. 203 Л о г а н о в а Л. В., Г о л о в а ш к и н Д. Л., С я г а й л о О. С. 9. Логанова Л. В. Параллельный алгоритм метода циклических прогонок // Вестн. Сам. гос. аэрокосм. ун-та, 2008. — №2(15). — C. 167–174. (
)
10. Golub G., van Loan Ch. Matrix Computations: 3rd edit., Johns Hopkins University Press,
1996. — 694 p.; русск. пер.: Голуб Дж., Ван Лоун Ч. Матричные вычисления. — М.:
Мир, 1999. — 548 с. Поступила в редакцию 03/X/2009;
в окончательном варианте — 03/III/2010. 10. Golub G., van Loan Ch. Matrix Computations: 3rd edit., Johns Hopkins University Press,
1996. — 694 p.; русск. пер.: Голуб Дж., Ван Лоун Ч. Матричные вычисления. — М.:
Мир, 1999. — 548 с. L. V. Loganova, D. L. Golovashkin, O. S. Syagailo 1 S. P. Korolyov Samara State Aerospace University,
34, Moskovskoe sh., Samara, 443086. 34, Moskovskoe sh., Samara, 443086. 2 2 Image Processing Systems Institute, 151, Molodogvardeyskaya str., Samara, 443001. E-mails: tk@smr.ru, dimitriy@smr.ru We report constructing a parallel algorithm based on the cyclic reduction method in the
boundary-value problem. Comparison with the familiar algorithms has been made. Re-
sults of the studies into the acceleration of the algorithm are discussed. The algorithm
is shown to be highly efficient. Key words: cyclic reduction method, parallel algorithm. Original article submitted 03/X/2009;
revision submitted 03/III/2010. Original article submitted 03/X/2009;
revision submitted 03/III/2010.
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Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus
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Journal of virology
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IMPORTANCE During the frameshift, the PRF typically involve a “slippery” heptanucleotide sequence,
where the shift in reading frame actually occurs, and a down-
stream RNA stem-loop or pseudoknot structure. The consensus
motif for the shift site sequence is X_XXY_YYZ, where XXX nor-
mally represents any three identical nucleotides (though certain
exceptions occur, including GGU), YYY represents AAA or UUU,
Z represents any nucleotide except G, and underscores separate
codons in the initial reading frame (12). During the frameshift, the T Received 21 April 2015 Accepted 1 June 2015
Accepted manuscript posted online 10 June 2015
Citation Finch LK, Ling R, Napthine S, Olspert A, Michiels T, Lardinois C, Bell S,
Loughran G, Brierley I, Firth AE. 2015. Characterization of ribosomal frameshifting
in Theiler’s murine encephalomyelitis virus. J Virol 89:8580–8589. doi:10.1128/JVI.01043-15. Editor: A. Simon
Address correspondence to Andrew E. Firth, aef24@cam.ac.uk. Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JVI.01043-15. Copyright © 2015 Finch et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution 3.0 Unported license. doi:10.1128/JVI.01043-15 Received 21 April 2015 Accepted 1 June 2015
Accepted manuscript posted online 10 June 2015
Citation Finch LK, Ling R, Napthine S, Olspert A, Michiels T, Lardinois C, Bell S,
Loughran G, Brierley I, Firth AE. 2015. Characterization of ribosomal frameshifting
in Theiler’s murine encephalomyelitis virus. J Virol 89:8580–8589. doi:10.1128/JVI.01043-15. Editor: A. Simon
Address correspondence to Andrew E. Firth, aef24@cam.ac.uk. Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JVI.01043-15. Copyright © 2015 Finch et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution 3.0 Unported license. doi:10.1128/JVI.01043-15 Received 21 April 2015 Accepted 1 June 2015
Accepted manuscript posted online 10 June 2015
Citation Finch LK, Ling R, Napthine S, Olspert A, Michiels T, Lardinois C, Bell S,
Loughran G, Brierley I, Firth AE. 2015. Characterization of ribosomal frameshifting
in Theiler’s murine encephalomyelitis virus. J Virol 89:8580–8589. doi:10.1128/JVI.01043-15. Editor: A. Simon
Address correspondence to Andrew E. Firth, aef24@cam.ac.uk. Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JVI.01043-15. Copyright © 2015 Finch et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution 3.0 Unported license. doi:10.1128/JVI.01043-15 Many RNA viruses contain sequences that stimulate a propor-
tion of ribosomes to undergo a 1 frameshift and to continue
translating in an alternative reading frame. ABSTRACT Theiler’s murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of posi-
tive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis vi-
rus, a programmed 1 ribosomal frameshift (1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding
region of the polyprotein open reading frame (ORF). Here we show that 1 PRF occurs at a similar site during translation of the
TMEV genome. In addition, we demonstrate that a predicted 3= RNA stem-loop structure at a noncanonical spacing downstream
of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the
G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque
phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the
highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient 1 PRF in TMEV provides a
mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of
picornaviruses. on October 2, 2015 by MRC LAB OF MOLECULAR BIO
http://jvi.asm.org/
m on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY Characterization of Ribosomal Frameshifting in Theiler’s Murine
Encephalomyelitis Virus on October 2, 2015 by M
http://jvi.asm.org/
Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
http://jvi.asm.org/
Downloaded from Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdoma; de Duve Institute, Université Catholique de
Louvain, Brussels, Belgiumb; School of Biochemistry and Cell Biology, Western Gateway Building, University College Cork, Cork, Irelandc 8580
jvi.asm.org IMPORTANCE Many viruses utilize programmed 1 ribosomal frameshifting (1 PRF) to produce different protein products at a defined ra-
tio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, 1 PRF occurs on specific “slippery” hep-
tanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of 1 PRF in Theiler’s murine encephalomyelitis virus (TMEV) that is extraordinarily efficient
(74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of 1 PRF, is depen-
dent upon a stem-loop structure beginning 14 nt downstream of the slippery site. Furthermore, in TMEV-based reporter con-
structs in transfected cells, efficient frameshifting is critically dependent upon virus infection. We suggest that TMEV evolved
frameshifting as a novel mechanism for removing ribosomes from the message (a “ribosome sink”) to downregulate synthesis of
the 3=-encoded replication proteins. 2015 by MRC LAB OF MOLECULAR BIOLOGY T
he genus Cardiovirus of the family Picornaviridae (a family of
positive-sense single-stranded RNA viruses) currently con-
tains two species, Encephalomyocarditis virus (EMCV) and Thei-
lovirus. The latter encompasses a number of divergent viruses,
including Theiler’s murine encephalomyelitis virus (TMEV), rat
theilovirus (RTV), and Saffold virus (SAFV). The approximately
8-kb genome contains a long open reading frame (ORF) that is
translated as a polyprotein and subsequently processed by the vi-
rus-encoded 3C protease (1–3). Separation of the L-1ABCD-2A
and 2BC-3ABCD components, however, occurs cotranslationally
via a process termed “StopGo” or “Stop-Carry on” (4–6) (Fig. 1). This unusual proteolysis-independent but ribosome-dependent
mechanism is mediated by the amino acid motif D(V/I)ExNPG|P
(where the symbol “|” represents the junction between 2A and
2B), together with less conserved but nonetheless functionally im-
portant upstream amino acids, which prevent the formation of a
peptide bond between Gly and Pro but allow the continuation of
translation with up to near-100% efficiency (7–11). PRF typically involve a “slippery” heptanucleotide sequence,
where the shift in reading frame actually occurs, and a down-
stream RNA stem-loop or pseudoknot structure. The consensus
motif for the shift site sequence is X_XXY_YYZ, where XXX nor-
mally represents any three identical nucleotides (though certain
exceptions occur, including GGU), YYY represents AAA or UUU,
Z represents any nucleotide except G, and underscores separate
codons in the initial reading frame (12). IMPORTANCE Two PCR products were then generated using
primers overlapping these restriction sites at one end and primers span-
ning the TMEV sequence and part of the V5 or HA sequence at the other. These fragments fused the first codon following the StopGo junction with
the 5= terminus of the tag coding region and the 3= terminus of this region
with the rest of the 2B/2B* sequence. Different variants were produced to
allow the generation of tagged WT, SS, LVWT, and StopGo and shift site
mutant (LVSS) clones. The fragments were joined by overlap extension
PCR and subcloned back into the modified full-length clone containing
the extra restriction sites. The V5 tag, including a glycine-serine linker,
was GKPIPNPLLGLDSTGSGSGS, while the HA tag was YPYDVPDYA. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY 2015 by MRC LAB OF MOLECULAR BIOLOGY p
Recently, EMCV was shown to utilize 1 PRF at a conserved
G_GUU_UUU sequence positioned just downstream of the
StopGo site. Frameshifting results in the production of a novel
14-kDa “transframe” protein (termed 2B*) whose N-terminal 11
or 12 amino acids (depending on strain) are encoded within the
polyprotein open reading frame (ORF) and whose C-terminal 117
amino acids are encoded by an overlapping ORF in the 1 reading
frame (19). In theiloviruses, a G_GUU_UUU sequence is con-
served at a similar position in the genome, but a long overlapping
ORF in the 1 reading frame is lacking (Fig. 1). If 1 PRF were to
occur at this site in these viruses, the resulting transframe protein
would be only 14 or 15 amino acids in length. Dual-luciferase frameshift reporter plasmids were prepared by intro-
ducing annealed oligonucleotide pairs into XhoI/BglII-digested pIDluc. Plasmid pIDluc is a derivative of the pDluc vector with the mengovirus
internal ribosome entry site (IRES) (DQ294633, nt 256 to 770) inserted
downstream of the T7 promoter of pDluc such that the second codon of
the Renilla luciferase gene is fused to the first seven codons of the mengo-
virus polyprotein ORF, allowing cap-independent translation. Here we show that 1 PRF does indeed occur in TMEV and is
functionally important for virus propagation. Frameshifting in
TMEV is extraordinarily efficient (74 to 82%); to our knowledge,
this is the highest frameshifting efficiency reported to date for any
virus. IMPORTANCE on October 2, 2015 by MRC LAB OF MOLECULAR BIO
http://jvi.asm.org/
om P- and A-site tRNAs detach from the zero frame codons XXY and
YYZ and re-pair in the 1 frame to XXX and YYY. Frameshifting
on a suitable shift site is stimulated to high levels (typically to a
final 1 PRF efficiency of 5 to 45%, depending on the particular
frameshift sequence) by a downstream stimulatory element usu-
ally in the form of an RNA pseudoknot or a stem-loop structure,
separated from the shift site by a 5- to 9-nucleotide (nt) spacer (13,
14). RNA structures at this distance from the shift site are thought
to be located at the mRNA unwinding site of the mRNA entrance
channel of the ribosome when the shift site sequence is located
within the decoding center (15, 16). Failure to efficiently unwind
the RNA structure—perhaps due to the presentation of an un-
usual topology—is thought to interfere with ribosome progressiv-
ity and may also induce tension within the mRNA that leads to
uncoupling and subsequent realignment of the codon-anticodon
duplexes (15, 17, 18). The GDVII strain of TMEV generated from the full-length infectious
clone pSK-GDVII was used (a kind gift from the Robert Fujinami lab,
University of Utah). The sequence of this clone is identical to GenBank
accession no. NC_001366.1 except for three nucleotide differences:
G2241A (serine to isoleucine in VP2), A2390G (synonymous change in
VP3), and G4437A (lysine to glutamine in 2B). Nucleotide coordinates
herein are given with respect to NC_001366.1 (20). Recombinant viruses and plasmids. QuikChange mutagenesis (Agi-
lent Technologies) was performed on a template containing the full-
length viral insert to create the full-length recombinant TMEV shift site
mutant (SS), stop codon mutant (SCM), and StopGo mutant (LVWT). All constructs were verified by sequencing of the complete virus genome. Recombinant viruses and plasmids. QuikChange mutagenesis (Agi-
lent Technologies) was performed on a template containing the full-
length viral insert to create the full-length recombinant TMEV shift site
mutant (SS), stop codon mutant (SCM), and StopGo mutant (LVWT). All constructs were verified by sequencing of the complete virus genome. V5 and HA-tagged virus variants were created as follows. First, two
unique restriction sites (a BsiWI site starting at nt 4166 and a SalI site
starting at nt 4536) were introduced (using synonymous changes) into a
subclone by site-directed mutagenesis, and the fragment was cloned back
into the full-length clone. IMPORTANCE We also demonstrate that a 3= RNA stem-loop structure is
involved in frameshift stimulation but acts from a location further
downstream of the slippery sequence (14 nt) than is typical (5 to 9
nt). As in EMCV, but in contrast to nearly all other known cases,
frameshifting in TMEV depends on virus infection, suggesting the
involvement of a frameshift-stimulatory trans-acting factor. In vitro transcription and generation of recombinant virus. Plas-
mids were linearized with XbaI and transcribed with T7 RNA polymerase
(Ambion) for 3 h at 37°C as recommended by the manufacturer. RNA
integrity was confirmed by electrophoresis, and RNA was transfected into
BHK-21 cells using DMRIE-C (Invitrogen) according to the manufactur-
er’s recommendations. Once cytopathic effect was observed, cells were
subjected to three rounds of freeze-thawing, followed by centrifugation
for 5 min at 5,000 rpm to pellet cell debris. Virus-containing supernatant
was titrated and stored at 80°C. IMPORTANCE Where functionally
utilized, this is termed programmed 1 ribosomal frameshifting
(1 PRF). In eukaryotic systems, the stimulatory elements for 1 8580
jvi.asm.org August 2015
Volume 89
Number 16 Journal of Virology Ribosomal Frameshifting in TMEV FIG 1 Schematic representation of the TMEV genome. The predicted 1 PRF site is situated at codons 5 and 6 downstream of the junction between the 2A and
2B coding sequences. Frameshift translation would yield a 14-aa transframe protein termed 2B*, whose N terminus would be encoded in the polyprotein frame
(blue) and whose C terminus would be encoded in the 1 frame (pink). on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
http://jvi.asm.org/
Downloaded from on October 2, 2015 by M
http://jvi.asm.org/
Downloaded from FIG 1 Schematic representation of the TMEV genome. The predicted 1 PRF site is situated at codons 5 and 6 downstream of the junction between the 2A and
2B coding sequences. Frameshift translation would yield a 14-aa transframe protein termed 2B*, whose N terminus would be encoded in the polyprotein frame
(blue) and whose C terminus would be encoded in the 1 frame (pink). virus was removed, and cells were overlaid with DMEM supplemented
with 2% FBS for various times. P- and A-site tRNAs detach from the zero frame codons XXY and
YYZ and re-pair in the 1 frame to XXX and YYY. Frameshifting
on a suitable shift site is stimulated to high levels (typically to a
final 1 PRF efficiency of 5 to 45%, depending on the particular
frameshift sequence) by a downstream stimulatory element usu-
ally in the form of an RNA pseudoknot or a stem-loop structure,
separated from the shift site by a 5- to 9-nucleotide (nt) spacer (13,
14). RNA structures at this distance from the shift site are thought
to be located at the mRNA unwinding site of the mRNA entrance
channel of the ribosome when the shift site sequence is located
within the decoding center (15, 16). Failure to efficiently unwind
the RNA structure—perhaps due to the presentation of an un-
usual topology—is thought to interfere with ribosome progressiv-
ity and may also induce tension within the mRNA that leads to
uncoupling and subsequent realignment of the codon-anticodon
duplexes (15, 17, 18). August 2015
Volume 89
Number 16 RESULTS 2015 by MRC LAB OF MOLECULAR BIOLOGY Mutating the predicted frameshift site attenuates virus growth. In the GDVII strain of TMEV, the mass of the predicted 14-ami-
no-acid (aa) transframe fusion protein that would arise from 1
PRF is only 1.4 kDa. Products of such small size are inherently
difficult to detect by SDS-PAGE and/or Western blotting. There-
fore, we decided to first seek genetic evidence for the functional
importance of the predicted frameshift site in TMEV through mu-
tagenesis of an infectious clone. Three mutants were generated
(Fig. 2A): (i) G_GUU_UUU to A_GUG_UUU (mutations are in
bold) at nt 4244 to 4250 to prevent frameshifting at the predicted
shift site (SS); (ii) ACU_AAA to ACA_AAA at nt 4272 to 4277 to
remove the UAA stop codon of the predicted 2B* product, thus
extendingitbyafurther21aainlength(SCM); and (iii) GGC_CCU
to CUG_GUC at nt 4230 to 4235 to inhibit StopGo by introducing
GP-to-LV amino acid changes at the junction between 2A and 2B
(LVWT). This mutation was shown previously to inhibit StopGo
from occurring, thereby fusing the 2A and 2B (or 2B*) proteins
together, and this mutant was constructed to assess the relative
importance of StopGo and frameshifting for TMEV replication
and to assess whether StopGo affects frameshifting. The SS and
SCM mutations do not change the polyprotein amino acid se-
quence. Antibodies. The antibody (Ab) against TMEV protein VP1 (mouse
monoclonal) has been described previously (21). Rat monoclonal anti-
tubulin Ab, mouse monoclonal anti-HA Ab, and agarose-conjugated V5
and HA antibodies used for the immunoprecipitation of tagged viral pro-
teins were from Sigma. Mouse monoclonal anti-V5 Ab was from Life
Technologies, and IRDye-conjugated secondary antibodies used for im-
munoblotting were from Li-Cor. LAB OF MOLECULAR BIOLOGY Immunoblotting. BHK-21 cells were infected with TMEV WT or mu-
tants thereof at an MOI of 10. Infected cells were lysed directly in Laem-
mli’s sample buffer and heated to 95°C for 5 min. Proteins were separated
on gels as indicated in the figure legends and transferred to nitrocellulose
membranes. These were blocked for 30 to 60 min with 5% powdered milk
(Marvel) in PBS containing 0.1% Tween 20 (PBST) and probed at 4°C
overnight with primary antibody. Membranes were washed in PBST prior
to incubation in the dark with an IRDye-conjugated secondary antibody
in PBST. Blots were scanned using an Odyssey infrared imaging system
(Li-Cor). Mass spectrometry. MATERIALS AND METHODS Viruses and cell culture. BHK-21 cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 1 mM L-glutamine, and antibiotics. In virus infections,
BHK-21 cells were washed with DMEM containing no serum (SFM) and
overlaid with wild-type (WT) TMEV or mutant derivatives at the multi-
plicities of infection (MOIs) stated below. After a 1-h adsorption period, Plaque assays. BHK-21 cells at 70 to 80% confluence in 6-well plates
were infected with 10-fold dilutions of virus for 1 h at 37°C. Cells were
overlaid with DMEM supplemented with carboxymethyl cellulose (CMC;
0.35% each of low- and high-viscosity CMC) and 2% FBS and incu- Journal of Virology jvi.asm.org 8581 Finch et al. bated at 37°C for 48 h. After incubation, cells were fixed using formal
saline and stained using 0.1% toluidine blue. trophoresis on a 12% bis-Tris urea gel run in morpholineethanesulfonic
acid (MES) buffer (Invitrogen) at 30 to 40 mA for optimal resolution. The
gel was washed and stained using colloidal Coomassie blue, and bands
were excised for mass spectrometry. One-step growth curves. BHK-21 cells were infected with WT, SS,
SCM or LVWT TMEV at an MOI of 10 and incubated at 37°C for 1 h. Cells
were washed twice in ice-cold phosphate-buffered saline (PBS) and over-
laid with 2% DMEM. Virus was harvested at time points indicated below,
and the titers were determined using a plaque assay. One-step growth curves. BHK-21 cells were infected with WT, SS,
SCM or LVWT TMEV at an MOI of 10 and incubated at 37°C for 1 h. Cells
were washed twice in ice-cold phosphate-buffered saline (PBS) and over-
laid with 2% DMEM. Virus was harvested at time points indicated below,
and the titers were determined using a plaque assay. Coomassie blue-stained products were excised from the gel and sub-
jected to in-gel trypsin digestion. Peptides were extracted and analyzed by
liquid chromatography-tandem mass spectrometry (LC-MS/MS) using
an Agilent 1200 series nanoflow system (Agilent Technologies) connected
to a LTQ Orbitrap mass spectrometer (Thermo Electron) equipped with a
nanoelectrospray ion source (Proxeon). Fragment MS/MS spectra were
searched with the Mascot 2.3 search engine (Matrix Science) against a
protein sequence database composed of expected viral target sequences
and common contaminant proteins such as trypsin and keratins. MATERIALS AND METHODS Search
parameters included a 5-ppm precursor mass tolerance and 0.6-Da
MS/MS mass tolerance, three missed trypsin cleavages, and trypsin cleav-
age before proline plus a number of variable modifications, such as oxi-
dation (M), oxidation (HW), phospho (ST), and phospho (Y). For the
figures, spectra were autoannotated with xiSPEC (http://spectrumviewer
.org). Competition assays. BHK-21 cells in six-well plates were infected
with either WT and SS or WT and SCM at an MOI of 0.1. After adsorp-
tion, cells were washed with PBS and overlaid with 2% DMEM (2 ml) and
incubated for 24 h, after which virus was harvested (passage 1) and 250 l
was used to reinfect fresh BHK-21 cells. This was repeated five times. RNA
was extracted from virus harvested at each passage using TRIzol-LS
(Sigma) according to the manufacturer’s instructions. A total of 1,500 ng
of RNA was reverse transcribed using avian myeloblastosis virus (AMV)
reverse transcriptase (Promega) for 1 h at 42°C. The cDNA generated was
used as the template for PCR amplification of a region encompassing the
frameshift site using specifically designed primers. PCR products were
purified using the Promega PCR purification kit and subsequently se-
quenced. on October 2, 2015 by M
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om Dual-luciferase assays. BHK-21 cells were transfected in triplicate
with the relevant plasmid using Lipofectamine 2000 reagent (Invitrogen)
in a 24-well plate. Transfected cells were incubated at 37°C for 18 h prior
to infection with WT TMEV at an MOI of 10. Luciferase activities were
determined using the dual-luciferase Stop & Glo reporter assay system
(Promega) at 7 h postinfection (p.i.). Transfected cells were washed once
with PBS and lysed in 100 l of 1 passive lysis buffer, and light emission
was measured following injection of 50 l of either Renilla or firefly lucif-
erase substrate. Firefly luciferase activity was calculated relative to the
activity of Renilla luciferase, and frameshifting efficiencies were deter-
mined by comparing the ratio of firefly to Renilla enzymatic activities in
parallel cell cultures transfected with either the test construct or an in-
frame control (IFC), as described previously (24). q
Metabolic labeling. BHK-21 cells at 90 to 100% confluence in 24-well
plates were infected with WT, SS, SCM, or LVWT TMEV at an MOI of 10. MATERIALS AND METHODS After 60 min adsorption at 37°C, virus inocula were removed by aspira-
tion and replaced with 2% DMEM, and the cells were incubated at 37°C
for 6 h. Cells were subsequently washed once with methionine-free
DMEM and incubated in 1 ml of methionine-free DMEM for 1 h. Cells
were labeled with [35S]methionine (200 Ci/ml) at 37°C for 1 h. Cells
were scraped into 0.4 ml ice-cold PBS and spun at 5,000 rpm for 5 min. Cell pellets were washed with ice-cold PBS, resuspended in 70 l Laem-
mli’s sample buffer, heated at 95°C for 5 min, and analyzed by SDS-PAGE
(6 to 15%). After electrophoresis, gels were fixed, dried, and subjected to
phosphorimaging. Frameshifting efficiencies were estimated from band
intensities quantified using the software ImageQuant TL, as described in
Results. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY 8582
jvi.asm.org RESULTS Infected BHK-21 cells from a 25-cm2 flask were
washed in cold PBS and lysed in radioimmunoprecipitation assay (RIPA)
buffer (50 mM Tris HCl [pH 8], 150 mM sodium chloride, 1% NP-40
substitute, 0.5% sodium deoxycholate, 0.1% SDS) plus protease inhibi-
tors and Benzonase (Merck) on ice for 20 min. Cell debris was removed by
centrifugation at 21,000 g at 4°C for 10 min. Cell lysates were incubated
with 50 l of protein A Sepharose in RIPA buffer and 0.5 l of an irrele-
vant goat antibody for 1 h at 4°C with gentle mixing. Lysates were centri-
fuged through SigmaPrep columns at 8,200 g at 4°C for 1 min, and the
supernatant was incubated overnight with 200 l of anti-V5 conjugated
agarose beads (Abcam) on an end-over-end mixer at 4°C. Beads were
washed three times in a high-salt wash (0.5 M LiCl, 0.1 M Tris-HCl [pH
8.5]) before resuspension in Laemmli’s sample buffer and boiling for 7
min to remove protein from the beads. Proteins were separated by elec- RNA transcribed from the WT, SS, SCM, and LVWT molecu-
lar clones was transfected into BHK-21 cells to generate virus
stocks which were analyzed by plaque assay. While the StopGo
mutant (LVWT) and 2B* extension mutant (SCM) produced
plaque sizes similar to those of the WT, the shift site mutant (SS)
gave significantly smaller plaques (Fig. 2B). The growth pheno-
types of WT, SS, SCM, and LVWT viruses were analyzed further 8582
jvi.asm.org Journal of Virology August 2015
Volume 89
Number 16 Ribosomal Frameshifting in TMEV FIG 2 Analysis of TMEV mutants. (A) Schematic representation of mutations. SS, shift site mutant; SCM, 2B* extension mutant; LVWT, StopGo mutant. The
shift site sequence is in blue, and the 2B* stop codon is in red. (B) Mean plaque sizes for TMEV WT and mutant viruses. Mean plaque sizes are the averages for
100 representative plaques; error bars indicate standard deviations. **, SS is significantly different from WT (two-tailed t test, P 0.001). (C) One-step growth
curves. BHK-21 cells were infected with WT, SS, SCM, or LVWT viruses at an MOI of 10 and harvested at the indicated time points. Titers were measured by
plaque assay. At least two biological repeats were performed for each virus; error bars indicate standard deviations. (D) Competition assay. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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/ , 2015 by MRC LAB OF MOLECULAR BIOLOGY FIG 2 Analysis of TMEV mutants. (A) Schematic representation of mutations. SS, shift site mutant; SCM, 2B* extension mutant; LVWT, StopGo mutant. The
shift site sequence is in blue, and the 2B* stop codon is in red. (B) Mean plaque sizes for TMEV WT and mutant viruses. Mean plaque sizes are the averages for
100 representative plaques; error bars indicate standard deviations. **, SS is significantly different from WT (two-tailed t test, P 0.001). (C) One-step growth
curves. BHK-21 cells were infected with WT, SS, SCM, or LVWT viruses at an MOI of 10 and harvested at the indicated time points. Titers were measured by
plaque assay. At least two biological repeats were performed for each virus; error bars indicate standard deviations. (D) Competition assay. BHK-21 cells were
infected with a mixture of an MOI of 0.1 of either SS plus WT or SCM plus WT viruses. At 24 h p.i., virus was harvested and used to reinfect BHK-21 cells. Passaging was repeated five times. RNA was extracted from passages 0, 1, and 5, and a region of 1,000 nt encompassing the mutated region was sequenced. Frameshiftingintheviralcontextishighlyefficient. Ifframe-
shifting does indeed occur in TMEV, then, since ribosomes that
frameshift would terminate and dissociate from the message at the
2B* stop codon (Fig. 1), there should be fewer ribosomes synthe-
sizing polyprotein products encoded downstream of the frame-
shift site (2BC-3ABCD) than polyprotein products encoded up-
stream of the frameshift site (L-1ABCD-2A). To investigate this,
we used metabolic labeling to quantify viral protein production. In TMEV, the frameshifting efficiency cannot be robustly esti-
mated simply by analyzing the ratio of upstream and downstream
products for WT virus, due to the presence of intermediate poly-
protein processing products, as well as potential differences in
protein turnover rates. However, by normalizing WT protein lev-
els to the corresponding levels for the SS mutant—in which pro-
teins encoded upstream and downstream of the shift site are ex-
pected to be produced in equimolar amounts—we can at least
partly correct for these confounding factors. 35 using one-step growth curve analysis (Fig. 2C). As in the plaque
assays, SS exhibited a marked defect in growth kinetics, with peak
titers 1.5 log lower than those of the WT. August 2015
Volume 89
Number 16 RESULTS BHK-21 cells were
infected with a mixture of an MOI of 0.1 of either SS plus WT or SCM plus WT viruses. At 24 h p.i., virus was harvested and used to reinfect BHK-21 cells. Passaging was repeated five times. RNA was extracted from passages 0, 1, and 5, and a region of 1,000 nt encompassing the mutated region was sequenced. on October 2, 2015 by MRC LA
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Downloaded from This is most obvious when the intensities of
the 2C and VP1 proteins are compared. The intensity of each band
depends on the number of methionines present and the abun-
dance of the protein. There is clearly much more of the down-
stream product 2C relative to VP1 in the SS sample than in the
WT, LVWT, and SCM samples. (Fig. 3A), indicating that a substantial proportion of ribosomes
leave the polyprotein frame in the vicinity of the frameshift site
and that this departure is mediated by the sequence of the pre-
dicted frameshift site. This is most obvious when the intensities of
the 2C and VP1 proteins are compared. The intensity of each band
depends on the number of methionines present and the abun-
dance of the protein. There is clearly much more of the down-
stream product 2C relative to VP1 in the SS sample than in the
WT, LVWT, and SCM samples. In order to calculate frameshifting efficiencies, the intensity of
each of the VP0, VP3, VP1, and 2C bands from WT, LVWT, and
SCM was normalized first by methionine content, then by the
mean of these values for VP0, VP1, and VP3 (to control for lane
loading), and then by the corresponding similarly normalized val-
ues for the SS mutant as described above. Then, the value for 2C
(downstream product) was divided by the average of the values for
VP0, VP3, and VP1 (upstream products). (2A and 3D were ex-
cluded, as these products were not always clearly resolved and
quantifiable.) This provides an estimate of the fraction of ribo-
somes that avoid a 1 PRF. Subtracting this value from 1 and
multiplying the result by 100 gives the percent frameshifting effi-
ciency (Fig. 3C). Using this method, the frameshifting efficiency
for WT virus was estimated as 74 to 82% (95% confidence inter-
val; n 5) (Fig. 3C). With the exception of one outlier, LVWT
behaved similarly to the WT virus (n 5) (Fig. 3C). on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from SCM exhibited a slight
attenuation in replication, reaching peak titers around half a log
lower than those observed for WT. In agreement with results re-
ported by Loughran and colleagues (22), the LVWT growth curve
closely mirrored that of the WT, with only a slight lag in replica-
tion kinetics. To further investigate fitness of the SCM and SS mutants, a
competition assay was performed, whereby a mixture of equal
amounts of WT and mutant virus was used to infect BHK-21 cells
and the sequences of the resulting progeny were determined fol-
lowing RNA extraction. At various passage numbers, we assessed
whether and how quickly the WT virus was able to outcompete
either mutant. Consistent with the single-cycle growth curves de-
scribed above, WT virus was able to outcompete SS within one
passage (approximately two full WT replication cycles), while for
SCM, after one passage, the ratio of SCM to WT RNA was approx-
imately 1:10. At later passages, the SCM sequence disappeared
completely, leaving only the WT sequence (Fig. 2D). Together,
these results provide genetic evidence for the functional impor-
tance of the predicted frameshift site in TMEV. [35S]methionine-labeled proteins from infected cell lysates
were separated by electrophoresis and quantified by phosphorim-
aging. The expression levels of products encoded downstream of
the predicted shift site were observed to be much lower, relative
to the upstream products, for WT virus than for the SS mutant Journal of Virology jvi.asm.org
8583 jvi.asm.org Finch et al. G 3 Analysis of frameshifting in the viral context. (A) Radiolabeled TMEV translation products. BHK-21 cells were infected with either WT, SCM, SS, or
WT viruses at an MOI of 8 or mock infected. Cells were labeled from 6 to 7 h p.i. and harvested at 7 h p.i., and proteins were separated by SDS-PAGE. Note
t the more slowly migrating 2A band for LVWT may contain both 2A-2B* and a 2A-2B cleavage product. All samples were run on the same gel; an irrelevant
e has been excised. (B) Relative amounts of TMEV proteins. Individual band intensities for WT, LVWT, and SCM were normalized first by methionine
ntent, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized band for SS. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from Then the value for 2C (downstream product) was divided by the average of the values for VP0, VP3, and VP1 (upstream products). Subtracting this value from
1 and multiplying the result by 100 gives the percent frameshifting efficiency. Each bar represents the mean value ( standard deviation) from five, five, and two
biological repeats for WT, LVWT, and SCM viruses, respectively. FIG 3 Analysis of frameshifting in the viral context. (A) Radiolabeled TMEV translation products. BHK-21 cells were infected with either WT, SCM, SS, or
LVWT viruses at an MOI of 8 or mock infected. Cells were labeled from 6 to 7 h p.i. and harvested at 7 h p.i., and proteins were separated by SDS-PAGE. Note
that the more slowly migrating 2A band for LVWT may contain both 2A-2B* and a 2A-2B cleavage product. All samples were run on the same gel; an irrelevant
lane has been excised. (B) Relative amounts of TMEV proteins. Individual band intensities for WT, LVWT, and SCM were normalized first by methionine
content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized band for SS. Each bar represents the mean ( standard deviation) from all biological repeats in which the corresponding band could be resolved and quantified (see the text). (C) Frameshifting efficiency. The intensity in each of the VP0, VP3, VP1, and 2C bands for WT, LVWT, and SCM viruses was normalized first by methionine
content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized values for
SS. Then the value for 2C (downstream product) was divided by the average of the values for VP0, VP3, and VP1 (upstream products). Subtracting this value from
1 and multiplying the result by 100 gives the percent frameshifting efficiency. Each bar represents the mean value ( standard deviation) from five, five, and two
biological repeats for WT, LVWT, and SCM viruses, respectively. 2015 by MRC LAB OF MOLECULAR BIOLOGY LAB OF MOLECULAR BIOLOGY ative expression levels are clearly much lower than for the SS mu-
tant (Fig. 3A). (Fig. 3A), indicating that a substantial proportion of ribosomes
leave the polyprotein frame in the vicinity of the frameshift site
and that this departure is mediated by the sequence of the pre-
dicted frameshift site. 8584
jvi.asm.org on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from ch bar represents the mean ( standard deviation) from all biological repeats in which the corresponding band could be resolved and quantified (see the text). Frameshifting efficiency. The intensity in each of the VP0, VP3, VP1, and 2C bands for WT, LVWT, and SCM viruses was normalized first by methionine
ntent, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized values for
Then the value for 2C (downstream product) was divided by the average of the values for VP0, VP3, and VP1 (upstream products). Subtracting this value from
nd multiplying the result by 100 gives the percent frameshifting efficiency. Each bar represents the mean value ( standard deviation) from five, five, and two
logical repeats for WT, LVWT, and SCM viruses, respectively. on October 2, 2015 by MRC LAB
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Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY FIG 3 Analysis of frameshifting in the viral context. (A) Radiolabeled TMEV translation products. BHK-21 cells were infected with either WT, SCM, SS, or
LVWT viruses at an MOI of 8 or mock infected. Cells were labeled from 6 to 7 h p.i. and harvested at 7 h p.i., and proteins were separated by SDS-PAGE. Note
that the more slowly migrating 2A band for LVWT may contain both 2A-2B* and a 2A-2B cleavage product. All samples were run on the same gel; an irrelevant
lane has been excised. (B) Relative amounts of TMEV proteins. Individual band intensities for WT, LVWT, and SCM were normalized first by methionine
content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized band for SS. Each bar represents the mean ( standard deviation) from all biological repeats in which the corresponding band could be resolved and quantified (see the text). (C) Frameshifting efficiency. The intensity in each of the VP0, VP3, VP1, and 2C bands for WT, LVWT, and SCM viruses was normalized first by methionine
content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized values for
SS. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from SS with LV-SS in Fig. 4E), suggesting that this band actually
represents N-terminally cleaved 2B. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY Following this initial characterization, BHK-21 cells were in-
fected with V5-tagged WT, SS, LVWT, or LVSS viruses and lysed
when cytopathic effect was extensive. Proteins were separated by
SDS-PAGE, and V5-tagged products were detected by Western
blotting (Fig. 4C). For V5-tagged WT virus, no product was ob-
served migrating at a size compatible with that expected for V5-
tagged 2B* (3.3 kDa), suggesting that this product may be rapidly
degraded (data not shown). The predicted size of the V5-tagged
2A-2B* fusion that would be produced when StopGo is inhibited
in LVWT virus is 18.8 kDa. A doublet (possible explanation be-
low) migrating at a position consistent with this size was observed
for both the V5-tagged WT and LVWT viruses, whereas for the
V5-tagged shift site mutant viruses (SS and LVSS), only a single
band was observed to migrate at this position. We hypothesized
that the additional band seen in WT and LVWT, but not in the
shift site mutants, represented the frameshift product 2A-V5-2B*
(18.8 kDa) (produced at a low level in V5-tagged WT virus only
when StopGo separation fails; see below). 2015 by MRC LAB OF MOLECULAR BIOLOGY Frameshifting is stimulated by virus infection and requires a
3= stem-loop structure. Previously, frameshifting in the related
EMCV was shown to depend upon virus infection and on at least
50 nt of the 3= sequence (19). The EMCV 3= sequence is predicted
to form a stem-loop at an unusual distance from the shift site. However, the role of the predicted structure in EMCV frameshift-
ing remains uncertain, as all mutations tested in a reporter sys-
tem—even ones predicted to restore the stem-loop structure but
with an altered sequence—inhibited frameshifting (19). A stem-
loop with similar spacing (14 nt) is also predicted in TMEV (Fig. 2A). To investigate the important elements for TMEV frameshift-
ing, we cloned the relevant region into a dual-luciferase reporter
construct, pIDluc, which is a modified version of pDluc (24, 25). As picornavirus infection results in host translational shutoff,
pDluc was adapted to contain the EMCV IRES to allow cap-inde-
pendent translation during viral infection. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from Purification of WT 2B* proved to be im-
practicable given its small size (1.4 kDa) and poor predicted
antigenicity. Therefore, we generated viruses expressing V5-
tagged 2B*. Sequences encoding a V5 tag and linker were inserted
just after the last codon of the StopGo sequence (proline in the WT
virus and valine in the LV mutants) in the WT, SS, LVWT, and
LVSS (a poorly growing mutant in which both StopGo and the
shift site were disabled) viruses (Fig. 4A). In this location, V5 is
expected to tag both 2B and 2B* near their N termini. Frameshift-
ing efficiencies were estimated from radiolabeled products as
described above and were found to be similar to those of the re-
spective untagged viruses (Fig. 4B). These data indicate that the
V5-tagged viruses mimic their untagged counterparts during vi-
rus replication in cell culture. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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ownloaded from A sequence encoding an HA tag was also inserted into the
LVWT backbone, and anti-HA immunoprecipitation was used to
purify 2A-HA-2B* for LC-MS/MS analysis. The sequence data
obtained were consistent with that of the V5-tagged viruses, with
high coverage of the 2A-HA-2B* product and recovery of the shift
site peptide, again verifying the site and direction of frameshifting
(Fig. 4D; also, see Fig. S1C in the supplemental material). These
experiments confirm that 1 PRF does indeed occur on the
G_GUU_UUU motif during translation of the TMEV genome. on October 2, 2015 by MRC LAB OF MOLECULAR BIO
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om These findings also help explain the migration patterns ob-
served for the low-molecular-mass radiolabeled products of the
eight viruses (Fig. 4E). In the untagged StopGo mutant viruses, a
product migrating slightly more slowly than 2A may represent
comigrating 2A-2B* and 2A-2BN in LVWT and 2A-2BN alone in
LVSS. In the tagged StopGo mutant viruses, this was replaced with
a still more slowly migrating band, consistent with 2A-V5-2B* or
2A-V5-2BN (the three prolines in the V5 tag may explain their
aberrant migration relative to 2A and 2A-2B*/2A-2BN). In con-
trast, the band presumed to correspond to 2B (note that the other
downstream low-mass viral products, 3A and 3AB, lack methio-
nines) did not shift between untagged and tagged viruses (e.g., SS)
(Fig. 4E), nor between wild-type and mutant StopGo viruses (e.g.,
cf. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from SCM had a
frameshifting efficiency of 53 to 75% (95% confidence interval;
n 2) which was significantly lower than the WT frameshifting The relative abundances of individual TMEV proteins were
calculated by normalizing the intensity of each band first by the
number of methionines in the corresponding product, then by the
means of these values for VP0, VP1, and VP3 (to control for lane
loading), and finally by the corresponding similarly normalized
band for the SS mutant (Fig. 3B). This highlights the substantial
fall in relative protein levels between the upstream products (VP0,
VP3, VP1, and 2A) and the downstream products (2C and 3D). Due to the relatively high background and low intensity of the 2B
and 3C bands, it was not possible to reliably estimate the levels of
these proteins for the WT and LVWT viruses, although their rel- 8584
jvi.asm.org August 2015
Volume 89
Number 16 Journal of Virology Ribosomal Frameshifting in TMEV produce a V5-tagged product comprising 2A fused to V5 and the
N-terminal 9 aa of 2B. This product (2A-V5-2BN) has a predicted
mass of 18.4 kDa and would migrate close to 2A-V5-2B*. Peptides
confirming usage of this cleavage site were observed during mass
spectrometry of both bands (Fig. 4D; also, see Fig. S1B in the
supplemental material). Thus, the doublet seen in the Western
blot for V5-tagged LVWT may comprise both 2A-V5-2B* and
2A-V5-2BN, with the detection of both products in both bands
simply indicating that the doublet was not resolved well enough to
avoid contamination between the two gel slices. This doublet was
also observed for V5-tagged WT virus, and this likely results from
a proportion of ribosomes reading through the StopGo cassette
without cotranslational separation occurring, leaving a propor-
tion of 2A fused to V5 and 2B* or 2BN. efficiency (P 0.003, 2-tailed pooled variance t test), indicating
that the SCM mutation may have partly interfered with a frame-
shift-stimulating element. It should be noted that this calculation
assumes that ribosomes either frameshift or continue translating
in the polyprotein frame; however, it is also possible that a pro-
portion of ribosomes prematurely terminate at the frameshift site
(23). Mass-spectrometric confirmation of 2B* expression and the
site of frameshifting. To confirm 1 PRF at the predicted frame-
shift site and translation of the predicted 2B* peptide, mass spec-
trometry was utilized. August 2015
Volume 89
Number 16 on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from The TMEV WT shift
site sequence (G_GUU_UUU) or mutated shift site sequence (SS;
A_GUG_UUU), together with 6 nt of upstream sequence and 92
nt of downstream sequence (including the predicted stem-loop
structure), was cloned between the Renilla and firefly luciferase
ORFs in pIDluc such that frameshifting is required for expression
of the firefly ORF. Two additional changes were introduced: a Next, we performed an anti-V5 immunoprecipitation using
lysate from cells infected with V5-tagged LVWT. Immunoprecipi-
tates were separated by SDS-PAGE and stained with colloidal
Coomassie blue. Again, a doublet was observed migrating at the
appropriate size. Both bands were excised and digested with tryp-
sin, and the resulting peptides were analyzed by liquid chroma-
tography-tandem mass spectrometry (LC-MS/MS). Peptides cov-
ering 98% or 96% of the predicted 2A-V5-2B* fusion were
identified from each band in the doublet (although some of these
peptides may have arisen from comigrating nonframeshift prod-
ucts; see below). Importantly, however, the peptide encoded by
the shift site sequence was identified, thus confirming both the site
and the direction (1) of the frameshift (Fig. 4D; also, see Fig. S1A
in the supplemental material). Peptides were also mapped to a portion of the 2B protein. Near
the N-terminal end of 2B, there is a potential 3C-Pro cleavage site,
QG, that is conserved in theiloviruses and EMCV (19). When
StopGo fails (WT) or is inhibited (LV), cleavage at this site would jvi.asm.org
8585 jvi.asm.org 8585 Journal of Virology Finch et al. Characterization of tagged viruses. (A) Schematic representation of the tagged viruses. A V5 tag was inserted just after the first proline of 2B i
nce (after valine in the GP-to-LV StopGo-mutated viruses), to tag products containing 2B or 2B*. (B) Frameshifting efficiencies of tagged and on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from n October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY 2015 by MRC LAB OF MOLECULAR BIOLOGY FIG 4 Characterization of tagged viruses. (A) Schematic representation of the tagged viruses. A V5 tag was inserted just after the first proline of 2B in the WT
sequence (after valine in the GP-to-LV StopGo-mutated viruses), to tag products containing 2B or 2B*. (B) Frameshifting efficiencies of tagged and untagged
viruses. BHK-21 cells were infected with WT, SS, LVWT, or LVSS viruses or their tagged equivalents, and frameshifting efficiencies were calculated from
radiolabeled products as described in Fig. 3C. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from All viruses were normalized by SS; thus, the frameshifting efficiency for SS is zero by definition. Negative
frameshifting efficiencies are likely an artifact of measurement errors and/or biological variability. Each bar represents the mean value ( standard deviation)
from three biological repeats (the three untagged WT and LVWT data points are also used in Fig. 3C). (C) Western blot of virus-infected cell lysates. BHK-21 cells
were infected with V5-tagged viruses at MOI of 1 for LVSS, 5 for SS, and 10 for WT and LVWT, and lysates were prepared when cytopathic effect was extensive. Fivefold-smaller amounts of the LVWT and LVSS samples were loaded to give band intensities similar to those of the WT and SS samples, where only proteins
generated by StopGo failure are detected. The proteins with the GP-to-LV mutation may migrate slightly faster than the wild-type ones. Samples were run on
bis-Tris gels with 6 M urea and MOPS buffer, which results in altered mobility of the prestained 14-kDa and 17-kDa markers. (D) Mass spectrometric analysis
of tagged products. Lysates from BHK-21 cells infected with V5- or HA-tagged TMEV LVWT were immunoprecipitated with V5 or HA antibodies, respectively,
and immunoprecipitates were separated by SDS-PAGE. Products migrating at the expected size for 2A-tag-2B* were subjected to in-gel trypsin digest, and
peptides were analyzed by LC-MS/MS. Fragmentation ions are shown for the shift site peptide derived from V5-tagged 2A-2B* (top), a peptide consistent with
3C-Pro cleavage of tagged 2A-2B at the conserved Q|G encoded just downstream of the frameshift site (middle), and the shift site peptide derived from HA-tagged
2A-2B* (bottom). Amino acids derived from the V5 or HA tag are in green. The b- and y-series ions correspond to N- and C-terminal fragments. See Fig. S1 in
the supplemental material for the fragmentation spectra. Below, the nucleotide sequence in the vicinity of the shift site G_GUU_UUU is shown, with conceptual
amino acid translations in all three reading frames. The C-terminal end of the frameshift tryptic peptide is underlined in green, and the C-terminal end of the 2B
cleavage peptide is in blue. (E) Low-molecular-mass radiolabeled TMEV translation products. BHK-21 cells were infected with WT, SS, LVWT, or LVSS viruses
or their tagged equivalents at an MOI of 10 or mock infected. Cells were labeled from 8 to 9 h p.i. and harvested at 9 h p.i., and proteins were separated by
SDS-PAGE. Journal of Virology August 2015
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Number 16 on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from A reduced
frameshifting efficiency would perhaps also be observed if the du-
al-luciferase reporter mRNA and “virus transactivator” were not
appropriately localized in the cell or if the reporter was present at
an inappropriate molar ratio. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY Canonical eukaryotic 1 PRF is stimulated by a stable 3= RNA
secondary structure separated from the shift site by a 5- to 9-nt
spacer sequence. However, the predicted stem-loop in EMCV and
TMEV is separated from the shift site by 13 or 14 nt (19). In order
to evaluate whether the predicted stem-loop is involved in the
stimulation of frameshifting in TMEV, three mutants were gener-
ated (Fig. 5C): (i) pIDluc SL5=, where the first three bases of the 5=
half of the stem were mutated (5=-GGU-3= to 5=-CCA-3=); (ii)
pIDluc SL3=, where the last three bases of the 3= half of the stem
were mutated (5=-ACC-3= to 5=-UGG-3=); and (iii) pIDluc
SL5= 3=, where both sets of mutations were combined so as to
restore the predicted structure but with reversed base pairings. For
each mutant, a corresponding IFC was constructed. Frameshifting
efficiencies for both pIDluc SL3= and pIDluc SL5= were found to
be negligible even with TMEV infection (Fig. 5D). In contrast, the
frameshifting efficiency for the restoration mutant (pIDluc
SL5= 3=) was 19% when stimulated by TMEV infection, similar
to that for pIDluc WT. This strongly indicates that the predicted 3=
RNA stem-loop structure does indeed form and plays a critical
role in the stimulation of frameshifting in TMEV. 2015 by MRC LAB OF MOLECULAR BIOLOGY Frameshifting at the same genomic location was previously
demonstrated in the related cardiovirus, EMCV (19). However, in
EMCV, frameshifting results in the production of a much larger
transframe 2B* protein (128 or 129 aa, depending on the isolate)
than in TMEV (14 aa). In EMCV, a mutation (PTC2) that trun-
cated 2B*, but that was not expected to interfere with frameshift-
ing, produced an intermediate phenotype, suggesting that both
the 2B* protein and frameshifting per se are functionally impor-
tant (19). In TMEV, frameshifting is critical for efficient viral
growth in cell culture, but it is unclear whether the 14-aa 2B* has
a function in its own right or whether it is simply the by-product of
a functionally important frameshift. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from Plasmids pIDluc SS, pIDluc WT, and pIDluc IFC were trans-
fected into BHK-21 cells 18 h prior to either mock infection or
infection with WT virus at an MOI of 10. Frameshifting efficien-
cies were measured to be 1% for pIDluc WT in mock-infected
cells but 12 to 13% in TMEV-infected cells, showing that, as in
EMCV, frameshifting on the TMEV frameshift sequence is depen-
dent on virus infection (Fig. 5A). As expected, pIDluc SS failed to
support frameshifting either with or without virus infection. To
test whether frameshift stimulation by TMEV infection is re-
stricted to the TMEV frameshift signal, two other dual-luciferase
constructs harboring unrelated frameshift signals from infectious
bronchitis coronavirus (pDluc IBV) and human immunodefi-
ciency virus type 1 (p2luc HIV) (26), and their corresponding
IFCs were tested alongside the TMEV constructs. In these con-
structs, frameshifting occurred in mock-infected cells at levels
consistent with previous work (26), and the frameshifting effi-
ciencies were not increased by TMEV infection (Fig. 5A and B). on October 2, 2015 by MRC LAB OF MOLECULAR BIO
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om Frameshifting efficiencies in the virus context were estimated
from polyprotein processing products quantified by metabolic la-
beling. Although we cannot rule out the possibility that the kinet-
ics of polyprotein processing may be modified in the mutants, the
very high efficiency of the TMEV frameshifting signal is consistent
with recent metabolic labeling and ribosome profiling data for the
cardiovirus EMCV, where frameshifting was found to be 50 to
70% efficient (R. Ling , J. D. Jones, I. Brierley, and A. E. Firth,
unpublished data). Why frameshifting is noticeably less efficient
in the context of the dual-luciferase reporter system (although still
TMEV infection dependent) is uncertain, although similar obser-
vations have been reported for EMCV (7% in pDluc [20]). One
possibility is that distal frameshift-stimulatory elements exist in
the virus genome that are not present in the dual-luciferase re-
porter vector. A more likely explanation, however, is that the re-
duced frameshifting efficiency is a consequence of a relative
buildup of the nonframeshift product (Renilla luciferase) in cells
prior to the onset of virus-stimulated frameshifting. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from Journal of Virology August 2015
Volume 89
Number 16 8586
jvi.asm.org 8586
jvi.asm.org 8586
jv Ribosomal Frameshifting in TMEV U-to-C mutation (removing the 1 frame stop codon) was intro-
duced into all plasmids to allow 1 frame translation to continue
into the downstream firefly luciferase, and a CAA-to-UAA muta-
tion was introduced to generate a stop codon in the zero frame at
the third codon after the shift site. The latter mutation was intro-
duced as preliminary experiments indicated that, without it, the
extended C-terminal tail that is appended to the Renilla luciferase
when frameshifting failed to occur was inhibiting its enzymatic
activity. An in-frame control (IFC) in which the firefly ORF was
placed in the same frame as the Renilla ORF by inserting an extra
U at the end of the G_GUU_UUU shift site was also constructed. teins using metabolic labeling, comparing WT virus with the SS
mutant revealed that frameshifting efficiency in the viral context is
exceptionally high, at 74 to 82%. When the TMEV frameshift cassette was cloned into a dual-
luciferase reporter, frameshifting was found to be dependent
upon viral coinfection, indicating that a virus-stimulated trans-
acting factor may be required for efficient frameshifting. Frame-
shift stimulation also involves a downstream RNA stem-loop
structure at a noncanonical spacing (14 nt) from the frameshift
site. While canonical 1 PRF-stimulatory RNA structures (5- to
9-nt spacing) are expected to be positioned partly within the
mRNA entrance channel at the onset of frameshifting, the cardio-
virus stem-loop structure will be located close to the leading edge
of the ribosome when the P and A sites are positioned on the shift
site sequence. One possible explanation for the observations may
be that a virus protein, or virus-stimulated host protein, binds to
the stem-loop structure and that this RNA-protein complex is able
to mimic a canonical 1 PRF-stimulatory RNA structure at a
different spacing or otherwise interact with the ribosome to pro-
mote 1 PRF. Recent work on Porcine reproductive and respiratory
syndrome virus (family Arteriviridae) has demonstrated that in at
least one other case of ribosomal frameshifting, the 3= stimulator
involves mRNA-protein interactions (27). on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY
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Downloaded from Frameshifting in TMEV di-
verts 74 to 82% of ribosomes out of the polyprotein reading frame,
thus greatly reducing expression of the 3=-encoded replication
proteins relative to the 5=-encoded structural proteins. This, in August 2015
Volume 89
Number 16 Journal of Virology DISCUSSION We have demonstrated that TMEV utilizes 1 PRF at a conserved
G_GUU_UUU sequence in the 2B-encoding region of the poly-
protein ORF. A virus (the SS mutant) with the predicted frame-
shift site disabled by mutations synonymous in the polyprotein
frame exhibited a small-plaque phenotype and attenuated growth
kinetics compared to WT virus. Moreover, using a V5-tagged
StopGo mutant virus, a product of the expected size for frameshift
translation was detected by anti-V5 Western blot, and the site and
direction of frameshifting confirmed by mass spectrometry of this
product. Analysis of the ratio of structural to nonstructural pro- jvi.asm.org
8587 jvi.asm.org Journal of Virology Finch et al. FIG 5 Analysis of frameshift stimulators. (A) Frameshifting efficiencies measured using dual-luciferase constructs. BHK-21 cells were transfected with frameshift
reporterconstructsand18hlaterwereeitherinfectedwithWTvirusatanMOIof10ormockinfected.Lysateswereharvestedat7hp.i.andassayedforRenillaandfirefly
luciferase activity. Frameshift efficiencies were determined by comparing luciferase activities to an in-frame control (IFC) construct. Mean values and standard
deviations are shown, each based on nine separate transfections. (B) Western blot verifying infection of infected samples. Aliquots of each of the cell lysates were
separated on a 10 to 20% Tris-Tricine gradient gel and probed with rat monoclonal anti-tubulin (red, IRDye 700-labeled secondary) and mouse monoclonal
anti-VP1 (TMEV capsid protein) (green, IRDye 800-labeled secondary) antibodies. Note that the rat monoclonal primary cross-reacts with both the secondary
antibodies. (C) Schematic representation of the fragments cloned into the pIDluc vector. All constructs contain the U-to-C mutation removing the 1 frame
UAA stop codon (red) to allow expression of the downstream luciferase and the C-to-U mutation to introduce a zero-frame UAA stop codon just 3= of the shift
site (see the text). pIDluc IFC contains an extra U in the G_GUU_UUU shift site sequence (blue). (D) Frameshifting efficiencies of dual-luciferase constructs
containing stem-loop mutants. See the description of panel A for details. Mean values and standard deviations are shown, each based on nine separate
transfections. on October 2, 2015 by MRC LAB OF M
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Downloaded from October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY FIG 5 Analysis of frameshift stimulators. (A) Frameshifting efficiencies measured using dual-luciferase constructs. BHK-21 cells were transfected with frameshift
reporterconstructsand18hlaterwereeitherinfectedwithWTvirusatanMOIof10ormockinfected.Lysateswereharvestedat7hp.i.andassayedforRenillaandfirefly
luciferase activity. Frameshift efficiencies were determined by comparing luciferase activities to an in-frame control (IFC) construct. Mean values and standard
deviations are shown, each based on nine separate transfections. DISCUSSION (B) Western blot verifying infection of infected samples. Aliquots of each of the cell lysates were
separated on a 10 to 20% Tris-Tricine gradient gel and probed with rat monoclonal anti-tubulin (red, IRDye 700-labeled secondary) and mouse monoclonal
anti-VP1 (TMEV capsid protein) (green, IRDye 800-labeled secondary) antibodies. Note that the rat monoclonal primary cross-reacts with both the secondary
antibodies. (C) Schematic representation of the fragments cloned into the pIDluc vector. All constructs contain the U-to-C mutation removing the 1 frame
UAA stop codon (red) to allow expression of the downstream luciferase and the C-to-U mutation to introduce a zero-frame UAA stop codon just 3= of the shift
site (see the text). pIDluc IFC contains an extra U in the G_GUU_UUU shift site sequence (blue). (D) Frameshifting efficiencies of dual-luciferase constructs
containing stem-loop mutants. See the description of panel A for details. Mean values and standard deviations are shown, each based on nine separate
transfections. 15 by MRC LAB OF MOLECULAR BIOLOGY efficiency of ribosomal frameshifting (Fig. 3C). It has been sug-
gested that the apparent redundancy of StopGo in TMEV may
partly stem from the presence of a potential 3C-Pro cleavage site
within 2B (QG, encoded just downstream from the shift site; also
conserved in EMCV) (22). Cleavage at QG in a StopGo mutant
would yield 2A fused to the N-terminal 9 amino acids of 2B (2A-
2BN) and a separate N-terminally truncated 2B. Consistent with
this, no product of the expected size of 2A-2B (30 kDa) was
observed in the radiolabeling experiment using LVWT virus. In-
stead, a product (or products) migrating slightly more slowly than
WT 2A was observed, consistent with either 2A-2B* or 2A-2BN. Mass-spectrometric analysis of V5-tagged LVWT confirmed that
V5-tagged products migrating at this size comprise both V5-
tagged 2A-2B* and V5-tagged 2A-2BN. If N-terminally truncated
2B can carry out all necessary 2B functions, then it would appear
that the importance of StopGo is to produce the correct C termi-
nus of 2A and/or the correct N terminus of 2B*. The differing efficiency of ribosomal frameshifting (Fig. 3C). It has been sug-
gested that the apparent redundancy of StopGo in TMEV may
partly stem from the presence of a potential 3C-Pro cleavage site
within 2B (QG, encoded just downstream from the shift site; also
conserved in EMCV) (22). 8588
jvi.asm.org DISCUSSION Cleavage at QG in a StopGo mutant
would yield 2A fused to the N-terminal 9 amino acids of 2B (2A-
2BN) and a separate N-terminally truncated 2B. Consistent with
this, no product of the expected size of 2A-2B (30 kDa) was
observed in the radiolabeling experiment using LVWT virus. In-
stead, a product (or products) migrating slightly more slowly than
WT 2A was observed, consistent with either 2A-2B* or 2A-2BN. Mass-spectrometric analysis of V5-tagged LVWT confirmed that
V5-tagged products migrating at this size comprise both V5-
tagged 2A-2B* and V5-tagged 2A-2BN. If N-terminally truncated
2B can carry out all necessary 2B functions, then it would appear
that the importance of StopGo is to produce the correct C termi-
nus of 2A and/or the correct N terminus of 2B*. The differing itself, may provide a selective advantage, as the structural proteins
are required in much greater quantities than the replication pro-
teins. Although the mutant with 2B* extended (the SCM mutant)
exhibited a slight defect in growth kinetics compared to WT, this
could be due to a reduction in frameshifting efficiency since the
mutation is within the frameshift-stimulatory stem-loop struc-
ture and radiolabeling indicated that this mutant had a decreased
frameshifting efficiency (Fig. 3C). At this time, however, a func-
tional role for TMEV 2B* cannot be ruled out. The StopGo process that cotranslationally separates the poly-
protein between 2A and 2B is conserved between TMEV and
EMCV and is essential for efficient replication in EMCV (8). How-
ever, consistent with previous results (22), our data show that
StopGo in TMEV is not crucial for efficient viral growth in cell
culture, as judged by the similar growth kinetics and plaque phe-
notypes exhibited by LVWT and WT viruses. In addition, inhib-
iting StopGo had no or only a slight detrimental effect on the 8588
jvi.asm.org August 2015
Volume 89
Number 16 Journal of Virology Ribosomal Frameshifting in TMEV cleavage activities of aphthovirus and cardiovirus 2A proteins. J Gen Virol
78:13–21. susceptibilities of EMCV and TMEV to StopGo inhibition may be
due to 2B* being functional in EMCV but not in TMEV, or per-
haps due to the 2B* that is appended to 2A having a greater inhib-
itory effect on 2A function or L-1ABCD-2A2B* polyprotein pro-
cessing in EMCV due to the larger size of EMCV 2B* (cf. the
polyprotein processing studies in reference 8). 10. Hahn H, Palmenberg AC. 2001. DISCUSSION Deletion mapping of the encephalomyo-
carditis virus primary cleavage site. J Virol 75:7215–7218. http://dx.doi
.org/10.1128/JVI.75.15.7215-7218.2001. 11. Doronina VA, Wu C, de Felipe P, Sachs MS, Ryan MD, Brown JD. 2008. Site-specific release of nascent chains from ribosomes at a sense
codon. Mol Cell Biol 28:4227–4239. http://dx.doi.org/10.1128/MCB
.00421-08. Frameshifting downregulates production of the TMEV non-
structural proteins 2BC-3ABCD by 74 to 82%. During the course
of viral infection, the structural proteins are required in much
greater quantities than the enzymatic proteins, in particular the
viral polymerase. In many RNA viruses, transcriptional or trans-
lational control mechanisms are used to downregulate production
of the latter relative to the former (reviewed in reference 28). However, in viruses that use a single-polyprotein expression strat-
egy, such mechanisms would appear to be unavailable, and any
regulation of relative protein levels must normally occur post-
translationally, e.g., via polyprotein processing, protein turnover,
the production of inactive conformers, or sequestration of enzy-
matic proteins in inclusion bodies or the nucleus. We suggest that
highly efficient 1 PRF in TMEV provides a mechanism to escape
the confines of a single-polyprotein expression strategy, allowing
efficient downregulation of replication protein synthesis while si-
multaneously releasing translational resources for enhanced
structural protein synthesis. 12. Brierley I, Jenner AJ, Inglis SC. 1992. Mutational analysis of the “slippery-
sequence” component of a coronavirus ribosomal frameshifting signal. J Mol
Biol 227:463–479. http://dx.doi.org/10.1016/0022-2836(92)90901-U. on October 2, 2015 by M
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decoding rules enriches gene expression. Springer, Heidelberg, Germany. 14. Miller W, Giedroc D. 2010. Ribosomal frameshifting in decoding plant
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7. Ryan MD, Drew J. ACKNOWLEDGMENTS 19. Loughran G, Firth AE, Atkins JF. 2011. Ribosomal frameshifting into an
overlapping gene in the 2B-encoding region of the cardiovirus genome. Proc Natl Acad Sci U S A 108:E1111–E1119. http://dx.doi.org/10.1073
/pnas.1102932108. Work in the A.E.F. lab is supported by the Wellcome Trust (088789 and
106207) and the Biotechnology and Biological Sciences Research Council
(BB/J007072/1). L.F. is supported by a Biotechnology and Biological Sci-
ences Research Council Ph.D. studentship. 20. Law KM, Brown TD. 1990. The complete nucleotide sequence of the
GDVII strain of Theiler’s murine encephalomyelitis virus (TMEV). Nu-
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thank John Atkins, Lawrence Tiley, David Brown, and Frédéric Sorgeloos
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/journal.ppat.1003474. DISCUSSION Namy O, Moran SJ, Stuart DI, Gilbert RJC, Brierley I. 2006. A mechanical
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8. Hahn H, Palmenberg AC. 1996. Mutational analysis of the encephalo-
myocarditis virus primary cleavage. J Virol 70:6870–6875. 28. Ahlquist P. 2006. Parallels among positive-strand RNA viruses, reverse-
transcribing viruses and double-stranded RNA viruses. Nat Rev Microbiol
4:371–382. http://dx.doi.org/10.1038/nrmicro1389. 9. Donnelly ML, Gani D, Flint M, Monaghan S, Ryan MD. 1997. The jvi.asm.org
8589 jvi.asm.org
8589 jvi.asm.org August 2015
Volume 89
Number 16 jvi.asm.org
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Gender Mainstreaming im Europäischen Parlament: Geschichte, Institutionalisierung, Hindernisse
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Gender Mainstreaming im Europäischen Parlament:
Geschichte, Institutionalisierung, Hindernisse
Ahrens, Petra
Veröffentlichungsversion / Published Version
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Verlag Barbara Budrich
Empfohlene Zitierung / Suggested Citation:
Ahrens, P. (2021). Gender Mainstreaming im Europäischen Parlament: Geschichte, Institutionalisierung, Hindernisse.
GENDER - Zeitschrift für Geschlecht, Kultur und Gesellschaft, 13(1), 11-26. https://doi.org/10.3224/gender.v13i1.02
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Schwerpunkt
Petra Ahrens
Gender Mainstreaming im Europäischen Parlament:
Geschichte, Institutionalisierung, Hindernisse
Zusammenfassung
Summary
Das Europäische Parlament (EP) hat sich als
eines der wenigen Parlamente weltweit seit
2003 in insgesamt sechs Entschließungen zur
Umsetzung von Gender Mainstreaming (GM)
verpflichtet. Ausgehend von dem Ansatz des
Feministischen Institutionalismus analysiert
dieser Beitrag chronologisch die potenzielle Institutionalisierung von GM und fragt, inwieweit sich neue Regeln und Normen in bestehende formale und informelle Regeln einflechten lassen. Hierfür werden zentrale EPDokumente für den Zeitraum 2003 bis 2019
sowie leitfadengestützte Interviews mit MEPs
verschiedener Fraktionen und EP-Beschäftigten aus 2019 und 2020 analysiert. Insgesamt
kann von einer GM-Institutionalisierung gesprochen werden, wenn sie auch heterogen
verläuft.
Gender mainstreaming in the European Parliament: History, institutionalization, obstacles
Schlüsselwörter
Europäisches Parlament, Gender Mainstreaming, Institutionalisierung, Mikropolitiken, Frauenrechts- und Gleichstellungsausschuss
The European Parliament (EP) is one of the
few parliaments worldwide to have adopted a
total of six resolutions in the period since
2003 in which it pledges to implement gender mainstreaming (GM). Applying Feminist
Institutionalism, the article analyzes the potential institutionalization of GM from a
chrono
logical perspective. It investigates to
what extent new rules and norms can be incorporated into existing formal and informal
rules. Examining key EP documents from the
period between 2003 and 2019 and semistructured interviews conducted with Members of the EP (MEPs) from different political
groups and with EP staff in 2019 and 2020,
the article provides an analysis of GM institutionalization. Overall, one can say that GM
has become institutionalized over time, al
though implementation is quite heterogenous.
Keywords
European Parliament, gender mainstreaming,
institutionalization, micro-politics, Committee on Women’s Rights and Gender Equality
1
Einleitung
Während im Zuge der Pekinger Aktionsplattform der UN-Weltfrauenkonferenz 1995
viele Regierungen Gender Mainstreaming (GM) als gleichstellungspolitische Strategie
verabschiedeten, blieben ähnliche Initiativen in den meisten Parlamenten aus. Eine herausstechende Ausnahme ist das Europäische Parlament (EP), welches sich seit 2003
mittels Entschließungen und Berichten selbst zu GM verpflichtet. Diese kontinuierliche
Bestrebung, GM im EP zu institutionalisieren, erscheint erstaunlich angesichts der Tatsache, dass GM in der Politik der Europäischen Union erheblich an Boden verloren hat
und sich erst mit der neuen Europäischen Kommission ein Wandel abzeichnet (Abels
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09.02.2021 16:22:49
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Petra Ahrens
2020; Ahrens 2018; Jacquot 2015). Bemerkenswert ist die Selbstverpflichtung auch,
weil im EP vertretene rechtskonservative und -extreme Parteien gegen Gleichstellungspolitik und GM opponieren und es als „Genderideologie“ diskreditieren (Korolczuk/
Graff 2018; Verloo/Paternotte 2018). Trotz der Machtzunahme des EP und des damit
potenziell wachsenden Einflusses auf supranationale Gleichstellungspolitik ist die langjährige Entwicklung im EP in der Forschung kaum adressiert worden (vgl. ausführlich
Ahrens/Rolandsen Agustín 2019).
Dieser Beitrag schließt diese Lücke, indem die GM-Umsetzungsprozesse im EP
chronologisch analysiert werden. Hierfür wird ausgehend vom Ansatz des Feministischen Institutionalismus analysiert, inwieweit sich neue Normen und Praxen (hier Gender Mainstreaming) in formale und informelle Regeln einflechten lassen angesichts des
konstanten Wandels des politischen EU-Systems und der Selbstwahrnehmung des EP
als Gleichstellungsmotor (Van der Vleuten 2019).
Die chronologische Analyse entsprechender EP-Entschließungen, Berichte, Abstimmungsergebnisse zu GM sowie weiterer Dokumente im Zusammenhang mit den
formalen parlamentarischen Verfahren deutet auf eine zunehmend stabile Institutionalisierung von GM hin. Dass diese nicht friktionslos verläuft, wird am Beispiel der Berücksichtigung von Genderaspekten in Ausschüssen während der letzten beiden Legislaturperioden illustriert. Dazu werden rund 120 leitfadengestützte Interviews mit MEPs
verschiedener Fraktionen und mit EP-Beschäftigten aus den Jahren 2019 und 2020 herangezogen.
2
GM auf supranationaler Ebene und Theorieansatz
Gender Mainstreaming (GM) zielt als politische Strategie darauf ab, institutionelle
Strukturen zu hinterfragen und dahingehend zu transformieren, dass Gleichstellungsförderung zu einem alle Ebenen durchdringenden Anspruch wird (Lombardo/Meier/
Verloo 2009; Minto/Mergaert 2018). Auf EU-Ebene wurde die Strategie durch ein institutionenübergreifendes feministisches Akteur*innennetzwerk in Artikel 3,2 des Amsterdamer Vertrags verankert (Hubert/Stratigaki 2016; Jacquot 2015). Wenn auch die vorherige Begrenzung der EU auf Beschäftigungspolitik durchbrochen wurde, enttäuschte
die praktische Umsetzung: Gleichstellungsaspekte wurden in bestehende Politiken und
Prozesse nur integriert oder kooptiert; selten wurden Politiken und Prozesse grundlegend transformiert (Lomazzi/Crespi 2019; Lombardo/Meier/Verloo 2009). Insgesamt
fehl(t)en kohärente Gleichstellungsziele, sodass Gleichstellung ein „empty signifier“ und eher als technische denn inhaltliche Anforderung verstanden wurde (Minto/
Mergaert 2018; Verloo 2005), da alltägliche Verwaltungsroutinen unangetastet blieben
(Jacquot 2015). Zudem entpolitisierte die Art und Weise der GM-Umsetzung in der
Europäischen Kommission Gleichstellungspolitik, schwächte die ursprüngliche institutionelle Gleichstellungsstruktur und untergrub die zuvor gute institutionenübergreifende
Zusammenarbeit feministischer Akteur*innen (Ahrens 2018; Jacquot 2015).
Die Rolle des EP für EU-Gleichstellungspolitik wurde in der Forschung öfter beleuchtet (Ahrens 2018; Jacquot 2015; Rolandsen Agustín 2013; Van der Vleuten 2012),
ebenso wie die Sitzverteilung der Fraktionen (auch nach Geschlecht), aber Studien zu
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Gender Mainstreaming im Europäischen Parlament
13
Gleichstellung innerhalb der EP-Prozesse, in den Fraktionen und in verschiedenen Organisationsebenen sind jüngerer Natur (vgl. ausführlicher Ahrens/Rolandsen Agustín
2019). Wichtig ist es, die einzelnen Fraktionen zu betrachten, denn deren gleichstellungspolitische Schwerpunkte und fraktionsinterne Gleichstellungsförderung unterscheiden sich deutlich (Kantola/Rolandsen Agustín 2016, 2019). Die Differenz verläuft
hier zwar teilweise entlang nationaler Delegationen, aber insgesamt folgt sie einem
Rechts-Links-Spektrum. EP-Entscheidungen erfordern stets Mehrheiten und die jeweilige Fähigkeit, entsprechende Koalitionen zu formen, beeinflusst die politische Ausrichtung im EP (Warasin et al. 2019).
Um zu verstehen, warum und wie sich GM in Organisationen (nicht) umsetzen
lässt, wird in diesem Beitrag Feministischer Institutionalismus aus einer analytischen
Perspektive genutzt. Es wird analysiert, wie Geschlechterverhältnisse in Organisationen formale und informelle Regeln und Normen sowie das Handeln von Frauen* und
Männern* prägen (Mackay/Kenny/Chappell 2010; Mackay 2014). Alle Institutionalismustheorien sehen Strukturen und Akteur*innen als ko-konstitutiv an: Institutionen
formen Verhalten mittels Regeln, Normen und Prozessen; Akteur*innen initiieren oder
blockieren institutionellen Wandel (Mackay/Kenny/Chappell 2010: 573). Ebenso setzen
sich alle mit formalen und informellen Institutionen, also den ‚gesellschaftlichen Spielregeln‘, sowie Entstehen, Stabilität und Wandel von Institutionen auseinander – wenn
auch mit unterschiedlichen Schwerpunkten (Mackay/Kenny/Chappell 2010: 576ff.).
Dennoch sind die meisten Institutionalismustheorien blind gegenüber der Kategorie
Gender als einem konstitutiven Element von Institutionen. Hier setzt Feministischer
Institutionalismus an, der schon länger in der Genderforschung genutzt wird, wenn auch
oft nicht unter diesem Label (Mackay/Kenny/Chappell 2010; MacRae/Weiner 2017).
Theorien des (Feministischen) Institutionalismus und deren Anwendung auf supra
nationale Institutionen und Politiken (MacRae/Weiner 2017) legen nahe, dass die GMUmsetzung im EP davon abhängt, wie sich GM in formale und informelle Regeln einflechten lässt, ob diese transformiert werden und ob GM zur Norm wird. Das hilfreiche,
von Mackay (2014) geprägte Konzept der „nested newness“ setzt daran an, dass auch
neue Institutionen – Organisationen wie Regeln – an frühere institutionelle Kontexte
anknüpfen und sich in der neuen Institution widerspiegeln (Mackay 2009, 2014). Durch
die „Nestedness“ werden neue Regeln und Normen als früheren Verfahren widersprechend wahrgenommen und dadurch (un)bewusst von Akteur*innen abgelehnt. Das trifft
vor allem auf oft unhinterfragte Regeln zum Geschlechterverhältnis zu, die sehr veränderungspersistent sind, wie Mackay für das schottische Parlament aufzeigt (Mackey
2009, 2014). Beim EP kommt einerseits hinzu, dass Akteur*innen mit dem Wissen ihrer früheren (nationalen) Institutionen eintreffen und (neue) gleichstellungsbezogene
Regeln zu jenen gehören, die leicht „vergessen“ werden (Mackay 2009). Andererseits
lassen das EP-Selbstverständnis als Gleichstellungsförderer*in (Van der Vleuten 2019)
und der ohnehin beständige Wandel durch Vertragsänderungen vermuten, dass sich GM
in EP-Prozessen verankern ließe.
Laut Minto und Mergaert, die die Umsetzung von GM und dessen Evaluation in der
Europäischen Kommission verglichen haben, kann der GM-Institutionalisierungsstand
in fünf Dimensionen gemessen werden: 1) formale Verankerung (inkl. Top-down-Unterstützung), 2) standardisierte Strukturen und Prozesse mit Zuständigen, 3) Qualitäts-
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Petra Ahrens
sicherung z. B. durch Ressourcen und Trainings, 4) transparente Verantwortlichkeiten
und Evaluation und 5) langfristige Stabilität (Minto/Mergaert 2018: 209f.). Wenn für
alle Dimensionen messbare Ergebnisse vorliegen, kann von einer Institutionalisierung
ausgegangen werden. Die chronologische Analyse der EP-Entschließungen wird diese
fünf Dimensionen für das EP überprüfen.
Der multikulturelle, multinationale und konsensorientierte Kontext macht das EP
zu einem Spezialfall, um konkurrierende Ideen zu Gleichstellungsförderung zu untersuchen. Angenommen wird, dass dieser Prozess nicht friktionsfrei verläuft, da GM sich
nicht ohne Widerstand ‚einbetten‘ lässt und je nach politischer Couleur Berichterstatter*innen inhaltliche Schwerpunkte setzen können. Parlamentarische Institutionen, die
erfordern, dass Regeln, Normen und politische Zielsetzungen beständig ausgehandelt
und gelernt werden müssen, ermöglichen es zu untersuchen, wie dennoch gerade Geschlechternormen und -machtverhältnisse reproduziert werden (Haastrup/Kenny 2016).
Zentral sind somit die Akteur*innen, die Regeln und Normen ‚erlernen‘ und Politikziele
definieren, da diese zentral für institutionelle Veränderungsprozesse sind.
3
Datenmaterial und Methoden
Die Analyse stützt sich vorrangig auf EP-Primärtexte wie Resolutionen, Plenardebattenprotokolle, Ausschussdokumente und weitere Publikationen mit Bezug zur GM-Umsetzung, die im Rahmen von sechs EP-Resolutionen entstanden sind (siehe Quellenverzeichnis). Die Resolutionen und alle anhängigen Texte1 erlauben es, narrative und historische Sequenzen der GM-Umsetzung sowie Änderungen nachzuvollziehen (Vromen
2010). Analysiert wurde, auf welche EP-Regeln explizit oder implizit Bezug genommen
und wo neue Vorgaben angedockt wurden. Konkreter: Wie wurde GM formal angelegt,
welche verschiedenen Akteur*innen – FEMM-Ausschuss, individuelle MEPs, Fraktionen – spielten welche Rolle? Was lässt sich daraus für die GM-Umsetzung ableiten?
Die verschiedenen Dokumente wurden jeweils für die entsprechende Legislaturperiode in ihrer zeitlichen Abfolge systematisiert und dann miteinander verglichen, um
Textänderungen, die zwischen verschiedenen Entwurfsstadien und der Endfassung bestehen, herauszufiltern. Diese Elemente wurden dahingehend beleuchtet, ob die Änderung für die GM-Umsetzung hemmend oder fördernd sein würde. Ergänzend wurden
Stellungnahmen von MEPs, Fraktionen und anderen Akteur*innen herangezogen, um
Begründungslogiken nachvollziehen zu können. Für Plenardebatten wurde geprüft,
welche Elemente von welcher Fraktion oder von welchem MEP kommentiert wurden
und ggf. wogegen opponiert wurde.
Die chronologische Institutionalisierungsanalyse wird ergänzt durch eine Momentaufnahme der aktuellen Umsetzung. Dafür wird auf Interviewmaterial zugegriffen, das
im Rahmen des Forschungsprojekts „Gender, party politics and democracy in Europe:
A study of European Parliament’s party groups“ (EUGenDem) erhoben wurde. Vor der
EP-Wahl 2019 wurden rund 50 Interviews und danach rund 70 Interviews mit MEPs
1
Pro Entschließung sind dies zehn bis 30 Dokumente. Die Anzahl anhängiger Dokumente nimmt
über die Zeit zu.
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Gender Mainstreaming im Europäischen Parlament
15
aller Fraktionen und EP-Beschäftigten zu Genderaspekten der Fraktionsarbeit geführt,
u. a. auch zu GM. Das Interviewmaterial wurde vom Forschungsteam, angelehnt an die
Grounded Theory (Glaser/Strauss 1967), computergestützt mit AtlasTi induktiv und deduktiv kodiert (vgl. auch Ahrens 2018: 253ff.). Die Interviews fragten u. a. nach der
alltäglichen Arbeit und den (Inter-)Aktionen der Interviewten und ermöglichen es, eine
„thick description“ des Politikprozesses aus feministischer, interpretativer Perspektive zu entwickeln (Vromen 2010: 249). Für diesen Beitrag wurden die Codes ‚Gender
Mainstreaming‘, ‚FEMM-Ausschuss‘, ‚Opposition gegen Gleichstellung‘ und ‚Ausschüsse‘ sowohl übergreifend als auch entlang der Fraktionen ausgewertet. Die Interviewauswertung ermöglicht es nachzuzeichnen, wie Akteur*innen die GM-Umsetzung
im EP aktuell beurteilen.
4
GM-Entschließungen und Berichte im Überblick
Das EP legt in seiner Geschäftsordnung (EP-GO) die Regeln für alle parlamentarischen
Verfahren fest, darunter für EP-Entschließungen und Berichte. Jeder Beschluss zu GM
musste hier ansetzen und regelkonform sein. Das EP unterscheidet legislative und
nicht-legislative Berichte sowie Initiativberichte (Kap. 2, EP-GO).2 Ausschüsse können
nach § 54 Initiativberichte beantragen, sie müssen aber von der Konferenz der Präsidenten3 genehmigt werden. Zum Zeitpunkt der ersten GM-Entschließung war die Zustimmung für Eigeninitiativberichte und -resolutionen alles andere als gesichert, im Gegenteil: Anträge wurden häufig als eine Form der Agendakontrolle abgelehnt (Mamadouh/
Raunio 2003: 335). Unterstützung für die erste GM-Entschließung zu sichern und sie in
standardisierte EP-Verfahren einzubetten, war demnach für alle weiteren Institutionalisierungsschritte von entscheidender Bedeutung.
Der erste FEMM-Antrag im Jahr 2002 für einen Initiativbericht und eine EP-Entschließung zu GM musste diese Hürde nehmen. Die damalige Präsidialgruppe bestand
aus acht Männern und einer Frau4, wobei spätere Presseberichte vermuten lassen, dass
der EP-Präsident Pat Cox (Europäische Liberale, Demokraten und Reformer) das Anliegen unterstützte (EP-News 2004). Am 14. März 2002 wurde FEMM mit dem ersten
Eigeninitiativbericht zu GM in Form einer Entschließung beauftragt. Mit Lissy Gröner
(Progressive Allianz der Sozialdemokraten, S&D) wurde am 27. März 2002 eine sehr
bekannte und etablierte EP-Gleichstellungspolitikerin als Berichterstatterin ernannt.
Interessanterweise wurde der Ausschuss für Recht und Binnenmarkt (JURI) für eine
Stellungnahme nominiert, was bei Eigeninitiativberichten ungewöhnlich ist. Warum der
FEMM-Initiativberichtantrag genehmigt wurde, bleibt mangels entsprechender Dokumentation offen.
2
3
4
Legislative sind Kommissionsvorschläge für Richtlinien oder Verordnungen, die seit dem LissabonVertrag mehrheitlich durch Rat und EP gemeinsam beschlossen werden. Nicht-legislative Berichte
wie Stellungnahmen und Entschließungen beantragt der zuständige Ausschuss nach § 46 der EPGO zu Themen mit EP-Initiativrecht.
Die Konferenz der Präsidenten (sic!) besteht aus EP-Präsident*in und Fraktionsvorsitzenden.
Mit Ausnahme der Doppelfraktionsspitze aus Heidi Hautala und Paul Lannoye (Fraktion der
Grünen/Freie Europäische Allianz) gab es nur männliche Fraktionsvorsitzende nebst EP-Präsident
Cox.
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Petra Ahrens
Werden die Entschließungen verglichen, hat die Fraktionszugehörigkeit des/der Berichterstatter*in nicht den Verabschiedungsprozess beeinflusst: Alle Resolutionen wurden als EP-Standardverfahren im FEMM inhaltlich erarbeitet, abgestimmt und dann in
einer Plenarsitzung beschlossen. Die Texte begründen die Notwendigkeit von GM mit
dem EU-Rechtsrahmen und entsprechenden Vertragsartikeln5 (z. B. Gleichstellung der
Geschlechter (EUV § 2 und § 3 Abs. 3), Nichtdiskriminierung (AEUV § 19) und gleiches Entgelt (AEUV § 157)), weiteren EP-Resolutionen sowie den Aktivitäten anderer
supranationaler Institutionen wie der Europäischen Kommission. Alle erläutern, warum
Gleichstellungsförderung nach wie vor ein notwendiges Ziel sei, bewerten die bisherige
GM-Umsetzung im EP und formulieren zukünftige Schritte.
Seit 2003 sind die GM-Entschließungen ähnlich strukturiert und werden vom
FEMM gestaltet. Im FEMM erhielten alle – trotz steigender Gegenstimmen – eine klare
Mehrheit, während in den Plenarsitzungen nur die 2009er-Entschließung breit unterstützt wurde, die erste im Jahr 2003 und alle weiteren nach 2009 waren umstrittener.
Tabelle 1 zeigt Berichterstatter*innen, Beschlussdaten und Abstimmungsergebnisse.
Tabelle 1: Gender Mainstreaming Entschließungen im EP
2003
2007
2009
2011
2016
2019
An FEMM
vergeben
EP-Beschluss
Berichterstatter*in
14.3.02
13.3.03
Lissy Gröner, S&D
15.6.06
23.9.08
7.7.11
10.9.15
5.7.18
18.1.07
22.4.09
17.11.11
8.3.16
15.1.19
Anna Záborská, EVP
Anna Záborská, EVP
Abstimmung
FEMM
Abstimmung
Plenum
+
23
255
-
1
186
0
0
+
30
-
0
15
Handzeichen
ohne Zählung
0
1
+
23
603
-
0
12
0
1
64
Mikael Gustafsson,
GUE/NGL
+
21
378
-
4
154
0
0
40
Angelika Mlinar,
ALDE
+
23
453
-
6
173
0
1
79
Angelika Mlinar,
ALDE
+
19
492
-
6
126
0
0
75
Erläuterung: + = Zustimmung, - = Ablehnung, 0 = Enthaltung
Quelle: Öffentliches Dokumentenregister EP6; Zusammenstellung Petra Ahrens.
5
6
Artikelnummerierungen ändern sich oft mit neuen Verträgen. Hier wird die Nummerierung aus
dem Lissabon-Vertrag verwendet.
Siehe https://www.europarl.europa.eu/RegistreWeb/home/welcome.htm?language=DE [Zugriff:
08.01.2021].
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Gender Mainstreaming im Europäischen Parlament
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Während die formale Verankerung der Entschließungen relativ einfach erschien, blieb
die in ihnen integrierte Berichterstattung zur GM-Implementierung eine herausfordernde Aufgabe, wie verschiedene Texte erkennen lassen. Normalerweise präsentieren EPEntschließungen lediglich zukünftige Handlungsoptionen und -anforderungen, doch die
zu GM fungierten auch als rückblickende GM-Berichterstattung. In diesen lässt sich
erkennen, wie der FEMM-Ausschuss und der/die jeweilige Berichterstatter*in EPSpielräume nutzte, um institutionellen Wandel zu initiieren und GM schrittweise formal
einzubetten. Auch ist erkennbar, welche geplanten neuen Regeln verworfen wurden.
Die Institutionalisierung verlief somit nicht linear, sondern wurde zwischen den Fraktionen im FEMM-Ausschuss ausgehandelt.
Die Umsetzungsergebnisse entlang der bereits 2003 definierten GM-Implementierungsschritte generierte der FEMM-Ausschuss mittels parlamentarischer Abfragen bei
anderen Ausschüssen und EP-Organen. Außerdem lud FEMM die für GM zuständigen
(stellvertretenden) Vorsitzenden anderer Ausschüsse regelmäßig zur Berichterstattung
ein und organisierte gemeinsame Sitzungen des GM-Netzwerks, das auf der Basis der
ersten Entschließung eingerichtet wurde. In den ersten vier Entschließungen bewerteten
die Berichterstatter*innen die Entwicklung der GM-Umsetzung im EP seit der vorherigen Entschließung, jedoch fehlte diese Bewertung in der 2016er-Entschließung. Die
2019er-Entschließung enthielt dann eine politische Erklärung, in der gleichzeitig die
Gesamtergebnisse zusammengefasst und – angesichts der Umsetzungsbemühungen seit
2003 – ernsthaftere Umsetzungsschritte angemahnt wurden. Damit beinhalten bereits
die ersten Entschließungen viele Elemente der ersten vier Dimensionen von Minto und
Mergaert (2018) und weisen auf eine messbare Institutionalisierung hin.
4.1 Vorsichtig-selektive GM-Blaupausen-Institutionalisierung – 2003
Die erste Entschließung wurde von den Debatten zur Umsetzung des Amsterdamer
Vertrages mit seinem GM-Artikel sowie von der bevorstehenden EU-Erweiterung
auf Mittel- und Osteuropa beeinflusst. FEMM knüpfte darin strategisch an den GMAnsatz der Europäischen Kommission an und präsentierte diesen als bewährte Methode und Blaupause für das EP (Europäisches Parlament 2003: 18). Zudem wurden
die 1998er-Empfehlungen der Expert*innengruppe des Europarates auf das EP umgemünzt: Top-down-Verankerung durch eine Hochrangige Gruppe für Gleichstellung im
EP, feste GM-Zuständigkeit eines Ausschusses, pro Ausschuss und Delegation ein*e
zuständige*r MEP, die ein GM-Netzwerk bilden, Beginn in Schlüsselbereichen, jährliches Arbeitsprogramm und Monitoring der Ausschüsse und Delegationen zu GM,
Überprüfung und Veränderung des EP-Verwaltungshandelns (Europäisches Parlament
2003: 19ff.). Jedes dieser Elemente gab es in ähnlicher Form in der Europäischen Kommission; es fehlten Gendertrainings und Konsultationen mit Interessengruppen. Gleichzeitig entsprachen z. B. Ausschusszuständigkeit und Berichtspflichten dem formalen
EP-Prozedere in anderen Politikbereichen; auch übergreifende Netzwerke parallel zu
Ausschüssen waren verbreitet.
In der Plenarsitzung betonte die Berichterstatterin, dass es angesichts der Erweiterung von 2004 notwendig sei, einen hohen Frauenanteil im Parlament zu gewährleisten,
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und der Bericht argumentierte, dass GM und die Gleichstellungsförderung zentrale demokratische Aufgaben des EP als „die den Bürgern am nächsten stehende europäische
Institution“ (Europäisches Parlament 2003: 19) seien.
Damals galt FEMM als feministisches Bollwerk (Jacquot 2015), kein anderer Ausschuss hatte jemals einen so hohen Anteil an weiblichen Mitgliedern (bis heute) bei
gleichzeitig rein freiwilliger Mitgliedschaft (d. h. zusätzlich zu einer weiteren „regulären“ Ausschusstätigkeit), relativer Machtlosigkeit und Umstrittenheit im EP (Ahrens
2016; Nugent 2019). Diese Zweiteilung ist aus institutionalistischer Perspektive bemerkenswert: Es gab Stimmen im EP, dass FEMM überflüssig sei, wenn alle EP-Organe
GM umsetzten (Ahrens 2016). In diesem Fall wäre es fatal gewesen, wenn dezidiert
FEMM für GM zuständig gewesen wäre. Eine klare Zuständigkeit vorzusehen, aber
offen zu lassen, welcher Ausschuss dies sei, sicherte GM ab, auch für den Fall, dass
FEMM in Zukunft als überflüssig erachtet und aufgelöst werden würde. FEMM als
zuständigen Ausschuss vorzuschlagen, monopolisierte wiederum dessen Kontrolle da
rüber, ob GM ausreichend umgesetzt würde. Die GM-Institutionalisierung wurde somit
zu einer Lebensversicherung für FEMM, indem es wiederkehrende Aufgaben und Berichtspflichten zuwies. Der mitberatende JURI-Ausschuss beschloss einstimmig eine
befürwortende Stellungnahme und bemängelte nur zu starre Vorgaben für Beförderungen von Frauen in der EP-Verwaltung (Europäisches Parlament 2003: 26f.).
4.2 Konservative Institutionalisierung – 2007
Nachdem sich das EP verpflichtet hatte, GM umzusetzen und alle zwei Jahre Bericht
zu erstatten, verzögerte die EP-Wahl 2004 dennoch den nächsten Bericht – er wurde
erst im Dezember 2006 vorgelegt und die Entschließung im Januar 2007 angenommen. Die Osterweiterung und der gescheiterte Verfassungsprozess veränderten die EPZusammensetzung, die Politikprozesse und damit auch die GM-Implementierung. Die
zentralen Vorschläge wurden zwar umgesetzt, aber die verschiedenen Berichtsentwürfe,
die verabschiedete Fassung und die Plenardebatte veranschaulichen die internen Probleme, GM in die bestehenden Verfahren einzubetten bzw. neue einzurichten und nicht nur
Lippenbekenntnisse zu produzieren.
Der Berichtsentwurf, der als Grundlage für die endgültige Entschließung angenommen wurde, enthielt konkrete Schritte wie z. B. die Notwendigkeit, dass die Hochrangige Gruppe für die Gleichstellung der Geschlechter verbindliche politische Prioritäten
für GM in den Bereichen Gesetzgebung, Kommunikations- und Informationspolitik
sowie Gender Budgeting und Personalpolitik festlegt. In dem Bericht wurde auch vorgeschlagen, verbindliche Quoten für weibliche Abgeordnete zu vereinbaren. Eine frühere Version forderte Gendertrainings für alle Abgeordneten. All diese neuen Elemente
verschwanden in der endgültigen Entschließung und zeigen die Schwierigkeit, GM in
einem neu zusammengesetzten EP weiter zu institutionalisieren.
In der Plenardebatte betonten mehrere FEMM-MEP, dass die EP-Ausschüsse GM
ablehnen, aber der Bericht selbst formuliert es viel vorsichtiger. Er entschuldigt die
klaren Mängel, weil „Ausschussmitglieder die jedem Vorgang innewohnende politische
und technische Dynamik nicht ignorieren [können]“, von der Dynamik „die Aufmerksamkeit abhängig [sei], die speziell dem Gender Mainstreaming in den einzelnen Aus-
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schüssen beigemessen wird“ und Mängel „keinesfalls zu einer moralischen Verurteilung
von Personen führen“ (Europäisches Parlament 2006: 10) dürfen. Die neu zusammengesetzten Ausschüsse schienen GM als Anforderung ‚vergessen‘ zu haben und (ver-)
zögerten außerdem entschlossene Umsetzungsschritte und -regeln.
Für diese erstaunliche Zurückhaltung des FEMM trug nicht zuletzt die EVP-Fraktion die Verantwortung, die mit der Slowakin Anna Záborská eine sehr konservative
Ausschussvorsitzende stellte (Jacquot 2015). Die von der EVP in weiten Teilen vertretenen konservativen Vorstellungen von Geschlechterverhältnissen und -binarität
spiegeln sich auch in Formulierungen wider, wie z. B. in der Forderung nach einer
„uneingeschränkte[n] Anerkennung des ‚Andersseins‘ und des ‚Sichergänzens‘ von
Männern und Frauen“ (Europäisches Parlament 2006: 8), die seitdem in jeder Legislaturperiode aufscheinen (Kantola/Rolandsen Agustín 2019).
Dennoch brachte der erste GM-Bericht im EP auch zwei neue informelle Regeln,
die sich mit weiteren Berichten verstetigten. So wurden erstens während des Berichtszeitraums regelmäßig die GM-Zuständigen anderer Ausschüsse zu einer Anhörung eingeladen, was langfristig den Druck zur GM-Umsetzung erhöhte, weil es unangenehm
wurde, nichts berichten zu können (Ahrens 2016: 790). Zweitens wurden mittels Fragebogen die Aktivitäten der Ausschüsse zu GM abgefragt, um damit den Bericht zu
bestücken (Europäisches Parlament 2006: 10). Beide Schritte waren für sich betrachtet
nicht ungewöhnlich im Austausch zwischen Ausschüssen; in der Breite jedoch – FEMM
kontaktiert alle Ausschüsse – verwiesen sie auf eine neue politische Praxis.
4.3 Naming, shaming und Stillstand zum Ende der Legislaturperiode – 2009
Die gegen Ende der Legislaturperiode verabschiedete Entschließung von 2009 ähnelte
bei den Entwürfen und dem angenommenen Text den beiden vorhergehenden. Der Berichtsteil änderte sich jedoch erheblich und beinhaltete anstatt allgemeiner Zusammenfassungen ein detailliertes ‚name-and-shame‘ der EP-Ausschüsse und -Delegationen mit
Einzelheiten zu (nicht vorhandenen) Tätigkeiten. 14 von 19 Ausschüssen gaben demnach
an, geschlechtsspezifische Aspekte nur in einigen (Nicht-)Gesetzgebungsakten aufgenommen zu haben, die anderen fünf meldeten gar nichts (Europäisches Parlament 2009). Da
rüber hinaus berichteten nur die Ausschüsse für Regionale Entwicklung (REGI), Fischerei
(PECH), Auswärtige Angelegenheiten (AFET) und deren Unterausschuss für Sicherheit
und Verteidigung (SEDE) über eine eigene interne Gleichstellungsstrategie. Nur wenige
Ausschüsse verwendeten entweder nach Geschlecht aufgeschlüsselte Daten, Indikatoren,
Folgenabschätzungen oder Gender Budgeting. Im Vergleich zu 2007, als kein einziger
Ausschuss eine interne Gleichstellungsstrategie hatte, stiegen die Aktivitäten jedoch und
indizieren damit, dass GM erste Schritte von formalen Vorgaben zu ‚rules in use‘ machte.
4.4 Verstetigung und neue (Minimal-)Schritte – 2011
Wie bei der Entschließung von 2007 spielten das neu gewählte EP und die durch den in
Kraft getretenen Vertrag von Lissabon erweiterten Befugnisse eine Rolle für die 2011erEntschließung. Wieder enthielt sie kaum Einzelheiten zu den verschiedenen Ausschüs-
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sen, die sich (nicht) aktiv an der GM-Umsetzung beteiligten. Das Prozedere bis zur
Verabschiedung war die kürzeste aller untersuchten Entschließungen und deutet auf
Routine im FEMM hin und damit auf eine wachsende Institutionalisierung.
Während bis dato die Mobilisierung anderer Ausschüsse zur GM-Umsetzung fokussiert wurde, signalisierte diese Entschließung eine Strategieänderung bei FEMM.
Der Bericht hob sog. „Gender Mainstreaming-Änderungsanträge“ (GMA7) als erfolgreichstes Instrument hervor. Änderungsanträge sind ein Standardinstrument im EP,
das Schattenberichterstatter*innen nutzen, um die Position ihrer Fraktion in die zu
verabschiedenden Texte einzubringen. Üblicherweise werden Änderungsanträge nur
in den zuständigen Ausschuss eingebracht oder ggf. zwischen zwei bis drei beteiligten Ausschüssen ausgetauscht, und nicht als ‚freies‘ Kommentarinstrument eines
Ausschusses gegenüber allen anderen. GMAs jedoch wurden im FEMM entworfen
und abgestimmt und dann an den federführenden Ausschuss weitergeleitet, der sie
annehmen konnte, aber nicht musste. In der Mehrheit der GMAs wurden in den meis
ten Fällen die Wörter „genderspezifisch“, „geschlechtsspezifisch“, „weiblich“ oder
„Frauen“ bei Kommissionsvorschlägen oder Initiativberichten hinzugefügt (Europäisches Parlament 2014: 84).
GMAs wurden zum informellen neuen GM-Standard, und in der Entschließung von
2011 wurde vorgeschlagen, sie offiziell in die Geschäftsordnung aufzunehmen. Dies
wurde bis heute nicht umgesetzt. Seit der Legislaturperiode 2009–2014 nahmen die
Anteile euroskeptischer, populistischer und rechtsgerichteter Parteien in verschiedenen
Fraktionen zu, oder sie gründeten neue Fraktionen, wie z. B. die Europäischen Konservativen und Reformer oder Europa der Freiheit und der Demokratie. Viele dieser
nationalen Delegationen vertraten zudem anti-feministische, anti-LGBTQI und gleichstellungsfeindliche Positionen (Verloo/Paternotte 2018). Zugleich ergab sich eine Diskrepanz zwischen FEMM-Positionen, die eher von einer linksgerichteten Mehrheit aus
S&D, Grünen, ALDE und GUE/NGL geprägt waren, und der eher rechtskonservativen
Mehrheit in Plenarsitzungen (Warasin et al. 2019). Während bis zu dieser Legislaturperiode FEMM-MEP gemeinsam agierten (Ahrens 2016; Nugent 2019), verstärkte sich
seit 2009 der Einfluss der Fraktionen auf die Debatten (Kantola/Rolandsen Agustín
2016; Warasin et al. 2019). Die daraus resultierenden Machtverhältnisse verhinderten
u. U. die formale Absicherung der GMAs, denn über die Änderung der Geschäftsordnung entscheiden die Fraktionen, und hier gab es im zentralen Gremium offensichtlich
keine Mehrheit, um GM weiter zu formalisieren – neue ‚rules in use‘ wurden nicht zu
‚rules in form‘. Dass in dieser Legislaturperiode keine weitere GM-Entschließung verabschiedet wurde, spricht für sich selbst.
4.5 GM-Wiederbelebung und Ausdehnung – 2016
Die 2016er-Entschließung zeigt, dass die Institutionalisierung von GM im EP sich nicht
nahtlos mit bestehenden Regeln und Normen verbinden lässt bzw. die EP-Organe durch
den FEMM kontinuierlich daran erinnert werden müssen, da sie sonst ‚vergessen‘ werden. Dennoch ist diese Entschließung beispielhaft dafür, wie routiniert und beharrlich
7
Englisch: Gender-Mainstreaming-Amendments, daher GMA.
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FEMM Brüche zwischen GM und eben diesen etablierten Regeln und Normen benennt
und neue Schritte vorschlägt.
FEMM schlug zusätzliche Maßnahmen vor, um GM im EP besser einzubetten
(Europäisches Parlament 2016: 8ff.) und um die verschiedenen Dimensionen einer erfolgreichen GM-Implementierung zu verbessern und zu verbinden (Minto/Mergaert
2018). So wurde z. B. für die weitere formale Verankerung gefordert, obligatorische
(anstatt fakultative) Genderaktionspläne für alle Ausschüsse mit einem in der EP-GO
festgelegten Verfahren zu etablieren. Diese Genderaktionspläne sowie die Forderung,
GM-Anhörungen im Zweijahresrhythmus parallel zur FEMM-Berichterstellung in allen Ausschüssen abzuhalten, dienen auch einer besseren Evaluation. Ein institutioneller
Koordinierungs- und Überwachungsmechanismus, der für die GM-Akteur*innen eingerichtet werden solle, um die Hochrangige Gruppe für Gleichstellung der Geschlechter
und Vielfalt, FEMM, das GM-Netzwerk und die EP-Verwaltung zu vernetzen, würde neue Strukturen und Zuständigkeiten etablieren. Dies gilt auch für das bestehende
GM-Netzwerk, welches erweitert werden soll, indem Delegationen einbezogen und
Adminstrator*innen in den Ausschusssekretariaten ernannt werden sollen. Auch ein
gemeinsamer Vorsitz durch FEMM und ein Mitglied der anderen Ausschüsse auf Rotationsbasis sollte eingerichtet werden. Der Vorschlag eine*r ständige*n GM-Berichterstatter*in zielt auf mehr formale Verankerung und standardisierte Zuständigkeit ab.
Neben Forderungen mit einem direkten Bezug zur GM-Umsetzung schlug FEMM
weitere gleichstellungspolitische Schritte vor. So solle ein ausgewogenes Geschlechterverhältnis (mind. 40 % bis 2020) bei Ausschuss- und Fraktionsvorsitzenden und den
Führungspositionen in der EP-Verwaltung sowie die gezielte Ernennung von MEPs
des bisher unterrepräsentierten Geschlechts in jedem Ausschuss (inkl. FEMM) erreicht
werden. Erstmals bezog FEMM auch LGBTQI-Themen ein, forderte erneut Gender
Budgeting und Gendertrainings ein und verlangte eine stärkere Einbeziehung des Europäischen Instituts für Gleichstellung und der EP-Forschungsdienste. Zudem zeigt die
Entschließung, dass rückblickend die Berichterstattung durch Abfragen, die neu geschaffenen Organe wie die Hochrangige Präsidialgruppe und das GM-Netzwerk sowie
GMAs fest institutionalisiert waren und auch Effekte zeitigten, da die Aufmerksamkeit
für geschlechtsspezifische Aspekte in einer Reihe von Ausschüssen zunahm (Europäisches Parlament 2016).
4.6 Gegen das Vergessen neuer Regeln – 2019
Die jüngste Entschließung wiederholte die Kernelemente der 2016er-Resolution und
verdeutlicht Institutionalisierungsfortschritte. Angelika Mlinar (ALDE) wurde 2016
die erste „Ständige Berichterstatterin für Gender Mainstreaming“. Zusätzlich zum
GM-Netzwerk ernannte das EP für jeden Ausschuss eine*n weitere GM-Netzwerkadministrator*in. Mit Ausnahme von FEMM erstellten alle anderen 21 (Unter-)Ausschüsse
einen öffentlich auf der FEMM-Website einsehbaren Aktionsplan. Außerdem publizierte das EP-Präsidium 2017 den Bericht „Gleichstellung von Männern und Frauen im
Generalsekretariat des Europäischen Parlaments – Bestandsaufnahme und Perspektiven
2017–2019“ mit quantifizierten Zielen für verschiedene Führungspositionen (European
Parliament 2018). Es bleibt abzuwarten, wie beständig diese neuen ‚rules in use‘ sein
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werden. Tatsächlich ist es FEMM gelungen, GM als ‚rule in use‘ zu einer ‚rule in form‘
weiterzubefördern, also messbare Erfolge für eine erweiterte formale Verankerung zu
erzielen. Anknüpfend an den sog. Corbett-Bericht wurde 2019 mit Artikel 228a8 erstmals GM in der EP-Geschäftsordnung verankert.
Inhaltlich betont die Entschließung Diversität als wichtiges Element zur Gleichstellungsförderung und bezieht sich auf LGBTQI und Geschlechtsidentität sowie auf
die Bedeutung männlicher Vorbilder für Gleichstellung (Europäisches Parlament 2019).
Zudem wurde vorgeschlagen, Regeln gegen sexuelle Belästigung und sexuellen Missbrauch in die Geschäftsordnung des EP aufzunehmen und entsprechende Fortbildungsmaßnahmen anzubieten.
4.7 Aktuelle Umsetzung in der Praxis
Gegenüber dieser formalen Absicherung und der Umsetzung vieler Vorschläge erlaubt
die Interviewanalyse eine qualitativ differenzierte Einschätzung, die die heterogene und
widersprüchliche GM-Umsetzung in der Praxis illustriert. Ein Großteil der Interviewten war überrascht von der Frage nach GM und antwortete nicht oder ausweichend.
Die besser informierten Interviewten waren oft (frühere) Mitglieder von FEMM oder
zuvor national engagiert in Sachen Gleichstellungspolitik. Ihre folgenden Einblicke
zeichnen ein kritisches Bild. Sie verwiesen auf die doppelte Zuständigkeit von FEMM
für 1) strukturelle Fragen wie Monitoring und neue Regelvorschläge und 2) inhaltliche Arbeit durch GMAs oder Stellungnahmen für andere Ausschüsse und benannten
zwei Kernprobleme. Erstens sei es angesichts der schieren Anzahl von EP-Vorgängen
und begrenzter Ressourcen unmöglich, in ausreichendem Maße GMAs und Stellungnahmen abzugeben, weshalb Ausschüsse ohne Gesetzgebungskompetenz wie der für
konstitutionelle EP-Fragen (AFCO) außen vor blieben (Interviews Grüne/EFA 10.3.20,
EPP 6.3.20). Zweitens sei der Umgang mit inhaltlichen Vorschlägen unterschiedlich
zwischen den Ausschüssen trotz objektiv gleicher Umsetzungsschritte, wie z. B. dem
Genderaktionsplan und GM-Zuständige MEP und Administrator*innen. Viele der Interviewten aus anderen Ausschüssen wussten nicht, dass es beides gab, und gaben an, dass
GM in ihrem Ausschuss nicht sichtbar sei (Interviews S&D 15.5.19, Renew 13.12.19,
EPP 4.3.20). Es hing von der Eigeninitiative der GM-Zuständigen ab, ob Genderaspekte
im Ausschuss auftauchen (Interview S&D 27.1.20).
Ebenso zeigt sich die Doppelaufgabe von FEMM-MEP in anderen Ausschüssen: Sie
bringen GMAs ein, auch wenn sie nicht im Ausschuss für GM zuständig sind (Interviews
Greens/EFA 13.3.20, S&D 26.2.20, Renew 4.3.20). GMAs werden ähnlich wie GM in
den Ausschüssen verschieden aufgenommen: Manche nehmen sie an, andere wie ECON
lehnen sie oft ab (Interviews Renew 4.3.20, S&D 4.3.20; vgl. European Parliament
2018). Viele der Interviewten führen das auf den höheren Status von ECON verglichen
mit FEMM zurück. Aber es wird in den Interviews auch klar, dass es generell wichtig ist,
welche Fraktion den Ausschussvorsitz hat und dass FEMM-Positionen oft als ‚zu links‘
8
Jetzt Artikel 239 – Gleichstellung von Männern und Frauen (Gender Mainstreaming): Das Präsidium nimmt einen Gleichstellungsaktionsplan an, damit der Gleichstellungsaspekt bei allen Tätigkeiten des Parlaments auf allen Ebenen und in allen Phasen berücksichtigt wird. Der Gleichstellungsaktionsplan wird zweimal jährlich kontrolliert und wenigstens alle fünf Jahre überarbeitet.
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gewertet werden. Andererseits betonten auch einige, dass viele Ausschüsse aufmerksamer für Genderaspekte geworden seien und dass diese auch dann diskutiert und in Texte
aufgenommen werden würden, wenn es zuvor keine FEMM-Initiative gegeben habe.
In den Entschließungen wurden die Fraktionen bisher nicht als Umsetzungselement
einbezogen, was aber angesichts ihrer Gatekeeper-Funktion wichtig wäre. Einige Fraktionen wie GUE/NGL und Grüne/EFA haben fraktionsinterne GM-Prozesse, andere wie
S&D und Renew gelten als offen und proaktiv, sind aber angewiesen auf engagierte MEP.
Alle anderen Fraktionen haben keine fraktionsinternen GM-Regeln. Die Annahme von
GMAs in den Ausschüssen hängt aber davon ab, welche Fraktion zuständig ist und welche
Mehrheiten sie erreichen kann. Werden Gender-Aspekte nicht als Priorität oder irrelevant
gesehen, fallen sie aus den abzustimmenden Texten heraus. Die Interviews weisen somit
darauf hin, dass die neuen GM-Regeln noch nicht ausreichend institutionalisiert sind.
5
Fazit
Mit diesem Beitrag wurden Geschichte, Institutionalisierung und Hindernisse von GM
im EP institutionentheoretisch analysiert. Minto und Mergaert entwickelten im Rahmen
von Feministischem Institutionalismus fünf Kerndimensionen zur GM-Umsetzungsmessung und scheinen eine grundsätzliche Institutionalisierung nahezulegen (Minto/
Mergaert 2018: 209f.). Bei einem oberflächlichen Blick sind die ersten vier (formale
Verankerung; klare Zuständigkeiten und standardisierte Prozesse; Qualitätssicherung;
transparente Verantwortlichkeiten und Evaluation) mit der ersten Entschließung 2003
beschlossen und dann umgesetzt worden. Die fünfte Dimension (langfristige Stabilität)
kann ebenfalls nach den zahlreichen Folge-Entschließungen bestätigt werden.
Dennoch zeigt die Analyse der Entschließungen, anhängigen Dokumente und rund
120 Interviews, dass die Kerndimensionen zur Institutionalisierung und damit auch der
Ansatz des Feministischen Institutionalismus’ weiter nuanciert werden müssen. Hinsichtlich der ersten Dimension ist GM zwar formal verankert, seit Neuestem sogar
mit Artikel 239 in der EP-GO von 2019 festgeschrieben, aber eine aktive Top-downUnterstützung ist trotz Hochrangiger Präsidialgruppe nicht sichtbar in den untersuchten
Dokumenten und Interviews. Die zweite und dritte Dimension stellen eine besondere
Herausforderung an Parlamente, deren Zusammensetzung sich mit Wahlen ändert, da
dann Zuständigkeiten und Ressourcen neu ausgehandelt werden. Mit der Zuständigkeit
von FEMM und den GM-Berichten ist die vierte Dimension vermutlich am vollständigs
ten erfüllt. Das vom – eher machtlosen – FEMM eingerichtete System hat es dem EP
aufgrund der kontinuierlichen Berichterstattung und zusätzlicher Elemente wie GMAs,
Genderaktionsplänen, GM-Netzwerken und einer ständigen GM-Berichterstatter*in erschwert, die neuen GM-Regeln zu vergessen (wenn auch vielleicht nicht überall umzusetzen). Ob die fünfte Dimension gegeben sein wird, hängt davon ab, wie sich das
EP nach Wahlen zusammensetzt und ob z. B. rechtskonservative und -extreme Parteien
stärker im EP oder im FEMM vertreten sein werden.
Angesichts der zentralen Rolle kritischer Akteur*innen sollte zukünftig insbesondere die innerparlamentarische Mikropolitik erforscht werden, um präziser die (informellen) Machtverhältnisse zwischen Ausschüssen sowie Fraktionen zu erfassen und zu
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Petra Ahrens
untersuchen, wie formale Regeln in der Praxis strategisch gehandhabt, (re)interpretiert
und letztlich verändert werden (Wiesner 2018). Mikropolitische Ansätze gehen davon
aus, dass der kontinuierliche und dynamische Wandel des EU-Politiksystems von innen
durch verschiedene Akteur*innen zielgerichtet vorangetrieben wird (Wiesner 2018). In
ihrer Studie zu inter-institutionellen Vereinbarungen zeichnet Wiesner (2018) z. B. nach,
wie das EP Anhörungen der nächsten Europäischen Kommission als ‚rules in use‘ etablierte, sie teilweise vertraglich absichern konnte und dadurch schrittweise an Macht
gewann. Die Unterscheidung von formal festgeschriebenen Regeln (‚rules in form‘)
und tatsächlich praktizierten Regeln (,rules in use‘) verweist hier auf die existierenden
Differenzen zwischen beiden und den damit verbundenen politischen Ergebnissen.
Solche konflikthaften Prozesse zeigen sich in alltäglichen Praxen, und da das EP
keine homogene Akteur*in ist (Wiesner 2018: 376), sondern einzelne MEPs, Ausschüsse, Fraktionen und nicht zuletzt die EP-Verwaltung strategisch (inter)agieren, wäre es
sinnvoll, die fördernden und blockierenden Elemente weiter auszuloten. Denn insgesamt hätte eine erfolgreiche GM-Umsetzung im EP potenziell erhebliche Auswirkungen, da dann das EP als Ko-Gesetzgeberin Gleichstellungsfragen in alle EU-Politiken
einflechten könnte.
Anmerkung
Dieser Beitrag wurde durch den European Research Council (ERC) im Rahmen des
Forschungsrahmenprogramms Horizont 2020 der Europäischen Union unter der Fördervereinbarung Nr. 771676 gefördert.
Literaturverzeichnis
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Ahrens, Petra (2016). The Committee on Women’s Rights and Gender Equality in the European
Parliament: Taking Advantage of Institutional Power Play. Parliamentary Affairs, 69(4),
778–793.
Ahrens, Petra (2018). Actors, Institutions and the Making of EU Gender Equality Programs.
Basingstoke: Palgrave Macmillan.
Ahrens, Petra & Rolandsen Agustín, Lise (Hrsg.). (2019). Gendering the European Parliament.
Structures, Policies, and Practices. London: Rowman & Littlefield.
EP-News (2004). Gender mainstreaming – a legacy for the next Parliament, 21.4.2004.
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TEXT+PRESS+NR-20040421-1+0+DOC+XML+V0//EN&language=EN#SECTION3.
Europäisches Parlament (2003). Bericht über Gender Mainstreaming im Europäischen Parlament
(2002/2025(INI)).
Europäisches Parlament (2006). Bericht über Gender Mainstreaming in der Arbeit der Ausschüsse
(2005/2149(INI)).
Europäisches Parlament (2009). Bericht über den integrierten Ansatz der Gleichstellung
von Männern und Frauen im Rahmen der Arbeit der Ausschüsse und Delegationen
(2008/2245(INI)).
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RegData/etudes/STUD/2014/493051/IPOL-FEMM_ET(2014)493051_DE.pdf.
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and Delegations of the European Parliament. Policy Department for Citizens’ Rights and
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eu/RegData/etudes/STUD/2018/608850/IPOL_STU(2018)608850_EN.pdf.
Glaser, Barney G. & Strauss, Anselm (1967). The Discovery of Grounded Theory. Strategies for
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Haastrup, Toni & Kenny, Meryl (2016). Gendering Institutionalism: A Feminist Institutionalist
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European Integration Theory (S. 197–216). Opladen, Berlin, Toronto: Verlag Barbara Budrich.
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Jacquot, Sophie (2015). Transformations in EU Gender Equality. From Emergence to Dismantling.
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Kantola, Johanna & Rolandsen Agustín, Lise (2016). Gendering Transnational Party Politics: The
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Kantola, Johanna & Rolandsen Agustín, Lise (2019). Gendering the representative work of the
European Parliament: A political analysis of women MEP’s perceptions of gender equality in
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Korolczuk, Elżbieta & Graff, Agnieszka (2018). Gender as ‘Ebola from Brussels’: The Anticolonial Frame and the Rise of Illiberal Populism. Signs: Journal of Women in Culture and
Society, 43(4), 797–821.
Lomazzi, Vera & Crespi, Isabella (2019). Gender Mainstreaming and Gender Equality in Europe.
Policies, Culture and Public Opinion. Bristol: Policy Press.
Lombardo, Emanuela; Meier, Petra & Verloo, Mieke (Hrsg.). (2009). The Discursive Politics of
Gender Equality: Stretching, Bending and Policymaking. London: Routledge.
Mackay, Fiona (2009). Travelling the distance? Equal Opportunities in the Scottish Parliament.
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Mackay, Fiona (2014). Nested Newness, Institutional Innovation, and the Gendered Limits of
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Gender Lens: Towards a Feminist Institutionalism? International Political Science Review,
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Mamadouh, Virginie & Raunio, Tapio (2003). The committee system: powers, appointments and
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Minto, Rachel & Mergaert, Lut (2018). Gender mainstreaming and evaluation in the EU:
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Petra Ahrens
Van der Vleuten, Anna (2019). The European Parliament as a Constant Promoter of Gender
Equality. Another European Myth? In Petra Ahrens & Lise Rolandsen Agustín (Hrsg.),
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Integration, 40(4), 375–391.
Quellenverzeichnis
Entschließung des Europäischen Parlaments zu Gender Mainstreaming im Europäischen
Parlament (2002/2025(INI))
Entschließung des Europäischen Parlaments zu Gender Mainstreaming in der Arbeit der
Ausschüsse (2005/2149(INI))
Entschließung des Europäischen Parlaments vom 22. April 2009 zu dem integrierten Ansatz
der Gleichstellung von Männern und Frauen im Rahmen der Arbeit der Ausschüsse und
Delegationen (2008/2245(INI))
Entschließung des Europäischen Parlaments vom 17. November 2011 zu dem Gender
Mainstreaming in der Arbeit des Europäischen Parlaments (2011/2151(INI))
Entschließung des Europäischen Parlaments vom 8. März 2016 zum Gender Mainstreaming in
der Arbeit des Europäischen Parlaments (2015/2230(INI))
Entschließung des Europäischen Parlaments vom 15. Januar 2019 zum Gender Mainstreaming im
Europäischen Parlament (2018/2162 (INI))
Zur Person
Petra Ahrens, Dr. phil., Senior Researcher, Tampere University, Finnland. Arbeitsschwerpunkte:
europäische Integration, Europäisches Parlament, Gleichstellungspolitiken in der Europäischen
Union und Deutschland, Zivilgesellschaft, politische Repräsentation.
Kontakt: Tampere University, Faculty of Social Sciences, Linna 5069, FI-33014 Tampere, Finnland
E-Mail: petra.ahrens@tuni.fi
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A “santa sabedoria” e a vanglória: dois textos da literatura sapiencial anglo-saxônica e sua tradução para o português
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Veredas da História
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1 Doutor em História Social pela Universidade de São Paulo – USP, professor de História Medieval da
Faculdade Sumaré (SP), membro da International Society of Anglo-Saxonists – ISAS e BRATHAIR – Grupo
de Estudos Celtas e Germânicos; e-mail: eosmedeiros@hotmail.com Elton Oliveira Souza de Medeiros
Faculdade Sumaré (SP)1 Resumo: As fontes literárias anglo-saxônicas estão entre as de maior importância da Alta
Idade Média ocidental. Nela podemos encontrar os tipos mais variados de assuntos e
temas, tanto em prosa quanto em verso. Dentre alguns desses tipos encontramos o que se
convencionou chamar de “literatura sapiencial”. O objetivo deste trabalho é apresentar
uma introdução a essa espécie de construção literária e, ao mesmo tempo, trazer pela
primeira vez em língua portuguesa o poema Vanglória e um dos capítulos da versão
anglo-saxônica da obra de Boécio, De Consolatio Philosophiae; ambos a partir dos
originais em inglês antigo. Palavras-chave: Inglês Antigo, Boécio, Anglo-Saxões, Sabedoria. Palavras-chave: Inglês Antigo, Boécio, Anglo-Saxões, Sabedoria. 5 5 Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 A “SANTA SABEDORIA” E A VANGLÓRIA: DOIS TEXTOS DA
LITERATURA SAPIENCIAL ANGLO-SAXÔNICA E SUA
TRADUÇÃO PARA O PORTUGUÊS. Elton Oliveira Souza de Medeiros
Faculdade Sumaré (SP)1 Keywords: Old English, Boethius, Anglo-Saxons, Wisdom. THE “HOLY WISDOM” AND THE VAINGLORY: TWO TEXTS OF THE ANGLO-
SAXON WISDOM LITERATURE AND THEIR TRANSLATION TO
PORTUGUESE. Abstract: The Anglo-Saxon literary sources can be considered among the most important
sources from the western Early Middle Ages. There can be found different sorts of
subjects and themes, in prose as well in verse. Among some of these types there is what
is conventionally called "wisdom literature". The aim of this paper is to present an
introduction to this kind of literary construction and, at the same time, bring for the first
time in Portuguese the poem Vainglory and one of the chapters of the Anglo-Saxon
version of the work of Boethius, De Consolatio Philosophiae; both from their originals
in Old English. Keywords: Old English, Boethius, Anglo-Saxons, Wisdom. 6
38 6 6
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 2 HANSEN, Elaine Tuttle. The Solomon Complex. Toronto: University of Toronto Press, 1988, p. 3 – 11.
3 MEDEIROS, Elton O. S. “Erudição e Poesia Encantatória na Inglaterra anglo-saxônica: Salomão e
Saturno I & o Encantamento das Nove Ervas”. Mirabilia 20 (1), 2015, p. 313 – 363. ( )
p
4 PULSIANO, Philip & TREHARNE, Elaine. A Companion to Anglo-Saxon Literature. Oxford:
Blackwell, 2001, p. 11 – 49; HUPPÉ, Bernard F. Doctrine and Poetry: Augustine’s Influence on Old
English Poetry. Nova York: State University of New York, 1959, p. 28 – 216. HANSEN, Elaine Tuttle. The Solomon Complex. Toronto: University of Toronto Press, 1988, p. 3 – 11. 2 HANSEN, Elaine Tuttle. The Solomon Complex. Toronto: University of Toronto Press, 1988, p. 3 – 11.
3 MEDEIROS, Elton O. S. “Erudição e Poesia Encantatória na Inglaterra anglo-saxônica: Salomão e
Saturno I & o Encantamento das Nove Ervas”. Mirabilia 20 (1), 2015, p. 313 – 363.
4 PULSIANO, Philip & TREHARNE, Elaine. A Companion to Anglo-Saxon Literature. Oxford:
Blackwell, 2001, p. 11 – 49; HUPPÉ, Bernard F. Doctrine and Poetry: Augustine’s Influence on Old
English Poetry. Nova York: State University of New York, 1959, p. 28 – 216. 3 MEDEIROS, Elton O. S. “Erudição e Poesia Encantatória na Inglaterra anglo-saxônica: Salomão
Saturno I & o Encantamento das Nove Ervas”. Mirabilia 20 (1), 2015, p. 313 – 363. Introdução “Literatura sapiencial” é uma terminologia utilizada – ao menos desde o século
XIX – por estudiosos no campo da História e estudos literários medievais ao se referir a
algumas das produções do período da Inglaterra anglo-saxônica que possuem elementos
temáticos muito semelhantes a textos do mundo clássico e do oriente próximo, textos
veterotestamentários (como os Salmos, Provérbios e Eclesiastes) e os diálogos de Platão. A terminologia não é um consenso e nem muito precisa ao tentar identificar quais textos
se enquadram dentro deste perfil. Contudo, de forma geral, existe um grupo de por volta
de quinze a vinte poemas e ao menos três textos em prosa, todos em inglês antigo, que
podem ser reconhecidos como de teor sapiencial.2 Essas obras dos tempos da Inglaterra anglo-saxônica (séculos V – XI)
compartilham de forma geral uma preocupação a respeito da condição, a existência e as
experiências humanas pelo mundo. Acompanhadas de argumentações e demonstração de
grandes verdades universais e, a exemplo de textos bíblicos e da antiguidade greco-latina,
por vezes adotam o estilo do “diálogo sapiencial”: onde uma personagem sábia (um
andarilho, um ancião ou até mesmo a personificação da própria Sabedoria – ainda que
implicitamente) passa seus ensinamentos à outra, geralmente representando a figura do
jovem discípulo. Tais obras na Inglaterra claramente apresentam uma forte influência da tradição
greco-latina através do viés da cristianização, desde o século V, assim como também da
tradição hiberno-latina.3 Na verdade será essa influência, que ao se adequar e assimilar
elementos locais da cultura bárbara anglo-saxônica, através da religião cristã como seu
catalisador, que teremos o florescimento de uma tradição cultural local nessa região das
Ilhas Britânicas.4 O mesmo tipo de construção literária e influência que pode ser
encontrado em outras regiões do norte europeu, como a Escandinávia medieval, em obras
como, por exemplo, o poema Hávamál e o Vafþrúðnismál – ambos na Edda Poética –
assim como o prólogo da Edda em Prosa de Snorri Sturluson. E, como no caso inglês,
temos a influência da tradição clássica através do estabelecimento da cultura cristã em
solo escandinavo (muito provavelmente por meio do trabalho de missionários anglo- 7 7
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 saxões e germânicos nos séculos IX – XI, não apenas no campo literário, mas também
político).5 Apesar dessa característica em comum, as obras da literatura sapiencial anglo-
saxônica variam muito em estilo e conteúdo. HOLLANDER, Lee M. The Poetic Edda. Austin: University of Texas Press, 2000, p. 4; LASSEN,
Annette. Odin på kristent pergament: En teksthistorisk studie. Copenhagen: Museum Tusculanums
Forlag, 2011; MEDEIROS, Elton. O. S. “A Linhagem Perdida de Sceaf: Genealogias Mítico-Históricas na
Inglaterra e Escandinávia & a Tradução do Prólogo da Edda de Snorri Sturluson”, Signum 16 (3), 2015, p.
46-77. 6 MEDEIROS, Elton O. S. “ráðna stafi, mjǫk stóra stafi, mjǫk stinna stafi: Tradução Comentada dos
Poemas Rúnicos Anglo-Saxão, Islandês, Norueguês e do Abecedarium Nordmannicum”. Medievalis 4 (1),
2015, p. 1 -31. , p
7 MEDEIROS, Elton O. S. “Erudição e Poesia Encantatória na Inglaterra anglo-saxônica: Salomão
Saturno I & o Encantamento das Nove Ervas”. Mirabilia 20 (1), 2015, p. 313 – 363. 5 HOLLANDER, Lee M. The Poetic Edda. Austin: University of Texas Press, 2000, p. 4; LASSEN, 9 Os demais poemas do manuscrito são (nomes traduzidos para o português, seguidos da forma mais
comumente encontrada em língua inglesa): Cristo I (O Advento) [Christ I], Cristo II (A Ascensão) [Christ
II], Cristo III (O Julgamento) [Christ III], Guthlac A [Guthlac A], Guthlac B [Guthlac B], Azarias [Azarias],
A Fênix [The Phoenix], Juliana [Juliana], O Andarilho [The Wanderer], Os Dons dos Homens [The Gifts
of Men], Preceitos [Precepts], O Navegante [The Seafarer], Vanglória [Vainglory], Widsith [Widsith], As
Fortunas dos Homens [The Fortunes of Men], Máximas I [Maxims I], A Ordem do Mundo [The Order of
the World], O Poema Rimado [The Rhyming Poem], A Pantera [The Panther], A Baleia [The Whale], A Introdução Em Os Diálogos de Salomão e
Saturno,8 como ocorre nas obras poéticas de mesmo nome, temos o diálogo entre a figura
do rei Salomão e de Saturno; mas desta vez a respeito de assuntos de teor apócrifo da
tradição bíblica (por exemplo, detalhes sobre a batalha celeste entre Lúcifer e o Senhor,
a longa linhagem dos filhos de Adão e Eva, entre outros temas). Assim como nos textos
poéticos, o diálogo entre as personagens dessas três obras em prosa – além de suas
especificidades – acaba por abordar os mais diversos aspectos da Criação, da natureza
divina, a conduta correta a se seguir durante a vida terrena e demais elementos
moralizantes, religiosos e, principalmente, de reflexão sobre os benefícios da Sabedoria. Especialmente a sabedoria de origem divina. reelaborações dos textos originais para o contexto da Inglaterra anglo-saxônica, e em
ambos encontramos o formato do diálogo sapiencial. No Froferboc este ocorre entre Mod
(“mente” em inglês antigo) e Wisdom (“sabedoria”) – no original tal diálogo é entre a
personagem do filósofo (representando o próprio Boécio) e a Filosofia – e nos Solilóquios
este ocorre entre a personagem de Agostinho e a Razão. Em Os Diálogos de Salomão e
Saturno,8 como ocorre nas obras poéticas de mesmo nome, temos o diálogo entre a figura
do rei Salomão e de Saturno; mas desta vez a respeito de assuntos de teor apócrifo da
tradição bíblica (por exemplo, detalhes sobre a batalha celeste entre Lúcifer e o Senhor,
a longa linhagem dos filhos de Adão e Eva, entre outros temas). Assim como nos textos
poéticos, o diálogo entre as personagens dessas três obras em prosa – além de suas
especificidades – acaba por abordar os mais diversos aspectos da Criação, da natureza
divina, a conduta correta a se seguir durante a vida terrena e demais elementos
moralizantes, religiosos e, principalmente, de reflexão sobre os benefícios da Sabedoria. Especialmente a sabedoria de origem divina. 8 KEMBLE, John. The Dialogue of Salomon and Saturnus. Londres: Ælfric Society, 1848. Introdução Nos poemas em inglês antigo Máximas I e
II, por exemplo, encontramos uma coleção heterogênea de sabedoria popular,
conhecimentos sobre a natureza e conselhos religiosos formulados como provérbios. No
poema Preceitos um ancião dá conselhos de natureza social e religiosa a um jovem
ouvinte. Enquanto o Poema Rúnico,6 por sua vez, oferece talvez uma das produções mais
enigmáticas do corpo literário anglo-saxônico, estruturado em torno das vinte nove runas
anglo-saxônicas e nos poderes mágicos que elas evocam. Em poemas como As Fortunas
dos Homens e Os Dons dos Homens vamos encontrar uma lista ou catálogo que identifica
os destinos e as dádivas que Deus concede à humanidade, contendo assim também um
teor moralizante e religioso. Já outros como A Ordem do Mundo se apresentam como
longas meditações onde um poeta-sábio discorre sobre as glórias e maravilhas da Criação;
poemas como Vanglória e Resignação exploram temas sobre os malefícios da soberba e
sobre o arrependimento, e temos também Os Diálogos Salomão e Saturno7 que retrata o
diálogo entre o rei veterotestamentário e a antiga divindade pagã sobre diversos assuntos,
desde aspectos mais mundanos da Criação até o poder mágico da oração do Pai-Nosso. Outros poemas em inglês antigo, apesar de não serem classificados exatamente
como tal, também possuem trechos que contêm características da literatura sapiencial,
como surge em algumas adivinhações, elegias, e trechos de poemas como O Andarilho,
O Navegante e Beowulf. Na prosa anglo-saxônica podemos citar como parte desse tipo produção o
Froferboc, os Solilóquios de Santo Agostinho e a versão em prosa de Os Diálogos de
Salomão e Saturno. Os dois primeiros são versões em inglês antigo de textos latinos:
sendo o Froferboc derivado da Consolatio Philosophiae de Boécio e os Solilóquios do
original de mesmo nome. Os dois textos apresentam como características interpolações e 8 8
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 reelaborações dos textos originais para o contexto da Inglaterra anglo-saxônica, e em
ambos encontramos o formato do diálogo sapiencial. No Froferboc este ocorre entre Mod
(“mente” em inglês antigo) e Wisdom (“sabedoria”) – no original tal diálogo é entre a
personagem do filósofo (representando o próprio Boécio) e a Filosofia – e nos Solilóquios
este ocorre entre a personagem de Agostinho e a Razão. 8 KEMBLE, John. The Dialogue of Salomon and Saturnus. Londres: Ælfric Society, 18 Vanglória e a Santa Sabedoria. Com a finalidade de apresentar de forma mais clara o teor das obras de conteúdo
sapiencial do cenário da produção literária anglo-saxônica (séculos V – XI), foi escolhido
um trecho do Froferboc – mais especificamente o capítulo 17 – e o poema em inglês
antigo conhecido como Vanglória. A escolha por esses dois textos se deu em função de
um ponto em comum. Além das óbvias referências aos benefícios do conhecimento e da
Sabedoria (aqui grafado em maiúsculo para diferenciar o conhecimento oriundo da
sabedoria terrena daquela advinda do logos divino, portanto, a “Santa Sabedoria”), é
possível também relacionarmos em ambos os textos os elementos contrários ao
comportamento sapiencial, movidos pela ignorância e a soberba e seus malefícios tanto
no campo da vida pessoal quanto político. O poema Vanglória é um dos textos que compõe o manuscrito conhecido como o
Livro de Exeter (MS. Exeter, Cathedral Library, 3501, fols. 8-130)9 sendo um dos maiores 9 9
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 do corpo poético anglo-saxônico. O manuscrito consiste numa coleção de poemas de
perfil tanto religioso quanto secular, e possivelmente confeccionado por um único autor,
que também teria sido o responsável por outros dois manuscritos em latim (MS. Londres,
Lambeth Palace Library, 149, fols. 1-139; e MS. Oxford, Bodleian Library, Bodley 319). O que pode servir de indício para entender que o manuscrito de Exeter, assim como os
outros dois manuscritos, se trata de uma cópia. A organização dos textos do Livro de
Exeter parece ter sido intencional, sendo os oito primeiros poemas os mais longos,
seguidos dos demais mais curtos.10 Uma das primeiras referências ao Livro de Exeter data do século XI no inventário
de livros entregues por Leofric, bispo de Devon e Cornwall, à catedral de Exeter pouco
antes de seu falecimento em c. 1072; contudo, não há qualquer indício de como o
manuscrito chegou a suas mãos. Através de análises paleográficas, o manuscrito teria sido
composto originariamente em meados do século X (c. 975). Isso seria muito antes que a
sé episcopal fosse estabelecida em Exeter em 1050. LAPIDGE, Michael. The Blackwell Encyclopaedia of Anglo-Saxon England. Oxford: Blackwell,
004, p. 177. BRADLEY, S.A.J. (trad. org.) Anglo Saxon poetry, Londres: Everyman, 2003, p. 201.
12 LAPIDGE, Michael. The Blackwell Encyclopaedia of Anglo-Saxon England. Oxford: Blackwe
2004, p. 177 – 178. Perdiz [The Partridge], Alma e Corpo II [Soul and Body II], Deor [Deor], Wulf e Eadwacer [Wulf and
Eadwacer], Adivinhações 1-59 [Riddles 1-59], Adivinhações 3, 5, 9, 25, 26, 29, 30a, 35, 38, 42-8, 53
[Riddles 3, 5, 9, 25, 26, 29, 30a, 35, 38, 42-8, 53], O Lamento da Esposa [The Wife’s Lament], O Dia do
Julgamento I [Judgment Day I], Resignação [Resignation], A Descida ao Inferno [The Descent into Hell],
A Doação de Donativos [Alms-giving], Faraó [Pharaoh], A Oração do Senhor I [The Lord’s Prayer I],
Fragmento Homilético II [Homiletic Fragment II], Adivinhações 30b [Riddles 30b], Adivinhações 60
[Riddles 60], A Mensagem do Marido [The Husband’s Message], A Ruína [The Ruin], Adivinhações 61-95
[Riddles 61-95], Adivinhações 61, 66, 69, 76, 86 [Riddles 61, 66, 69, 76, 86]; cf. KRAPP, George Philip
(ed.) & DOBBIE, Elliot van Kirk (ed.) The Anglo-Saxon Poetic Records III: The Book of Exeter. Nova
York: Columbia University Press 1936 ],
ç
,
,
,
,
[
,
,
,
,
];
,
g
p
d.) & DOBBIE, Elliot van Kirk (ed.) The Anglo-Saxon Poetic Records III: The Book of Exeter. Nova
ork: Columbia University Press, 1936. p
11 BRADLEY, S.A.J. (trad. org.) Anglo-Saxon poetry, Londres: Everyman, 2003, p. 201. Perdiz [The Partridge], Alma e Corpo II [Soul and Body II], Deor [Deor], Wulf e Eadwacer [Wulf an
Eadwacer], Adivinhações 1-59 [Riddles 1-59], Adivinhações 3, 5, 9, 25, 26, 29, 30a, 35, 38, 42-8, 5 Perdiz [The Partridge], Alma e Corpo II [Soul and Body II], Deor [Deor], Wulf e Eadwacer [Wulf and p
11 BRADLEY, S.A.J. (trad. org.) Anglo-Saxon poetry, Londres: Everyman, 2003, p. 201.
12 LAPIDGE, Michael. The Blackwell Encyclopaedia of Anglo-Saxon England. Oxford: Blackwell, Perdiz [The Partridge], Alma e Corpo II [Soul and Body II], Deor [Deor], Wulf e Eadwacer [Wulf and
Eadwacer], Adivinhações 1-59 [Riddles 1-59], Adivinhações 3, 5, 9, 25, 26, 29, 30a, 35, 38, 42-8, 53
[Riddles 3, 5, 9, 25, 26, 29, 30a, 35, 38, 42-8, 53], O Lamento da Esposa [The Wife’s Lament], O Dia do
Julgamento I [Judgment Day I], Resignação [Resignation], A Descida ao Inferno [The Descent into Hell],
A Doação de Donativos [Alms-giving], Faraó [Pharaoh], A Oração do Senhor I [The Lord’s Prayer I],
Fragmento Homilético II [Homiletic Fragment II], Adivinhações 30b [Riddles 30b], Adivinhações 60
[Riddles 60], A Mensagem do Marido [The Husband’s Message], A Ruína [The Ruin], Adivinhações 61-95
[Riddles 61-95], Adivinhações 61, 66, 69, 76, 86 [Riddles 61, 66, 69, 76, 86]; cf. KRAPP, George Philip
(ed.) & DOBBIE, Elliot van Kirk (ed.) The Anglo-Saxon Poetic Records III: The Book of Exeter. Nova
York: Columbia University Press, 1936.
10 LAPIDGE, Michael. The Blackwell Encyclopaedia of Anglo-Saxon England. Oxford: Blackwell,
2004, p. 177.
11 BRADLEY, S.A.J. (trad. org.) Anglo-Saxon poetry, Londres: Everyman, 2003, p. 201.
12 LAPIDGE, Michael. The Blackwell Encyclopaedia of Anglo-Saxon England. Oxford: Blackwell,
2004, p. 177 – 178. [Riddles 3, 5, 9, 25, 26, 29, 30a, 35, 38, 42 8, 53], O Lamento da Esposa [The Wife s Lament], O Dia d
Julgamento I [Judgment Day I], Resignação [Resignation], A Descida ao Inferno [The Descent into Hel
A Doação de Donativos [Alms-giving], Faraó [Pharaoh], A Oração do Senhor I [The Lord’s Prayer
Fragmento Homilético II [Homiletic Fragment II], Adivinhações 30b [Riddles 30b], Adivinhações 6
[Riddles 60], A Mensagem do Marido [The Husband’s Message], A Ruína [The Ruin], Adivinhações 61-9
[Riddles 61-95], Adivinhações 61, 66, 69, 76, 86 [Riddles 61, 66, 69, 76, 86]; cf. KRAPP, George Phil 15 O traidor, dentro do cenário literário do período, desempenha a figura modelar do pária, do proscrito;
aquele que traiu seu senhor – terreno ou divino – e que em função disso passa a viver exilado do convívio
de outras pessoas. Não fazendo mais parte do mundo dos homens ou da Criação por ter se voltado contra a
ordem universal. Os melhores exemplos desse tipo de personagem na poesia em inglês antigo é Lúcifer
(condenado às trevas do Inferno por sua traição), seguido por Caim (fratricida e que também se volta contra
Deus) e, talvez o mais famoso de todos, Grendel no poema Beowulf (por ser retratado como da linhagem
amaldiçoada de Caim e por desprezar o poder divino e a sociedade humana, o que o torna não apenas um
proscrito como um ser monstruoso e abominável). Para maiores informações sobre personagens da
literatura anglo-saxônica e a relação com o pecado da traição ver MEDEIROS, Elton O. S. “Mito e História
no Campo de Batalha: Apropriação e Interpretação do Passado pelo Medievo e como História Nacional”.
Revista de História Comparada 8 (2), 2014, p. 29 – 59. 13 Assim como o livro apócrifo de Enoque (I Enoque) outros textos apócrifos que podem ter influenciado
na produção literária anglo-saxônica são o Caverna dos Tesouros, Descida de Cristo ao Inferno e A
Vingança do Salvador, entre outros que podem ter sido levados à Inglaterra nos tempos de Teodoro,
arcebispo de Canterbury (c. 602 – 690). Teodoro era de origem grega e pode ter levado tais livros consigo
para a Inglaterra, pois, apesar de não terem sobrevivido aos nossos tempos, sabe-se da existência de tais
livros apócrifos na Inglaterra devido às evidências encontradas nos comentários e estudos do século VII da
“Escola de Canterbury” – estes sim preservados – que fazem referências aos mesmos. Para maiores
informações ver BISCHOFF, Bernhard & LAPIDGE, Michael. Biblical Commentaries from the
Canterbury School of Theodore and Hadrian. Cambridge: Cambridge University Press, 2007; KASKE,
R. E. “Beowulf and the Book of Enoch”. Speculum, 46 (3), 1971, p. 421-431; PROENÇA, Eduardo de
(org.) Apócrifos da Bíblia e pseudo-epígrafos. São Paulo: Fonte Ed., 2005. g )
p
p
p g
KRAPP, George Philip (ed.) The Anglo-Saxon Poettic Records I: The Junius Manuscript, Nova
ork: Columbia University Press, 1931. 13 Assim como o livro apócrifo de Enoque (I Enoque) outros textos apócrifos que podem ter influenciado
na produção literária anglo-saxônica são o Caverna dos Tesouros, Descida de Cristo ao Inferno e A
Vingança do Salvador, entre outros que podem ter sido levados à Inglaterra nos tempos de Teodoro,
arcebispo de Canterbury (c. 602 – 690). Teodoro era de origem grega e pode ter levado tais livros consigo
para a Inglaterra, pois, apesar de não terem sobrevivido aos nossos tempos, sabe-se da existência de tais
livros apócrifos na Inglaterra devido às evidências encontradas nos comentários e estudos do século VII da
“Escola de Canterbury” – estes sim preservados – que fazem referências aos mesmos. Para maiores
informações ver BISCHOFF, Bernhard & LAPIDGE, Michael. Biblical Commentaries from the
Canterbury School of Theodore and Hadrian. Cambridge: Cambridge University Press, 2007; KASKE,
R. E. “Beowulf and the Book of Enoch”. Speculum, 46 (3), 1971, p. 421-431; PROENÇA, Eduardo de
(org.) Apócrifos da Bíblia e pseudo-epígrafos. São Paulo: Fonte Ed., 2005.
14 KRAPP, George Philip (ed.) The Anglo-Saxon Poettic Records I: The Junius Manuscript, Nova
York: Columbia University Press, 1931.
15 O traidor, dentro do cenário literário do período, desempenha a figura modelar do pária, do proscrito;
aquele que traiu seu senhor – terreno ou divino – e que em função disso passa a viver exilado do convívio
de outras pessoas. Não fazendo mais parte do mundo dos homens ou da Criação por ter se voltado contra a
ordem universal. Os melhores exemplos desse tipo de personagem na poesia em inglês antigo é Lúcifer
(condenado às trevas do Inferno por sua traição), seguido por Caim (fratricida e que também se volta contra
Deus) e, talvez o mais famoso de todos, Grendel no poema Beowulf (por ser retratado como da linhagem
amaldiçoada de Caim e por desprezar o poder divino e a sociedade humana, o que o torna não apenas um
proscrito como um ser monstruoso e abominável). Para maiores informações sobre personagens da
literatura anglo-saxônica e a relação com o pecado da traição ver MEDEIROS, Elton O. S. “Mito e História
no Campo de Batalha: Apropriação e Interpretação do Passado pelo Medievo e como História Nacional”.
Revista de História Comparada 8 (2), 2014, p. 29 – 59. Vanglória e a Santa Sabedoria. Portanto, Leofric trouxe o manuscrito
pronto e já compilado como nós o conhecemos na atualidade, o que exclui seu nome como
um dos possíveis autores da obra.11 Sabe-se que mais tarde, em 1566, uma cópia desse inventário foi adicionada ao
manuscrito e entregue pelo diácono e capelão de Exeter a Matthew Parker, arcebispo de
Canterbury. Exemplar que já possuía a seguinte inscrição em inglês antigo: “i. mycel
englisc boc be gehwilcum þingum on leoðwisan geworht” [“um grande livro em inglês a
respeito de assuntos compostos em forma de verso”]. Apesar de sua existência ser
conhecida desde os tempos de Leofric, o reconhecimento de sua importância para os
estudos do inglês antigo e do período anglo-saxônico – assim como a publicação de seu
conteúdo – só veio a acontecer no século XIX.12 10 10
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 10
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 Voltando nossa atenção especificamente ao poema Vanglória, ele pode ser
identificado – como dissemos anteriormente – como parte da “literatura sapiencial”
anglo-saxônica. Com apenas 84 versos, o conteúdo principal do texto remete aos
ensinamentos dos Pais da Igreja a respeito dos pecados do orgulho e da soberba,
estabelecendo um paralelo entre o comportamento do humilde e o do orgulhoso, traçando
a origem deste mal à soberba de Lúcifer e sua recusa em servir a Deus. Temática
recorrente na literatura em inglês antigo e que possivelmente, dentre outras possíveis
fontes da época, teria sido introduzida na Inglaterra do período por meio do livro apócrifo
de Enoque (I Enoque, capítulos 1 – 16) 13 que narra justamente a rebelião de Lúcifer e
dos demais anjos, a guerra celestial e pôr fim a queda. Complementando assim a
passagem bíblica de Genesis 6, 1 – 4. Além de Vanglória, as referências sobre a insurreição e a queda dos anjos também
aparecem em outras obras importantes do corpo poético anglo-saxão. Temos, por
exemplo, os poemas Gênesis, Cristo & Satã (ambos parte do manuscrito Codex Junius
XI)14 e Descida ao Inferno (também do Livro de Exeter). A narrativa do primeiro é mais
próxima a de Vanglória, ao relatar a rebelião de Lúcifer, apresentando-o como um
guerreiro (um þegn) a serviço do Senhor e que, em consequência de sua soberba, acaba
por desempenhar um dos arquétipos mais negativos dentro do universo literário anglo-
saxônico que é a figura do traidor.15 O relato da batalha celestial também aparece nos dois 11 11
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 últimos poemas supracitados, mas de forma breve, apenas como forma de rememorar a
razão do aprisionamento dos anjos e da razão de Cristo – durante os três dias antes de sua
ressurreição – ter descido até o Inferno para libertar as almas ali cativas e mais uma vez
punir Lúcifer e seus seguidores. Além da temática apócrifa, em Vanglória encontraremos elementos moralizantes
que podemos identificar em outros textos do período e também do contexto do norte
europeu e da antiguidade latina. 16 Como, por exemplo, a sexta estrofe: “De sua inteligência o homem não deve se vangloriar, mas ser
cauteloso sobre os pensamentos; quando o sábio e silencioso vem até uma habitação, raramente o mal recai
sobre o cuidadoso, pois melhor amigo nenhum homem conseguirá do que a grande sabedoria”; cf.
MEDEIROS, Elton O. S. “Hávamál: Tradução Comentada do Nórdico Antigo para o Português. Mirabilia
13 (2), 2013, p. 558. 16 Como, por exemplo, a sexta estrofe: “De sua inteligência o homem não deve se vangloriar, mas s 10
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 O contraste que o narrador em Vanglória estabelece entre
o homem tomado pelo orgulho e aquele que é possuidor de humildade é muito semelhante
em Beowulf ao discurso que a personagem do rei Hrothgar direciona ao jovem Beowulf
após este último voltar vitorioso ao seu salão (Beowulf, versos 1700 – 1768). O velho rei
conta sobre o final trágico do antigo rei Heremod que, tomado pela arrogância e o orgulho,
trouxe grande tristeza e desgraça ao povo dinamarquês e aconselha a Beowulf que não
siga o mesmo caminho. Neste trecho do poema, através da fala de Hrothgar, é elaborada
uma alegoria onde um soberano permite que o orgulho e os vícios corrompam sua alma. Esta elaboração não apenas o aproxima da temática moralizante do texto de Vanglória
como também remonta à tradição da alegoria poética cristã do escritor latino Prudêncio,
em seu poema Psychomachia. Em sua obra, Prudêncio retrata o confronto entre os vícios
e as virtudes como se fosse um combate armado real e físico. Ambos os “exércitos” são
representados por figuras e utensílios do âmbito militar. Assim, em Beowulf tal alegoria
é apresentada da seguinte forma: o “guardião da alma” (sawele hyrde) é a razão ou a
consciência humana que adormece, e as “flechas do vício” penetram na alma, tornando-
a arrogante e fadada a um triste fim, como retratado em Vanglória, versos 26 – 31. Ao mesmo tempo, nos trinta primeiros versos de Vanglória, poderíamos dizer que
há a presença de elementos não apenas moralizantes e cristológicos, mas também voltados
a normas de conduta social. Sobre a postura dos guerreiros no salão, durante o banquete,
em como se portar ao falar aos demais convidados etc. O que pode nos fazer pensar em
outro texto importante do norte europeu e que também, como mencionado anteriormente,
se enquadra no mesmo tipo de literatura sapiencial que os textos anglo-saxônicos: o
poema Hávamál (do conjunto de textos islandeses da Edda Poética), principalmente suas
quinze primeiras estrofes.16 12 12
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 12
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 Em relação ao campo da escrita em prosa, nossa principal fonte para esse artigo
remonta aos tempos do rei Alfred o Grande (871 – 899) e ao que podemos chamar de
período alfrediano (séculos IX – IX). 17 Os demais textos que compõe os textos alfredianos seriam as versões para o inglês antigo de a Regula
Pastoralis (de Gregório Magno), os Solilóquios de Santo Agostinho, os cinquenta primeiros Salmos, além
das leis do rei Alfred. 18 “Livro da Consolação” em português. g
g
Versions of Boethius’s De Consolatione Philosophiae, Vol. I. Oxford: Oxford University Press, 2009, p.
9. 19 GODDEN, Malcom & IRVINE, Susan. The Old English Boethius: An Edition of the Old English
Versions of Boethius’s De Consolatione Philosophiae, Vol. I. Oxford: Oxford University Press, 2009, p.
9.
20 18 “Livro da Consolação” em português. ç
p
g
19 GODDEN, Malcom & IRVINE, Susan. The Old English Boethius: An Edition of the Old Englis ,
,
g
g
ersions of Boethius’s De Consolatione Philosophiae, Vol. I. Oxford: Oxford University Press, 2009, p.
Ibid., p. 18. 20 Ibid., p. 18. 10
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 Mais especificamente à documentação contida nos
textos alfredianos:17 a versão em inglês antigo da obra Consolatio Philosophiae de Boécio
(chamada em inglês antigo como Froferboc18). A escolha se dá uma vez que, entre as
demais obras do conjunto, é nela que se encontra de forma mais explícita o elemento
principal da produção literária sapiencial do período: a Sabedoria divina. A respeito das fontes sobre o Froferboc, é sabido da existência de dois textos
sobreviventes aos dias de hoje: os manuscritos Bodley 180 (2079) e o Cotton Otho A. VI. Conhecidos também como versões B (Bodley) e C (Cotton), estes dois manuscritos são
os mais bem conservados e mais completos da versão feita a partir do texto de Boécio nos
tempos alfredianos. O manuscrito B contém uma versão da Consolatio Philosophiae em inglês antigo
que se acredita ter sido escrito por um único autor, entre o final do século XI e começo
do XII.19 A origem do manuscrito não é muito clara. Sabe-se apenas que ele foi adquirido
pela Universidade de Oxford por volta de 1602, sendo identificado como Translatio
quaedam Alfrædi, através da doação feita por um homem chamado Thomas Draper no
ano anterior. O nome se deve ao texto começar com uma clara referência a figura do rei
Alfred como responsável pela tradução, mas sem explicitar qual seria esse texto. Contudo,
na terceira página há uma referência escrita à mão (supostamente do século XVI) onde se
lê: “Boecius de consolatione philosophie translated into the Saxonice tong by King
Alfred”. Juntamente com a versão do texto de Boécio, Draper também teria doado uma
grande quantidade de cartas de Thomas Becket (copiadas no fim do século XII-XIII), o
Auctor imperfecti operis in Mattaeum, fo. MS (uma cópia de comentários de pseudo-
Crisóstomo sobre o texto de São Mateus) e uma coleção identificada como Epistolae
Cantuarienses, escritas no começo do século XIII.20 Devido a todas essas fontes
apresentarem certa ligação com a sé de Canterbury, uma possibilidade seria de que a 13 13
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 versão B também tivesse sido elaborada em Canterbury. Entretanto, não existem maiores
indícios a esse respeito e a origem do manuscrito continua imprecisa. versão B também tivesse sido elaborada em Canterbury. Entretanto, não existem maiores
indícios a esse respeito e a origem do manuscrito continua imprecisa. p
22 Ibid., p. 46. 21 Ibid., p. 18 – 19. 21 Ibid., p. 18 – 19. 22 Ibid., p. 46. 10
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 A segunda hipótese seria de que o formato apenas em prosa é que
seria de fato a versão final e que esta circulou pela Inglaterra da época e posteriormente
elaborou-se uma versão alternativa em prosa e verso, tentando imitar o formato original
do texto de Boécio.23 Há um número maior de elementos que favorecem a segunda
hipótese: a) o autor omite ou resume drasticamente muitas passagens originais em verso
ao passá-las para o inglês antigo, fato que não ocorre com os textos em prosa (o que
poderia indicar uma maior preocupação com a estrutura das passagens em prosa do que
em verso); b) o formato da versão B está mais bem finalizado (contendo prefácio, índice,
um texto dividido e numerado em respectivos capítulos) e c) seu conteúdo circulou na
época (o texto da versão B era conhecido por Ælfric de Eynsham – final do século X e
início do XI – que o utilizou ao compor os textos de suas “Vidas dos Santos”, e pelo
arcebispo Wulfstan de York em suas homilias durante o mesmo período24). Já sobre o
texto da versão C, não há elementos suficientes que possam comprovar a circulação de
seu conteúdo durante o mesmo período na Inglaterra. O que nos chama a atenção para o texto da Consolatio Philosophiae em inglês
antigo e para os demais textos alfredianos não é sua existência em idioma vernáculo, mas
a maneira como se deu sua composição e as discrepâncias em relação aos textos originais. No caso do Froferboc, ele reproduz de forma geral o texto original de Boécio. Contudo,
ao longo dele podemos notar alterações e reinterpretações, paráfrases e interpolações que
por vezes diferem radicalmente da obra original (o que também se reflete nos demais
textos do conjunto documental alfrediano e encontra paralelos nas reconstruções poéticas
de inspiração bíblica do período, como o poema Judite,25 por exemplo). Os primeiros
estudos modernos sobre o texto em inglês antigo justificavam tais diferenças como erros
de tradução, resultado da falta de um conhecimento mais aprofundado de seu autor do
idioma latino e dos conceitos tratados por Boécio. Para tais críticos, o autor demonstrava
um despreparo que o levou a cometer tais equívocos.26 Entretanto, numa análise mais
atenta, podemos notar vários elementos que indicam que isso não é verdade. Um dos grandes exemplos de alteração interpretativa se dá através da imagem da
Fortuna. 10
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 Primeiramente, há um consenso de que teria existido uma versão original
em prosa da qual as duas versões atuais descendem.22 Uma primeira hipótese é de que
esta versão original em prosa seria um rascunho, usada como referência para a
composição de outra versão em prosa e verso (sendo esta a versão final desejada para sua 14 14
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 circulação no período). A segunda hipótese seria de que o formato apenas em prosa é que
seria de fato a versão final e que esta circulou pela Inglaterra da época e posteriormente
elaborou-se uma versão alternativa em prosa e verso, tentando imitar o formato original
do texto de Boécio.23 Há um número maior de elementos que favorecem a segunda
hipótese: a) o autor omite ou resume drasticamente muitas passagens originais em verso
ao passá-las para o inglês antigo, fato que não ocorre com os textos em prosa (o que
poderia indicar uma maior preocupação com a estrutura das passagens em prosa do que
em verso); b) o formato da versão B está mais bem finalizado (contendo prefácio, índice,
um texto dividido e numerado em respectivos capítulos) e c) seu conteúdo circulou na
época (o texto da versão B era conhecido por Ælfric de Eynsham – final do século X e
início do XI – que o utilizou ao compor os textos de suas “Vidas dos Santos”, e pelo
arcebispo Wulfstan de York em suas homilias durante o mesmo período24). Já sobre o
texto da versão C, não há elementos suficientes que possam comprovar a circulação de
seu conteúdo durante o mesmo período na Inglaterra. circulação no período). 10
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 O manuscrito da versão C, por sua vez, faz parte do volume conhecido como
Cotton Otho A. VI. Este volume é composto pela versão em inglês antigo do texto de
Boécio (datado do século X), uma cópia em latim da vida do rei Edward o Confessor (do
final do século XII), e uma narrativa a respeito da visita de um grupo de peregrinos ao
santuário de Edward em Westminster no ano de 1240 (datado do século XIII). Não se
sabe quando estes textos foram reunidos como tal, mas é possível que isso não tenha
ocorrido muito anterior ao final do século XVI-XVII, quando tal compilação chegou à
coleção de Sir Robert Cotton. As primeiras referências ao manuscrito datam da década
de trinta do século XVII nos catálogos da coleção Cotton. Devido a um incêndio em 1731,
que danificou e destruiu boa parte da coleção, o manuscrito apresenta falhas e páginas
parcialmente danificadas, principalmente nas bordas. Após ter sido levado para o Museu
Britânico e posteriormente a fazer parte do acervo da British Library, passou por um
processo de restauração durante a primeira metade do século XIX. Sobre sua origem,
assim como a versão B, não há muitas informações. Não existem indícios de como e onde
o manuscrito foi adquirido e sua procedência.21 Os dois manuscritos possuem características muito distintas entre si. A versão B
está inteiramente em prosa, contendo um prefácio, índice e uma introdução sobre Boécio
e Teodorico, seguida pela versão em inglês antigo da obra latina. O texto está dividido
em quarenta e dois capítulos, onde a introdução conta como sendo o primeiro. A versão
C se assemelha ao formato do texto latino original, começando por um prefácio (em
prosa), um segundo prefácio (em forma poética, mas sem um índice como na versão B),
seguido de passagens em verso e prosa sucessivamente até o final. O conteúdo dos trechos
em prosa é muito similar aos da versão B, enquanto que o mesmo não ocorre com os
trechos em forma poética. Atualmente existem duas principais hipóteses a respeito da existência dos
manuscritos. ,
( ),
, p
26 SEDGEFIELD, Walter John (trad.), King’s Alfred Old English Version of the Consolations
Boethius. Oxford: Clarendon Press 1900, p. 242. Ibid., p. 48.
25 MEDEIROS, Elton O. S. “A Corajosa Mulher: Representações Femininas de Poder na Inglaterra Angl
Saxônica”. Revista Poder e Cultura, 3 (5), 2016, p. 30 – 47. 23 Ibid., p. 45.
24 Ibid., p. 48.
25 MEDEIROS, Elton O. S. “A Corajosa Mulher: Representações Femininas de Poder na Inglaterra Anglo-
Saxônica”. Revista Poder e Cultura, 3 (5), 2016, p. 30 – 47.
26 SEDGEFIELD, Walter John (trad.), King’s Alfred Old English Version of the Consolations of
B
hi
O f
d Cl
d
1900
242 Ibid., p. 48.
MEDEIROS, Elton O. S. “A Corajosa Mulher: Representações Femininas de Poder na Inglaterra Anglo- 23 Ibid., p. 45. Saxônica”. Revista Poder e Cultura, 3 (5), 2016, p. 30 – 47.
26 SEDGEFIELD, Walter John (trad.), King’s Alfred Old English Version of the Consolations of
Boethius. Oxford: Clarendon Press 1900, p. 242. 24 Ibid., p. 48. 10
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 Em Boécio, bens, riquezas, poder e outras coisas, estariam sob o jugo da Fortuna 15 15
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 e ligados à ideia de efemeridade. No texto em inglês antigo não há esta presença da
Fortuna, como no original. No Froferboc, a Fortuna latina sai de cena, até mesmo o nome
desaparece, e a argumentação do texto é reconstruída em torno de dois novos termos em
inglês antigo: Wyrd e woruldsælða. Diferentemente do que ocorre em Boécio com a
Fortuna, nenhum desses dois termos são personificações. Woruldsælða seriam as coisas
materiais em si, os bens e as vantagens terrenas as quais os homens adquirem e mantêm
em função de sua busca pela Sabedoria. O termo Wyrd, por sua vez, remete a um antigo
conceito do mundo germânico, sendo algo próximo da ideia de destino. Wyrd, no
Froferboc, acaba se confundindo com a figura de Deus, pois, diferente da Fortuna, no
texto em inglês antigo Wyrd não tem poder para influenciar as vidas dos homens. Os bens
terrenos, a prosperidade e infortúnios, diferente da Fortuna de Boécio, são de
responsabilidade exclusiva de Deus. No Froferboc, ao invés de uma força independente,
Wyrd é na verdade a forma pela qual Deus manifesta sua vontade no mundo.27 e ligados à ideia de efemeridade. No texto em inglês antigo não há esta presença da
Fortuna, como no original. No Froferboc, a Fortuna latina sai de cena, até mesmo o nome
desaparece, e a argumentação do texto é reconstruída em torno de dois novos termos em
inglês antigo: Wyrd e woruldsælða. Diferentemente do que ocorre em Boécio com a
Fortuna, nenhum desses dois termos são personificações. Woruldsælða seriam as coisas
materiais em si, os bens e as vantagens terrenas as quais os homens adquirem e mantêm
em função de sua busca pela Sabedoria. O termo Wyrd, por sua vez, remete a um antigo
conceito do mundo germânico, sendo algo próximo da ideia de destino. Wyrd, no
Froferboc, acaba se confundindo com a figura de Deus, pois, diferente da Fortuna, no
texto em inglês antigo Wyrd não tem poder para influenciar as vidas dos homens. Os bens
terrenos, a prosperidade e infortúnios, diferente da Fortuna de Boécio, são de
responsabilidade exclusiva de Deus. 29 Algumas passagens bíblicas servem para reforçar isso, como 1 Reis 3, 9 – 14; 2 Crônicas 1, 7 – 12;
Sabedoria de Salomão 6, 1 – 25; 7, 7 – 14; 8, 9 – 16; Provérbios 8, 12 – 36 (em especial os versículos 12 –
21) e 9, 10; e também o Salmo 111 (110) capítulo 10; e Eclesiastes 1; 19, 18 – 21; 25, 13 – 15. 27 PAYNE, F. Anne. King Alfred & Boethius: An Analysis of the Old English Version of the
Consolation of Philosophy. Wisconsin: University of Wisconsin Press, 1968, pp. 87-92.
28 PRATT, David. The Political Thought of King Alfred the Great. Cambridge: Cambridge University
Press, 2007, p. 152 – 153. 30 KRAPP, George Philip (ed.) & DOBBIE, Elliot van Kirk (ed.) The Anglo-Saxon Poettic Records III:
The Book of Exeter. Nova York: Columbia University Press, 1936, p. 134 – 136, 140 – 142, 147 – 148;
The Anglo-Saxon Poetic Records IV: Beowulf and Judith. Nova York: Columbia University Press,
1953, p. 53 – 55; FULK, R. D. The Beowulf Manuscript. Cambridge: Harvard University Press, 2010, p.
199 – 205; MEDEIROS, Elton O. S. Beowulf, São Paulo: Ed. 34, 2016 (no prelo); PRATT, David. The
Political Thought of King Alfred the Great. Cambridge: Cambridge University Press, 2007, p. 153 –
154. onsolation of Philosophy. Wisconsin: University of Wisconsin Press, 1968, pp. 87-92.
PRATT, David. The Political Thought of King Alfred the Great. Cambridge: Cambridge University
ess, 2007, p. 152 – 153. 30 KRAPP, George Philip (ed.) & DOBBIE, Elliot van Kirk (ed.) The Anglo-Saxon Poettic Records III:
The Book of Exeter. Nova York: Columbia University Press, 1936, p. 134 – 136, 140 – 142, 147 – 148;
The Anglo-Saxon Poetic Records IV: Beowulf and Judith. Nova York: Columbia University Press,
1953, p. 53 – 55; FULK, R. D. The Beowulf Manuscript. Cambridge: Harvard University Press, 2010, p. PAYNE, F. Anne. King Alfred & Boethius: An Analysis of the Old English Version of the
onsolation of Philosophy. Wisconsin: University of Wisconsin Press, 1968, pp. 87-92. Political Thought of King Alfred the Great. Cambridge: Cambridge University Press, 2007, p. 153
154. )
,
;
(
)
p
;
;
,
;
,
KRAPP, George Philip (ed.) & DOBBIE, Elliot van Kirk (ed.) The Anglo-Saxon Poettic Records III:
he Book of Exeter. Nova York: Columbia University Press, 1936, p. 134 – 136, 140 – 142, 147 – 148; he Book of Exeter. Nova York: Columbia University Press, 1936, p. 134 – 136, 140 – 142, 147 – 148;
he Anglo-Saxon Poetic Records IV: Beowulf and Judith. Nova York: Columbia University Press,
53, p. 53 – 55; FULK, R. D. The Beowulf Manuscript. Cambridge: Harvard University Press, 2010, p. The Anglo-Saxon Poetic Records IV: Beowulf and Judith. Nova York: Columbia University Press,
1953, p. 53 – 55; FULK, R. D. The Beowulf Manuscript. Cambridge: Harvard University Press, 2010, p.
199 – 205; MEDEIROS, Elton O. S. Beowulf, São Paulo: Ed. 34, 2016 (no prelo); PRATT, David. The
Political Thought of King Alfred the Great. Cambridge: Cambridge University Press, 2007, p. 153 –
154. , p
;
,
p
g
y
,
, p
199 – 205; MEDEIROS, Elton O. S. Beowulf, São Paulo: Ed. 34, 2016 (no prelo); PRATT, David. The
Political Thought of King Alfred the Great. Cambridge: Cambridge University Press, 2007, p. 153 –
154 27 PAYNE, F. Anne. King Alfred & Boethius: An Analysis of the Old English Version of the
Consolation of Philosophy. Wisconsin: University of Wisconsin Press, 1968, pp. 87-92.
28 PRATT, David. The Political Thought of King Alfred the Great. Cambridge: Cambridge University
Press, 2007, p. 152 – 153.
29 Algumas passagens bíblicas servem para reforçar isso, como 1 Reis 3, 9 – 14; 2 Crônicas 1, 7 – 12;
Sabedoria de Salomão 6, 1 – 25; 7, 7 – 14; 8, 9 – 16; Provérbios 8, 12 – 36 (em especial os versículos 12 –
21) e 9, 10; e também o Salmo 111 (110) capítulo 10; e Eclesiastes 1; 19, 18 – 21; 25, 13 – 15.
30 KRAPP, George Philip (ed.) & DOBBIE, Elliot van Kirk (ed.) The Anglo-Saxon Poettic Records III:
The Book of Exeter. Nova York: Columbia University Press, 1936, p. 134 – 136, 140 – 142, 147 – 148;
The Anglo-Saxon Poetic Records IV: Beowulf and Judith. Nova York: Columbia University Press,
1953, p. 53 – 55; FULK, R. D. The Beowulf Manuscript. Cambridge: Harvard University Press, 2010, p.
199 – 205; MEDEIROS, Elton O. S. Beowulf, São Paulo: Ed. 34, 2016 (no prelo); PRATT, David. The
Political Thought of King Alfred the Great. Cambridge: Cambridge University Press, 2007, p. 153 –
154. ç
j
,
33 Supondo que a figura do rei Alfred o Grande tenha de fato alguma ligação direta com a composição do
texto, o diálogo sapiencial poderia ser interpretado também como representando um debate entre Cristo e
o próprio Alfred. 32 “Eu sou a Sabedoria, moro com a sagacidade, e possuo o conhecimento da reflexão (...) É por mim que
reinam os reis, e que os príncipes decretam a justiça; por mim governam os governadores, e os nobres dão
sentenças justas” - Provérbios 8, 12-16. 31 “Cristo é a Sabedoria de Deus”; AGOSTINHO, Civitate Dei, XVII, 20.
É 31 “Cristo é a Sabedoria de Deus”; AGOSTINHO, Civitate Dei, XVII, 20. 10
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 No Froferboc, ao invés de uma força independente,
Wyrd é na verdade a forma pela qual Deus manifesta sua vontade no mundo.27 Contudo, uma das principais alterações do texto de Boécio para o inglês antigo
está no papel que Wisdom (Sabedoria) desempenha do ponto de vista político e teológico:
o princípio da realeza. Esta alteração fornece uma das identificações mais claras com o
pensamento político alfrediano e com todo o conjunto de interpretações e mudanças
realizadas ao verter o texto de Boécio para o vernáculo, assim como sua relação com os
demais textos alfredianos. Em tais obras se torna de extrema importância o papel de tal
sabedoria, tendo como seu principal referencial bíblico o arquétipo salomônico.28 De
maneira que isso acaba se refletindo em uma associação dos conceitos de sabedoria,
governança e (consequentemente) aplicação da lei.29 Algo que, não surpreendentemente,
se reflete na produção poética do período, através da fala da personagem do rei Hrothgar
a Beowulf em Beowulf (vv. 1724 – 1785); assim como também, de forma mais evidente,
nos poemas O Andarilho, Preceitos e Vanglória.30 16 16
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 17
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 17
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 Este seria um ponto fundamental para o ideal régio cristão alfrediano e para a
legitimidade de sua autoridade do ponto de vista filosófico, teológico e principalmente
político; cujos reflexos interpretativos poderão ser encontrados ao longo do período
alfrediano tanto em textos em prosa (como na documentação legislativa e nos textos de
Ælfric de Eynsham e do arcebispo Wulfstan) como na poesia (em Beowulf, por exemplo,
a manifestação de termos como wyrd e wisdom ao longo do poema se dá exatamente como
interpretado no Froferboc). Ideal presente no discurso dos demais textos alfredianos e
que serviria aos propósitos da Casa de Wessex ao promover um novo tipo de prática de
poder que iria mudar as relações da organização social anglo-saxônica ao fortalecer a
autoridade da aristocracia (afetando diretamente a administração pública e influenciando
a reorganização militar), mas ao mesmo tempo (diferente do que acontecia antes do século
IX) restringindo sua autonomia por estar mais submetida a uma autoridade régia
centralizadora. O que contribuiria mais tarde para o processo de unificação da Inglaterra
nos tempos do governo do rei Athelstan (neto de Alfred), com o qual este ideal de poder
régio cristão anglo-saxônico atingiria seu clímax ao consolidar a imagem do monarca, do
soberano de toda a ilha.34 34 STENTON, Sir Frank M. Anglo-Saxon England. Oxford: Oxford University Press, 1943, p. 349;
MEDEIROS, Elton O. S. “Alfred o Grande e a linhagem sagrada de Wessex: A construção de um mito de
origem na Inglaterra anglo-saxônica”. Mirabilia 13, 2011, p. 134 – 172. g
g
g
,
, p
35 KRAPP, George Philip (ed.) & DOBBIE, Elliot van Kirk (ed.) The Anglo-Saxon Poettic Records II
The Book of Exeter. Nova York: Columbia University Press, 1936, p. 147 – 148. 34 STENTON, Sir Frank M. Anglo-Saxon England. Oxford: Oxford University Press, 1943, p. 349;
MEDEIROS Elt
O S “Alf d
G
d
li h
d d W
A
t
ã d
it d g
g
y
p
MEDEIROS, Elton O. S. “Alfred o Grande e a linhagem sagrada de Wessex: A construção de um mito de
origem na Inglaterra anglo-saxônica”. Mirabilia 13, 2011, p. 134 – 172. 16
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 16
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 Ao longo do Froferboc a Sabedoria é descrita como a mais alta das virtudes
(hehsta cræft), uma manifestação divina e, diferentemente do simples poder mundano,
possui a capacidade de conceder méritos e qualidades àqueles que a buscar e a amar. Desta forma, a Sabedoria estaria identificada com Deus, e ligada à ideia de um Deus que
é o governante supremo, detentor de um poder que está além da completa compreensão
humana. Wisdom representaria o poder de Deus de governar a Criação, o verdadeiro poder
dos reis, dos homens e de todos aqueles que detêm algum tipo de autoridade e bem a
administram. E através de Mod (Mente) esta Sabedoria divina é venerada como a fonte
do poder régio. A fonte do poder pelo qual é possível ser rei ao obter autoridade sobre os
elementos materiais e espirituais necessários para a manifestação desta habilidade (cræft). Diferente de Wyrd, a presença de Wisdom no Froferboc beira à personificação, remetendo
a elementos cristológicos31 e do Velho Testamento.32 Desta forma, a Sabedoria estaria vinculada ao ideal da figura do soberano e
atrelada à prática do poder. À qualidade da realeza, a que concede poder e que permite a
prosperidade terrena a seus seguidores. Enquanto na obra original de Boécio a Filosofia
(Philosophiae) assume uma postura ascética, desprezando riquezas, status, poder e fama,
a Sabedoria (Wisdom) valoriza esses mesmos elementos como instrumentos necessários
da vida humana e do governante. Através disso, a Sabedoria passa ao seu interlocutor
(Mod) o respeito e a responsabilidade que ele deve ter no uso dos materiais e das pessoas
deste mundo. Uma das principais qualidades que se espera de um rei. Podemos ver que
este se torna um ponto fundamental para a compreensão do texto em inglês antigo e do
discurso dos demais textos alfredianos: o poder e a autoridade que provém de Deus
através da Sabedoria. No Froferboc é mantido o padrão do diálogo sapiencial (assim
como nos Solilóquios); mas, enquanto no texto original este ocorre entre a Filosofia e o
filósofo (ou entre a Razão e Agostinho), no texto anglo-saxônico pode-se interpretar que
este ocorre entre a “Realeza” e o rei.33 Realeza que se manifesta através da Sabedoria,
uma força divina que controla e administra a Criação, e não mero conhecimento mundano
(cynescipe). 17 36 GODDEN, Malcom & IRVINE, Susan. The Old English Boethius: An Edition of the Old English
Versions of Boethius’s De Consolatione Philosophiae, Vol. I. Oxford: Oxford University Press, 2009, p.
277 – 278. Sobre a tradução atual Como podemos ver na produção literária anglo-saxônica, o campo de abrangência
da “literatura sapiencial” é vasto. Sendo assim, trazemos o poema Vanglória como uma
pequena amostra de como essa literatura pode proporcionar uma série de abordagens,
enfoques e relações com outros textos do mesmo período, tanto na Inglaterra quanto fora
dela. A tradução do poema para o português foi realizada a partir da transcrição do texto
original em inglês antigo presente na obra de George Philip Krapp e Elliot van Kirk
Dobbie, The Anglo-Saxon Poettic Records III: The Book of Exeter de 1936.35 A respeito da tradução a partir do Froferboc, como dissemos anteriormente, foi
escolhido o capítulo 17 da versão B, contido no manuscrito MS. Bodley 180 (2079). Para
essa tarefa nos baseamos na transcrição do original anglo-saxônico que se encontra no
primeiro volume da obra de Malcom Godden e Susan Irvine, The Old English Boethius: 18 18
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 An Edition of the Old English Versions of Boethius’s De Consolatione Philosophiae, de
2009.36 An Edition of the Old English Versions of Boethius’s De Consolatione Philosophiae, de
2009.36 Em ambos os casos, vale ressaltar (especialmente para a tradução de Vanglória),
a forma escolhida para suas versões finais em língua portuguesa foi a prosa – para assim
auxiliar sua leitura e para uma melhor compreensão – devido às particularidades do inglês
antigo e seu estilo poético. Ao mesmo tempo também houve a preocupação de manter os
textos o mais próximo possível de seu conteúdo original – visando facilitar o estudo e a
consulta junto ao texto em inglês antigo – realizando adaptações e alterações apenas
quando uma tradução mais literal se tornou inadequado em língua portuguesa. Sabedoria é tratada pelo pronome masculino “ele”.
38 Cræft: pode ser traduzida como “habilidade”, “virtude”, “arte”, “talento” e etc. Apesar de não existir
originalmente tal conceito na Inglaterra anglo-saxônica, poderíamos aproximar à ideia de mana – de forma
não totalmente satisfatória, devemos admitir. O termo vem da Antropologia, originalmente dos estudos
sobre as culturas polinésias de cujo idioma provém a palavra. Mana estaria relacionada com um tipo de
poder “sobrenatural” presente na sociedade e que estaria concentrado em objetos, palavras, atos e pessoas. 37 þa geswigode he: numa tradução mais literal, “então ele se silenciou”; em inglês antigo a personagem d
Sabedoria é tratada pelo pronome masculino “ele”. Traduções Consolation Philosophiae em Inglês Antigo (Froferboc)
[Versao B: Ms. Bodley 180 (2079)]
Capitulo 17 [Como a Mente disse que a ambição e a
cobiça nunca lhe atraíram muito, exceto
as ferramentas que ela buscou] [Hu þæt mod sæde þæt him næfre seo
mægþ and seo gitsung forwel ne licode
buton tola þe he tilade] Quando a Sabedoria cantou sua canção,
ela então ficou em silencio,37 e a Mente
respondeu e assim disse: “Ó Razão,
verdadeiramente você sabe que a cobiça e
o desejo por esse poder terreno nunca me
atraíram
muito,
nem
eu
ansiei
grandemente por essa autoridade terrena,
mas busquei pelas ferramentas e material
para a tarefa a qual fui incumbido de
cumprir; de forma que eu honradamente e
adequadamente possa guiar e conduzir
aquele poder que me foi confiado. Verdadeiramente você sabe que ninguém
pode demonstrar qualquer habilidade,38 Þa se wisdom þa þis leoð asungen hæfde,
þa geswigode he, and andsworede þæt
mod and þus cwæð. Eala gesceadwisnes,
hwæt þu wast þæt me næfre seo gitsung
and
seo
gemægð
þisses
eorðlican
anwealdes forwel ne licode, ne ic ealles
forswiðe ne girnde þisses eorðlican rices,
buton [tola] ic wilnode þeah and
andweorces to þam weorce þe me
beboden was to wyrcanne; þæt ic
unfracodlice and gerisenlice mihte steoran
and reccan þone anweald þe me befæst
wæs. Hwæt þu wast þæt nan mon ne mæg
nænne cræft cyþan ne nænne anweald 19 19
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 reccan ne stioran butan tolum and
andweorce. Þæt
bið
ælces
cræftes
andweorc þæt mon þone cræft buton
wyrcan ne mæg. Þæt bið þonne cyninges
andweorc and his tol mid to ricsianne þæt
he hæbbe his land fulmannod. He sceal
habban gebedmen and fyrdmen and
weorcmen. Hwæt þu wast þætte butan
þisum tolum nan cyning his cræft ne mæg
cyðan. Þæt is eac his andweorc þæt he
habban sceal to þam tolum þam þrim
geferscipum biwiste. Þæt is þonne heora
biwist: land to bugianne and gifta and
wæpnu and mete and ealo and claþas, and
gehwæt þæs ðe þa þre geferscipas
behofiað. Ne mæg he butan þisum þas tol
gehealdan, ne buton þisum tolum nan þara
þinga wyrcan þe him beboden is to
wyrcenne. Termo adotado pela academia britânica e que se revelou muito útil para o estudo da autoridade régia tanto
dentro do contexto da sociedade pré-cristã quanto da religiosidade cristã, assim como na compreensão de
como a liturgia funcionava relacionada a fórmulas medicinais, bênçãos e demais práticas de fundo mágico
e espiritual tolerados pela Igreja. Para uma melhor abordagem sobre o tema cf. MAUSS, Marcel. Sociologia
e Antropologia. São Paulo: Cosac Naify, 2003, p. 142-173. p
g
y
p
39 He sceal habban gebedmen and fyrdmen and weorcmen, temos aqui já a idealização das três ordens d
sociedade medieval. 40 Mete: “carne”, em um sentido mais literal. 41 Wordhord (“tesouro de palavras”): a boca. Traduções Todos
compreender isto,
10
se þe hine ne læteð on þas lænan tid
amyrran his gemyndum modes
[gælsan
ond on his dægrime druncen to rice,
þonne monige beoð mæþelhegendra,
wlonce wigsmiþas winburgum in,
aqueles que não per
este tempo passage
nublada por um esp
deixe levar pela beb
dias: quando muito
em assembléia, os o
em suas fortalezas d
15
sittaþ æt symble, soðgied wrecað,
wordum wrixlað, witan fundiaþ
hwylc æscstede inne in ræcede
mid werum wunige, þonne win
[hweteð
beornes breostsefan. Breahtem
[stigeð,
sentados ao banqu
pronunciam, trocand
que tipo de campo d
dentro do salão ent
habitam, quando o
coração dos guerreir
41 Wordhord (“tesouro de palavras”): a boca. Original em Inglês Antigo
Hwæt, me frod wita on fyrndagum
sægde, snottor ar, sundorwundra
fela. Wordhord onwreah witgan larum
beorn boca gleaw, bodan ærcwide,
5
Þæt ic soðlice siþþan meahte
ongitan bi þam gealdre godes agen
[bearn,
wilgest on wicum, ond þone wacran
[swa some,
scyldum bescyredne, on gescead
[witan. þæt mæg æghwylc mon eaþe
geþencan,
10
se þe hine ne læteð on þas lænan tid
amyrran his gemyndum modes
[gælsan
ond on his dægrime druncen to rice,
þonne monige beoð mæþelhegendra,
wlonce wigsmiþas winburgum in,
15
sittaþ æt symble, soðgied wrecað,
wordum wrixlað, witan fundiaþ
hwylc æscstede inne in ræcede
mid werum wunige, þonne win
[hweteð
beornes breostsefan. Breahtem
[stigeð, – sobre diversas maravilhas. Abriu seu
tesouro de palavras41 sobre a sabedoria
dos profetas, a prudência dos heróis
instruídos,
os
velhos
dizeres
dos
apóstolos, de forma que eu pudesse verdadeiramente
reconhecer por estes ensinamentos o
verdadeiro Filho de Deus, um convidado
bem-vindo; e assim como aquele exilado
eu devo reconhecer, banido por seus
pecados. Todos os homens podem
compreender isto, de forma que eu pudesse verdadeiramente
reconhecer por estes ensinamentos o
verdadeiro Filho de Deus, um convidado
bem-vindo; e assim como aquele exilado
eu devo reconhecer, banido por seus
pecados. Todos os homens podem
compreender isto, aqueles que não permitirem que durante
este tempo passageiro sua mente seja
nublada por um espírito de soberba e se
deixe levar pela bebida por todos os seus
dias: quando muitos homens se reúnem
em assembléia, os orgulhosos guerreiros
em suas fortalezas de vinho, sentados ao banquete, prontamente se
pronunciam, trocando palavras, pensando
que tipo de campo de batalha encontrarão
dentro do salão entre os homens que o
habitam, quando o vinho encharca o
coração dos guerreiros. Traduções Wordhord onwreah witgan larum
beorn boca gleaw, bodan ærcwide,
Ouçam, nos tempos de outrora um velho
conselheiro me disse – sábio mensageiro
– sobre diversas maravilhas. Abriu seu
tesouro de palavras41 sobre a sabedoria
dos profetas, a prudência dos heróis
instruídos,
os
velhos
dizeres
dos
apóstolos,
5
Þæt ic soðlice siþþan meahte
ongitan bi þam gealdre godes agen
[bearn,
wilgest on wicum, ond þone wacran
[swa some,
scyldum bescyredne, on gescead
[witan. þæt mæg æghwylc mon eaþe
geþencan,
de forma que eu pudesse verdadeiramente
reconhecer por estes ensinamentos o
verdadeiro Filho de Deus, um convidado
bem-vindo; e assim como aquele exilado
eu devo reconhecer, banido por seus
pecados. Todos os homens podem
compreender isto,
10
se þe hine ne læteð on þas lænan tid
amyrran his gemyndum modes
[gælsan
ond on his dægrime druncen to rice,
þonne monige beoð mæþelhegendra,
wlonce wigsmiþas winburgum in,
aqueles que não permitirem que durante
este tempo passageiro sua mente seja
nublada por um espírito de soberba e se
deixe levar pela bebida por todos os seus
dias: quando muitos homens se reúnem
em assembléia, os orgulhosos guerreiros
em suas fortalezas de vinho,
15
sittaþ æt symble, soðgied wrecað,
wordum wrixlað, witan fundiaþ
hwylc æscstede inne in ræcede
mid werum wunige, þonne win
[hweteð
beornes breostsefan. Breahtem
[stigeð,
sentados ao banquete, prontamente se
pronunciam, trocando palavras, pensando
que tipo de campo de batalha encontrarão
dentro do salão entre os homens que o
habitam, quando o vinho encharca o
coração dos guerreiros. Um grito se ergue, Vanglória
Livro de Exeter
[MS. Exeter, Cathedral 3501, fol. 83a – 84b] Livro de Exeter
[MS. Exeter, Cathedral 3501, fol. 83a – 84b
Original em Inglês Antigo
Trad
Hwæt, me frod wita on fyrndagum
sægde, snottor ar, sundorwundra
fela. Wordhord onwreah witgan larum
beorn boca gleaw, bodan ærcwide,
Ouçam, nos tempos
conselheiro me disse
– sobre diversas m
tesouro de palavras
dos profetas, a pr
instruídos,
os
v
apóstolos,
5
Þæt ic soðlice siþþan meahte
ongitan bi þam gealdre godes agen
[bearn,
wilgest on wicum, ond þone wacran
[swa some,
scyldum bescyredne, on gescead
[witan. þæt mæg æghwylc mon eaþe
geþencan,
de forma que eu pud
reconhecer por es
verdadeiro Filho de
bem-vindo; e assim
eu devo reconhece
pecados. Traduções Forþy ic wilnode andweorces
þone anweald mid to reccenne, þæt mine
cræftas and anweald ne wurde [forgiten]
and forholen, forþam ælc cræft and ælc
anweald
bið
sona
forealdod
and
forswugod, gif he bið buton wisdome;
forþam ne mæg non mon nænne cræft
forðbringan buton wisdome, forþam þe
swa hwæt swa þurh dysige gedon biþ ne
mæg hit mon næfre to cræfte gerecan. Þæt
is nu hraþost to secganne þæt ic wilnode
weorðfullice to libbanne þa hwile þe ic
lifede, and æfter minum life þam monnum
to læfanne [þe] æfter me wæren [min]
gemynd on godum weorccum. nem exercer ou conduzir nenhum poder, a
não ser com ferramentas e materiais. O
material de qualquer habilidade é aquele
sem o qual ninguém pode exercer tal
habilidade. Assim, o material para um rei
e suas ferramentas com as quais governa
são aquelas com as quais ele mantém sua
terra povoada. Ele deve ter homens de
oração e homens de armas e homens de
trabalho.39 Pois você sabe que sem essas
ferramentas nenhum rei pode exercer sua
habilidade. Assim também, seu material é
aquele que ele deve ter para o sustento das
ferramentas destes três grupos. Isto é
então o seu sustento: terra para habitar, e
presentes e armas e comida40 e cerveja e
roupas, e tudo aquilo que os três grupos
necessitam. Sem isso ele não pode manter
as
ferramentas,
nem
sem
essas
ferramentas realizar qualquer uma das
coisas que lhe é designado a realizar. Portanto, eu desejo material para então
exercer poder, de forma que minhas
habilidades e autoridade não sejam
esquecidas e escondidas, pois toda
habilidade e todo o poder serão logo
sobrepujados pelo tempo e silenciados se
forem sem sabedoria; pois ninguém pode
realizar
nenhuma
habilidade
sem
sabedoria, uma vez que qualquer coisa que
é feita através da estupidez não pode
nunca ser considerada uma habilidade. Digo agora isso sucintamente: eu desejo
viver honradamente o tanto quanto eu
viver, e depois de minha vida deixar para
aquelas pessoas que vier depois de mim
minha memória em boas obras. 20 20
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 Vanglória
Livro de Exeter
[MS. Exeter, Cathedral 3501, fol. 83a – 84b]
Original em Inglês Antigo
Tradução
Hwæt, me frod wita on fyrndagum
sægde, snottor ar, sundorwundra
fela. Traduções Um grito se ergue, sentados ao banquete, prontamente se
pronunciam, trocando palavras, pensando
que tipo de campo de batalha encontrarão
dentro do salão entre os homens que o
habitam, quando o vinho encharca o
coração dos guerreiros. Um grito se ergue, 21 21
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 21
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 20
cirm on corþre, cwide scralletaþ
missenlice. Swa beoþ modsefan
dalum gedæled, sindon dryhtguman
ungelice. Sum on oferhygdo
þrymme þringeð, þrinteð him in
[innan
um clamor na multidão, bradam diversas
palavras. Assim são as mentes, diversas e
divididas,
os
nobres
homens
são
diferentes (entre si). Alguém em sua
arrogância exalta sua força, nele flui
25
ungemedemad mod; sindan to
[monige þæt! Bið þæt æfþonca eal gefylled
feondes fligepilum, facensearwum;
breodað he ond bælceð, boð his
[sylfes
swiþor micle þonne se sella mon,
um espírito desagradável; muitos são
desta forma! Está assim todo cheio de
ódio, por dardos voadores42 e artimanhas
do demônio; ele grita e urra, vangloria-se
de si mesmo muito mais do que um
homem melhor (que ele),
30
þenceð þæt his wise welhwam þince
eal unforcuþ. Biþ þæs oþer swice,
þonne he þæs facnes fintan sceawað. pensa que seu modo de agir irá parecer a
todos como honrado. Isto será outra
mentira, quando ele vir o resultado desta
farsa. um clamor na multidão, bradam diversas
palavras. Assim são as mentes, diversas e
divididas,
os
nobres
homens
são
diferentes (entre si). Alguém em sua
arrogância exalta sua força, nele flui um clamor na multidão, bradam diversas
palavras. Assim são as mentes, diversas e
divididas,
os
nobres
homens
são
diferentes (entre si). Alguém em sua
arrogância exalta sua força, nele flui um espírito desagradável; muitos são
desta forma! Está assim todo cheio de
ódio, por dardos voadores42 e artimanhas
do demônio; ele grita e urra, vangloria-se
de si mesmo muito mais do que um
homem melhor (que ele), pensa que seu modo de agir irá parecer a
todos como honrado. Isto será outra
mentira, quando ele vir o resultado desta
farsa. pensa que seu modo de agir irá parecer a
todos como honrado. Isto será outra
mentira, quando ele vir o resultado desta
farsa. Referências
Fontes
ANLEZARK, Daniel. The Old English Dialogues of Solomon and Saturn, Cambridge:
D. S. Brewer, 2009. BRADLEY, S.A.J. (trad. org.) Anglo-Saxon poetry, Londres: Everyman, 2003. 42 Fligepilum (“dardos voadores”): dentre as crenças populares dos anglo-saxões estava a de que seres como
elfos e espíritos da natureza eram capazes de causar malefício às pessoas e animais ao atirar flechas e dardos
invisíveis. Com a cristianização essa crença passou também a atribuir a seres demoníacos tal prática,
causando males não apenas físicos como também espirituais; cf JOLLY, Karen. Popular Religion in Late p
p
;
,
p
g
Saxon England. Chapel Hill: University of North Carolina Press, 1996. Traduções CHARLES, R. H. (trad.) The Book of Enoch, Boston: Weiser Books, 2003. FULK, R. D. The Beowulf Manuscript. Cambridge: Harvard University Press, 2010. GODDEN, Malcom & IRVINE, Susan. The Old English Boethius: An Edition of the
Old English Versions of Boethius’s De Consolatione Philosophiae, Vol. I - II. Oxford: Oxford University Press, 2009. HOLLANDER, Lee M. The Poetic Edda. Austin: University of Texas Press, 2000. causando males não apenas físicos como também espirituais; cf JOLLY, Karen. Popular Religion in Late
Saxon England. Chapel Hill: University of North Carolina Press, 1996. PROENÇA, Eduardo de (org.) Apócrifos da Bíblia e pseudo-epígrafos. São Paulo:
Fonte Ed., 2005. PROENÇA, Eduardo de (org.) Apócrifos da Bíblia e pseudo-epígrafos. São Paulo:
Fonte Ed., 2005. PRUDENCIO. Psychomachia, Bryn Mawr: Bryn Mawr Commentaries, 2004. SEDGEFIELD, Walter John (ed.), King’s Alfred Old English Version of the
Consolations of Boethius. Oxford: Clarendon Press, 1882. SEDGEFIELD, Walter John (trad.), King’s Alfred Old English Version of the
Consolations of Boethius. Oxford: Clarendon Press, 1900. NLEZARK, Daniel. The Old English Dialogues of Solomon and Saturn, Cambridge:
D. S. Brewer, 2009. BRADLEY, S.A.J. (trad. org.) Anglo-Saxon poetry, Londres: Everyman, 2003. CHARLES, R. H. (trad.) The Book of Enoch, Boston: Weiser Books, 2003. FULK, R. D. The Beowulf Manuscript. Cambridge: Harvard University Press, 2010. GODDEN, Malcom & IRVINE, Susan. The Old English Boethius: An Edition of the
Old English Versions of Boethius’s De Consolatione Philosophiae, Vol. I - II. Oxford: Oxford University Press, 2009. GODDEN, Malcom & IRVINE, Susan. The Old English Boethius: An Edition of the
Old English Versions of Boethius’s De Consolatione Philosophiae, Vol. I - II. Oxford: Oxford University Press, 2009. HOLLANDER, Lee M. The Poetic Edda. Austin: University of Texas Press, 2000. 22 22
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 JÓNSSON,
Guðni. Hávamál,
Copenhagen,
1924. Disponível
em:
http://www.heimskringla.no/wiki/H%C3%A1vam%C3%A1l
(Acessado
em
18/07/2012). KEMBLE, John. The Dialogue of Salomon and Saturnus. Londres: Ælfric Society,
1848. KRAPP, George Philip (ed.) The Anglo-Saxon Poettic Records I: The Junius
Manuscript, Nova York: Columbia University Press, 1931. KRAPP, George Philip (ed.) & DOBBIE, Elliot van Kirk (ed.) The Anglo-Saxon Poettic
Records III: The Book of Exeter. Nova York: Columbia University Press, 1936. ______. The Anglo-Saxon Poetic Records IV: Beowulf and Judith. Nova York:
Columbia University Press, 1953. MEDEIROS, Elton O. S. “Hávamál: Tradução Comentada do Nórdico Antigo para o
Português”. Mirabilia 13 (2), 2013, p. 545 – 601. Disponível em: <
http://www.revistamirabilia.com/sites/default/files/pdfs/2013_02_23.pdf > Acesso
em: 03/05/2016. ______. “ráðna stafi, mjǫk stóra stafi, mjǫk stinna stafi”: Tradução Comentada dos
Poemas
Rúnicos
Anglo-Saxão,
Islandês,
Norueguês
e
do
Abecedarium
Nordmannicum”. Medievalis 4 (1), 2015, p. 1 – 31. Disponível em: <
http://medievalis.nielim.com/ojs/index.php/medievalis/article/view/69/59 > Acesso
em: 03/05/2016. ______. “Erudição e Poesia Encantatória na Inglaterra anglo-saxônica: Salomão e
Saturno I & o Encantamento das Nove Ervas”. Mirabilia 20 (1), 2015, p. 313 – 363. Disponível
em:
<
http://www.revistamirabilia.com/sites/default/files/pdfs/20-
17_0.pdf > Acesso em: 03/05/2016. ______. “A Linhagem Perdida de Sceaf: Genealogias Mítico-Históricas na Inglaterra e
Escandinávia & a Tradução do Prólogo da Edda de Snorri Sturluson”, Signum 16 (3),
2015,
p. 46
–
77. Disponível
em:
<
http://www.abrem.org.br/revistasignum/index.php/revistasignumn11/article/view/19
1/168 > Acesso em: 03/05/2016. ___. (trad. org.) Beowulf, São Paulo: Ed. 34, 2016 (no prelo). Bibliografia BISCHOFF, Bernhard & LAPIDGE, Michael. Biblical Commentaries from the
Canterbury School of Theodore and Hadrian. Cambridge: Cambridge University
Press, 2007. 23 23
Veredas da História, [online], v. 8, n. 2, 2015, p. 5-23, ISSN 1982-4238 FULK, R. D. & CAIN, Christopher M. A History of Old English Literature. Oxford:
Blackwell, 2005. GODDEN, Malcolm & LAPIDGE, Michael. The Cambridge Companion to Old
English Literature. Cambridge: Cambridge University Press, 1994. HANSEN, Elaine Tuttle. The Solomon Complex. Toronto: University of Toronto Press,
1988. HUPPÉ, Bernard F. Doctrine and Poetry: Augustine’s Influence on Old English
Poetry. Nova York: State University of New York, 1959. JOLLY, Karen Louise. Popular Religion in Late Saxon England: elf charms in
context. Chapel Hill: University of North Carolina Press, 1996. KASKE, R. E. “Beowulf and the Book of Enoch”. Speculum, 46 (3), 1971, p. 421-431 LAPIDGE, Michael. The Blackwell Encyclopaedia of Anglo-Saxon England. Oxford:
Blackwell, 2004. LASSEN, Annette. Odin på kristent pergament: En teksthistorisk studie. Copenhagen: Museum Tusculanums Forlag, 2011. MAUSS, Marcel. Sociologia e Antropologia. São Paulo: Cosac Naify, 2003. MAUSS, Marcel. Sociologia e Antropologia. São Paulo: Cosac Naify, 2003. MEDEIROS, Elton O. S. “Uma Introdução ao Estudo da Conversão e das Práticas
Mágicas na Inglaterra Anglo-Saxônica”, Brathair 10 (1), 2010, pp. 54 – 66. Disponível
em:
<
http://ppg.revistas.uema.br/index.php/brathair/article/view/437/378>
Acesso
em:
03/05/2016 ______. “Alfred o Grande e a linhagem sagrada de Wessex: A construção de um mito de
origem na Inglaterra anglo-saxônica”. Mirabilia 13, 2011, p. 134 – 172. Disponível
em: < http://www.revistamirabilia.com/sites/default/files/pdfs/2011_02_07.pdf >
Acesso em: 03/05/2016. ______. “Mito e História no Campo de Batalha: Apropriação e Interpretação do Passado
pelo Medievo e como História Nacional”. Revista de História Comparada 8 (2),
2014,
p. 29
–
59. Disponível
em:
<
http://www.hcomparada.historia.ufrj.br/revistahc/artigos/rhc_volume008_Num002_
002.pdf > Acesso em: 03/05/2016. _____. “Mito e História no Campo de Batalha: Apropriação e Interpretação do Passado
pelo Medievo e como História Nacional”. Revista de História Comparada 8 (2), ______. “A Corajosa Mulher: Representações Femininas de Poder na Inglaterra Anglo-
Saxônica”. Revista Poder e Cultura, 3 (5), 2016, p. 30 – 47. PAYNE, F. Anne. King Alfred & Boethius: An Analysis of the Old English Version
of the Consolation of Philosophy. Wisconsin: University of Wisconsin Press, 1968. PRATT, David. The Political Thought of King Alfred the Great. Cambridge:
Cambridge University Press, 2007. PULSIANO, Philip & TREHARNE, Elaine. A Companion to Anglo-Saxon Literature. Oxford: Blackwell, 2001. STENTON, Sir Frank M. Anglo-Saxon England. Oxford: Oxford University Press,
1943.
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https://openalex.org/W2934202356
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http://www.scielo.br/pdf/rsbmt/v52/1678-9849-rsbmt-52-e20180537.pdf
|
English
| null |
Asymptomatic Plasmodium infection in a residual malaria transmission area in the Atlantic Forest region: Implications for elimination
|
Revista da Sociedade Brasileira de Medicina Tropical
| 2,019
|
cc-by
| 7,611
|
Abstract Introduction: Elimination of malaria in areas of interrupted transmission warrants careful case assessment to avoid the
reintroduction of this disease. Occasional malaria cases are reported among visitors of the Atlantic Forest area of Brazil, while
data on residents of this area are scarce. Methods: A sectional study was carried out to examine 324 individuals living in a
municipality where autochthonous cases were detected. Results: Asymptomatic Plasmodium infections were detected in 2.8%
of the individuals by polymerase chain reaction (PCR), with one case of P. falciparum (0.3%), two cases of P. vivax (0.6%),
and six cases of P. malariae (1.9%). The thick blood smears were negative in all individuals. Serological tests performed in
314 subjects were reactive in 11.1%, with 3.5% for P. falciparum and 7.7% for P. vivax. A subsample of 42 reactive individuals
for any Plasmodium species showed P. malariae in 30.9% of specimens. Individuals who entered the Atlantic Forest region
were 2.7 times more likely to exhibit reactive serology for P. vivax compared with individuals who did not enter this region
(p<0.05). Children <15 years had a higher chance of reactive serology for P. falciparum and P. vivax than individuals ≥15 years
of age (p<0.05). Individuals living in the Paraiso district had a higher chance of reactive serology for P. vivax compared to other
districts (p<0.05). No associations were found between sex, past exposure to malaria, or serological response to antibodies of any
Plasmodium species. Conclusions: The implications of these results for the elimination of malaria were discussed. Keywords: Malaria. Plasmodium infection. Extra-Amazonian region. Elimination. Rio de Janeiro S Renata Bortolasse Miguel[1],[2], Hermano Gomes Albuquerque[1],[2], Maria Carmen Arroyo
Sanchez[3], José Rodrigues Coura[1],[2], Simone da Silva Santos[1], Sidnei da Silva[4],
Carlos José de Carvalho Moreira[1] and Martha Cecilia Suárez-Mutis[1],[2]
[1]. Laboratório de Doenças Parasitárias, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, RJ, Brasil.
[2]. Programa de Pós-graduação em Medicina Tropical, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, RJ, Brasil.
[3]. Laboratório de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical de São Paulo, São Paulo, SP, Brasil.
[4]. Laboratório de Parasitologia, Instituto Nacional de Infectologia, Rio de Janeiro, RJ, Brasil. Renata Bortolasse Miguel[1],[2], Hermano Gomes Albuquerque[1],[2], Maria Carmen Arroyo
Sanchez[3], José Rodrigues Coura[1],[2], Simone da Silva Santos[1], Sidnei da Silva[4],
Carlos José de Carvalho Moreira[1] and Martha Cecilia Suárez-Mutis[1],[2]
[1]. Laboratório de Doenças Parasitárias, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, RJ, Brasil. [2]. Programa de Pós-graduação em Medicina Tropical, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, RJ, Brasil. [3]. Laboratório de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical de São Paulo, São Paulo, SP, Brasil. [4]. Laboratório de Parasitologia, Instituto Nacional de Infectologia, Rio de Janeiro, RJ, Brasil. Revista da Sociedade Brasileira de Medicina Tropical
Journal of the Brazilian Society of Tropical Medicine
Vol.:52:e20180537: 2019
doi: 10.1590/0037-8682-0537-2018 Revista da Sociedade Brasileira de Medicina Tropical
Journal of the Brazilian Society of Tropical Medicine
Vol.:52:e20180537: 2019
doi: 10.1590/0037-8682-0537-2018 Revista da Sociedade Brasileira de Medicina Tropical
Journal of the Brazilian Society of Tropical Medicine
Vol.:52:e20180537: 2019
doi: 10.1590/0037-8682-0537-2018 Revista da Sociedade Brasileira de Medicina Tropical
Journal of the Brazilian Society of Tropical Medicine
Vol.:52:e20180537: 2019
doi: 10.1590/0037-8682-0537-2018 Major Article METHODS A cross-sectional study was carried out during the fall
of 2011 in Guapimirim, a municipality located 86 km away
from Rio de Janeiro, Brazil. Six localities were eligible for
participant recruitment because nearby autochthonous cases
were detected, the rural areas Garrafão, Orindi, and Paraíso
and the peri-urban areas Barreira, Caneca Fina, and Monte
Olivete (Figure 1). The required sample size was estimated
using an expected prevalence of 4% for Plasmodium infection
as a variable with an acceptable error of 2%. The final sample
size was calculated as at least 312 individuals. Residents of
both sexes older than 5 years of age who gave their informed
consent (for adults) or received consent from the legal guardian
(for children under 18 years) were randomly recruited. A semi-
structure questionnaire on personal data, exposure to malaria,
daily habits, knowledge about disease, clinical information,
and history of febrile symptoms in the month preceding the
study period was administered. Thick and thin blood smears
were collected from each participant according to standard
protocols of the Brazilian Ministry of Health. Total blood (5
mL) was collected in Vacutainer® tubes (Becton Dickinson
New Jersey, USA) containing ethylenediaminetetraacetic acid
(EDTA) for DNA extraction for parasitological diagnosis of
malaria by polymerase chain reaction (PCR). Ten milliliters
of blood were collected without anticoagulant for serological
studies. A malaria case was defined as an individual with any of the
typical malaria symptoms (fever, chills, sweating, headache)
at the time of the interview and with a positive thick blood
smear or PCR result. An asymptomatic Plasmodium carrier was
defined as an individual with Plasmodium species detected in
the thick blood smear and/or the PCR but without symptoms
30 days before and after sample collection and without using
antimalarial drugs. Serological analysis Strategy for Malaria 2016-2030”, with the goal of “preventing
a resurgence of malaria in all countries and areas that are
malaria-free”10. A recent malaria outbreak caused by P. simium
was described in tourists visiting some regions of the dense
and protected Atlantic Forest in Rio de Janeiro state9. Malaria
cases in this area are detected predominantly among visitors
but it is unclear whether the local residents are also affected. We therefore carried out a cross-sectional study to verify the
status of Plasmodium infections in residents of the Atlantic
Forest area of Rio de Janeiro state. The novel epidemiology
of malaria in interrupted transmission areas must be carefully
assessed, particularly considering worldwide efforts to eliminate
the disease2,10. Serological examinations were performed using enzyme-
linked immunosorbent assay (ELISA) with erythrocytic antigens
for P. falciparum and P. vivax and immunofluorescence assay
(IFA) for P. malariae. IgG antibodies were detected by ELISA
using a crude blood stage P. falciparum antigen extracted with
Zwittergent® (Calbiochem, Billerica, MA, USA)15 and MSP1-19
recombinant antigen of P. vivax16. Reactions were assessed by
measuring the absorbance at 492 nm using Titertek Multiskan
MCC/340 (Labsystems Diagnostics Group, Vantaa, Finland). To determine the cut-off for the ELISA using P. falciparum
and P. vivax antigens, receiver operating characteristic (ROC)
curves were constructed based on the absorbance of positive
and negative samples17 (Figure 2). The reactivity index
(RI=absorbance/cut-off) was calculated for all samples, and
samples with RI ≥1.1 were considered positive. Samples
with values between 0.9 and 1.1 (gray zone) were considered
inconclusive. IgG antibodies against P. malariae were detected
using the IFA protocol described by Ferreira and Sanchez16. As
the quantity of P. malariae antigen was low, this serological
test was performed only for the PCR-positive samples and
all reactive and inconclusive serologies. The experiments
were performed at the Laboratory of Seroepidemiology and
Immunobiology at the Institute of Tropical Medicine of São
Paulo, Brazil. Ethical considerations All the procedures followed the ethical standards of the
Ethics Committee of Research involving Human Subjects of
the Research Institute Evandro Chagas-INI/Fiocruz, Brazil
and in accordance with the Helsinki Declaration (Protocol
0229.0.000.009/10 approved). INTRODUCTION proportion has dropped to only 0.05% to date2. Rio de Janeiro
state, located in Southeastern Brazil, used to be considered a
highly endemic area for malaria with the reported presence of
An. darlingi3. Cases of infection by P. falciparum and P. vivax
occurred particularly in the lowlands (Baixada Fluminense
region) in the proximity of the capital of the state4. The Global
Malaria Eradication Campaign supported the implication of
control measures against vectors and the discovery of new drugs
for infected people, leading to the interruption of transmission by
1968 with An. darlingi now rarely found in this area5. In recent
years, a new malaria epidemiological scenario is emerging
in Rio de Janeiro, with the presence of cases imported from
endemic areas of Brazil and outside the country and occasional
autochthonous cases including outbreaks6,7,8,9. In 2016, the World
Health Organization (WHO) launched the “Global Technical Malaria endemic areas are restricted to the Amazonian
region of Brazil where Anopheles darlingi Root 1926 is the
main vector implicated in transmission1. An average of 1,296
cases are reported annually outside of this region, most of
which (89%) have been imported from elsewhere2. While 50%
of the malaria cases in the extra-Amazonian region of Brazil
were autochthonous in the middle of the 20th century, this Corresponding author: Dra. Martha Cecilia Suárez-Mutis. e-mail: marmutis@ioc.fiocruz.br / marthasuarezmutis@gmail.com
Orcid: 0000-0003-2809-6799
Received 17 December 2018
Accepted 19 February 2019 Corresponding author: Dra. Martha Cecilia Suárez-Mutis. e-mail: marmutis@ioc.fiocruz.br / marthasuarezmutis@gmail.com
Orcid: 0000-0003-2809-6799
Received 17 December 2018
Accepted 19 February 2019 1/9 Miguel RB et al. - Plasmodium infection in Rio de Janeiro Serological analysis Statistical analysis Thick blood smears were stained and 100 fields examined
at 1,000× magnification11. DNA was extracted for molecular
diagnosis from 200 μL of whole blood using a commercial
DNA Purification Kit (Illustrablood genomicPrep MiniSpin Kit),
according to the manufacturer’s instructions (GE Healthcare,
Pittsburgh,USA). PCR was performed following the nested
PCR protocol by Snounou et al. with minor modifications12,13. Agarose gel electrophoresis was performed, gels stained with
GelRedTMnucleic acid gel stain, Biotium, Fremont, CA, USA and
visualized under UV light. A sample was considered positive if
a 120, 144, or 205 bp PCR product (P. vivax, P. malariae, and
P. falciparum, respectively) was detected. A sensitivity level of
0.001% for PCR detection of parasitemia levels is appropriate
for the diagnosis of subpatent infections14. Positive results were
re-tested twice to verify the results. Exploratory analyses were performed using contingency
tables and chi-square and Fisher tests to verify possible
relationships between the dependent and independent variables. This relationship was modeled using a series of simple
binomial generalized linear models (GLM). Only variables with
p<0.2 in the bivariate analysis were evaluated in the model. Multiple binomial GLMs were used to model the effects of all
independent variables on each dependent variable. Interaction
effects were tested in each model formulation, but no significant
interaction was found. We employed the Firth’s Bias-Reduced
Logistic Model to account for numerical problems and model
convergence in the P. falciparum and P. malariae models. Data
were analyzed using the freely available EPI2000 statistical
program (Centers for Disease Control, Atlanta, Georgia, USA), 2/9 Rev Soc Bras Med Trop Vol.:52:e-20180537, 2019 FIGURE 1: Study locations and prevalence of asymptomatic Plasmodium infections in the municipality of Guapimirim, Rio de Janeiro, Brazil. FIGURE 1: Study locations and prevalence of asymptomatic Plasmodium infections in the municipality of Guapimirim, Rio de Janeiro, Brazil. 100
80
60
40
20
0
0 20 40 60 80 100
100% - Specificity %
Sensivity %
A
100
80
60
40
20
0
0 20 40 60 80 100
100% - Specificity %
B
Sensivity %
FIGURE 2: Receiver operating characteristic (ROC) curves to determine enzyme-linked immunosorbent assay (ELISA) performance for anti-PvMSP119 IgG
and anti-Pf IgG. Panel A shows the curve obtained for samples from P. vivax-infected patients (n=41) and healthy subjects (n=37). Panel B shows the curve
for samples from P. falciparum-infected patients (n=41) and healthy subjects (n=37). PvMSP119-and Pf -ELISAs considered a cut-off of 0.100 in the ROC
curves. Statistical analysis Anti-IgG PvMSP119 for P. vivax was detected in 100.0% (range: 90.5-100.0, CI: 95%) of P. vivax cases with a specificity of 97.6% (range: 87.1-99.9,
CI: 95%). Anti-IgG for P. falciparum was detected in 94.4% (range: 81.3-99.3, CI: 95%) with a specificity of 100.0% (range: 91.2-100.0, CI: 95%). 100
80
60
40
20
0
0 20 40 60 80 100
100% - Specificity %
Sensivity %
A 100
80
60
40
20
0
0 20 40 60 80 100
100% - Specificity %
B
Sensivity % A FIGURE 2: Receiver operating characteristic (ROC) curves to determine enzyme-linked immunosorbent assay (ELISA) performance for anti-PvMSP119 IgG
and anti-Pf IgG. Panel A shows the curve obtained for samples from P. vivax-infected patients (n=41) and healthy subjects (n=37). Panel B shows the curve
for samples from P. falciparum-infected patients (n=41) and healthy subjects (n=37). PvMSP119-and Pf -ELISAs considered a cut-off of 0.100 in the ROC
curves. Anti-IgG PvMSP119 for P. vivax was detected in 100.0% (range: 90.5-100.0, CI: 95%) of P. vivax cases with a specificity of 97.6% (range: 87.1-99.9,
CI: 95%). Anti-IgG for P. falciparum was detected in 94.4% (range: 81.3-99.3, CI: 95%) with a specificity of 100.0% (range: 91.2-100.0, CI: 95%). 3/9 Miguel RB et al. - Plasmodium infection in Rio de Janeiro 320 (98.8%) individuals. Of these, 9 (2.8%) were positive for
Plasmodium infections, 1 for P. falciparum (0.3%), 2 for P. vivax (0.6%), and 6 for P. malariae (1.9%). These 9 individuals
did not have symptoms suggestive of malaria and all had
negative smears. The individual with a positive PCR result
for P. falciparum was a man from the Orindi municipality, and
those participants with a positive PCR result for P. vivax were 2
women from the Paradise municipality. The 6 participants with
positive PCR results for P. malariae, 3 women and 3 men, were
residents of the Orindi, Garrafão, Paradise, and Monte Olivetti
locations (Table 1). Although the towns of Paradise and Orindi
showed a higher frequency of positive samples, there was no
statistically significant association. the R software, and the RStudio software with the Companion to
Applied Regression (CAR) package, R Foundation for Statistical
Computing, Vienna, Austria. DISCUSSION The prevalence of autochthonous malaria is low in
Southeastern Brazil and in the Southern states of the Atlantic
Forest region18,19,20. The characteristics of malaria in these areas
are different from those observed in the Amazon region18. The
extra-Amazonian malaria is often described as asymptomatic, Serology Serological tests were performed in 314 participants and 35
(11.1%) were reactive for any of the three Plasmodium species
studied. The P. falciparum IgG antibody test was reactive in
11 participants (3.5%) and inconclusive in 5 (1.6%), while
samples from 24 participants (7.7%) were reactive and 4 (1.3%)
inconclusive for P. vivax (anti-IgG PvMSP19 antibody). Due
to operational constraints, only 42 samples were tested for
P. malariae (all positive PCR samples and/or reactive serology
for P. vivax or P. falciparum). Thirteen samples were reactive
for P. malariae antibodies (30.9%, 13/42) and 11 samples
(29.7%) were reactive for more than one Plasmodium species. Table 2 summarizes the presence of Plasmodium species in
positive individuals. Table 3 shows the association between
positive PCR samples and serological reactivity for Plasmodium
species. Cohen’s kappa coefficient (κ) for agreement between
molecular and serological results was considered “moderate”
for P. malariae (κ=0.54, range: 0.27-0.82, confidence interval
[CI]: 95%) and “fair” for all Plasmodium species (κ=0.29, range:
0.11-0.5, CI: 95%). Previous malaria episodes Of the 324 participants, 316 (97.5%) never had malaria, 4
(1.2%) did not remember, and 4 (1.2%, 2 women and 2 men)
had previously suffered a single episode of malaria. One of
these women (age: 69 years) had malaria at the age of 27 years
while staying in Mozambique and the other (age: 56 years)
had malaria at the age of 6 while staying in the municipality of
Mage (state of Rio de Janeiro). One of the previously infected
men (age: 70 years) had malaria at the age of 6 years but did
not remember the place, while the other man (age: 89 years)
had malaria due to P. falciparum infection at the age of 59 years
while staying in Rondônia (Amazonian region in Brazil). None
of these individuals had symptoms associated with malaria in the
year preceding the survey and all of them had negative results
in the thick blood smear and PCR tests. Table 4 shows variables associated with IgG reactivity. Individuals who entered the Atlantic Forest region (for hunting,
leisure, collecting plants, or other activities) had a 2.7 times
increased probability of having a reactive serology for P. vivax
compared with individuals who did not enter the forest (p<0.05). On the other hand, children <15 years of age had a higher chance
of reactive serology for P. falciparum and P. vivax compared
with individuals ≥15 years of age (p<0.05). Individuals living
in the Paraiso district had a higher chance of reactive serology
for P. vivax and any Plasmodium species (but not specifically
for P. falciparum and P. malariae) than people living in other
districts (p<0.05). There were no associations between sex,
symptoms, or past exposure to malaria and serological response
to antibodies of any Plasmodium species. Individuals who
reported previous episodes of malaria were not reactive to any
species of Plasmodium. Demographics aspects We interviewed a total of 324 individuals [186 women
(57.4%) and 138 men (42.6%)], with a mean patient age of 32.6±
9.8 years without differences between women and men (p=0.16). Of these, 215 participants (66.4%) lived in rural areas and 109
(33.6%) in peri-urban areas. Most individuals were born in the
state of Rio de Janeiro (260/324, 81.8%). The mean educational
duration of the participants was 6.9±3.6 years, and 269 people
(85.4%) had attended school and could read and write. The
average time of residence in the Guapimirim municipality was
18.6±13.1 years, the time of residence in the respective locality
was 17.7±12.8 years, and that in the current home was 9.8±12
years. A total of 36 individuals (36/324, 11.1%) had changed
housing in the last 5 years, 20 of which (6.2%) moved within
the municipality. Exposure to malaria Seventy-eight participants (24.1%) had left the Guapimirim
municipality in the 6 months prior to the study, most of them
during the last 15 days (41 subjects, 12.7%), and 2 residents went
to the Amazon region. Only 10.8% of the participants (35/324)
used a mosquito net while sleeping, with 7.7% (25/324) using
it always. In terms of visiting the forest region for activities
such as collecting fruits, walking, working, or leisure, 75.9%
(246/324) of the participants said that they did not regularly enter
the forest, 3.1% (10/324) reported going occasionally (mountain
climbing or walking), 10.2% (33/324) reported going often,
9.9% (32/324) reported going daily because their homes were
in the forest, and 3 participants did not answer this question. Most of the participants (73%) had insufficient knowledge of
malaria transmission and prevention. Laboratory tests None of the subjects had a positive thick blood smear
result. Diagnostic PCR was performed with the samples from 4/9 Rev Soc Bras Med Trop Vol.:52:e-20180537, 2019 TABLE 1: Frequency of Plasmodium infections diagnosed by PCR in the residents of the municipality of Guapimirim, Rio de Janeiro, Brazil, according to
the different investigated variables. um infections diagnosed by PCR in the residents of the municipality of Guapimirim, Rio de Janeiro, Brazil, according to TABLE 1: Frequency of Plasmodium infections diagnosed by PCR in the residents of the municipality of Guapimirim, Rio de Janeiro, Brazil, according to
the different investigated variables. P. falciparum
P. vivax
P. malariae
Total
Pos
Neg
Pos
Neg
Pos
Neg
Pos
Neg
Total
%
p-value
Age
<15 years
0
80
0
80
1
79
1
79
80
1,3
0.558
≥15 years
1
239
2
238
5
235
8
232
240
3.3
Total
1
319
2
318
6
314
9
311
320
2.8
Gender
M
1
137
0
138
3
135
4
134
138
2.9
0.794
F
0
182
2
180
3
179
5
177
182
2.7
Total
1
319
2
318
6
314
9
311
320
2.8
Localities
Caneca Fina
0
39
0
39
0
39
0
39
39
0
0.08
Garrafão
0
58
0
58
1
57
1
57
58
1.7
Barreira
0
51
0
51
0
51
0
51
51
0
Monte Olivete
0
16
0
16
1
15
1
15
16
6.3
Paraíso
0
54
2
52
1
53
3
51
54
5.6
Orindi
1
101
0
102
3
99
4
98
102
3.9
Total
1
319
2
318
6
314
9
311
320
2.8
Entry to the Atlantic Forest region for hunting, leisure, or collecting plants
Yes
0
74
0
74
2
72
2
70
72
2.8
0.7005
No
1
244
2
244
4
242
7
241
248
2.8
Total
1
318
2
318
6
314
9
311
320
2.8
Symptoms in the month preceding the study
Yes
0
29
0
29
0
29
0
29
29
0
-
No
1
290
2
289
6
285
9
282
291
3.1
Total
1
319
2
318
6
314
9
311
320
2.8
Previous malaria
Yes
0
4
0
4
0
4
0
4
4
0
-
No
1
315
2
314
6
310
9
307
316
2.8
Total
1
319
2
318
6
314
9
311
320
2.8
Pos: positive; Neg: negative. Laboratory tests TABLE 2: Positive serological reactions for IgG anti-Plasmodium species
antibodies in the population of the municipality of Guapimirim. TABLE 2: Positive serological reactions for IgG anti-Plasmodium species
antibodies in the population of the municipality of Guapimirim. TABLE 2: Positive serological reactions for IgG anti-Plasmodium species
antibodies in the population of the municipality of Guapimirim. TABLE 2: Positive serological reactions for IgG anti-Plasmodium species
antibodies in the population of the municipality of Guapimirim. with low parasitemia and predominance of the species
P. vivax20,21,22,23. The municipality of Guapimirim is very near
to the mountain ecosystem of the Atlantic Forest region, and
our study was carried out in areas where malaria cases had
been reported. Our results showed that the majority of the
study population had lived in the municipality for the last 18
years and only 2 participants (0.6%) had a history of travel to
an endemic area but without acquiring malaria. Only 1.2% had
a single previous malaria episode in the past, but all of them
showed negative PCR, thick blood smear, and serology results. In contrast to our expectations only one of these people had
contracted malaria in the Amazon region Rondônia. Parasite
Number
%
P. vivax (Pv)
14
40.0
P. falciparum (Pf)
3
8.6
P. malariae (Pm)*
7
20.0
Pv+Pf
5
14.3
Pv+Pm*
3
8.6
Pm+Pf*
1
2.8
Pv+Pf+Pm*
2
5.7
Total
35
100.0
*IgG for P. malariae was assessed only for samples positive in the PCR or
serological tests (42 samples). We observed no human malaria symptomatic cases and all
thick and thin blood smears were negative for Plasmodium
species. These results are not unexpected because data from the
governmental surveillance system have demonstrated that cases 5/9 Miguel RB et al. - Plasmodium infection in Rio de Janeiro TABLE 3: Relationship between PCR and serology results of individuals in the municipality of Guapimirim, Rio de Janeiro, Brazil. PCR
IgG anti-PvMSP119
(n=312)
IgG anti-P falciparum
(n=313)
IgG anti-P malariae
(n=42)
Any Plasmodium species
(n=314)
Positive
Negative
Total
Positive
Negative
Total
Positive
Negative
Total
Positive
Negative
Total
P. vivax
Positive
1
1
2
1
1
2
1
1
2
1
1
2
Negative
23
287
310
10
301
311
12
28
40
34
278
312
Total
24
288
312
11
302
313
13
29
42
35
279
314
P. Laboratory tests It is important to note
the presence of asymptomatic infection in individuals with no
previous history of malaria, travel to endemic areas, or contact
with a previous malaria-infected person. We speculated that
these subclinical infections were probably autochthonous cases. Only a few studies on asymptomatic infection by Plasmodium
species in the extra-Amazon region have been carried out to
date. Curado et al.24 demonstrated the presence of asymptomatic
infections with P. falciparum and mixed malaria caused by
P. falciparum and P. vivax in the Atlantic Forest region in São
Paulo state. Cerutti et al.20 conducted a population-based study
in the Espírito Santo state and obtained similar results. No
parasites were detected by microscopy in 1,777 blood samples
from residents of the area with reported malaria cases, but
asymptomatic infections with P. vivax (1.5%), P. malariae
(0.9%), and P. falciparum (0.5%) were diagnosed by PCR. De Alencar et al.25 in Espírito Santo state found a prevalence
of 3.4% of P. malariae and P. vivax infections. Maselli et al26
revealed a prevalence of P. falciparum (5.14%) and P. vivax
(2.26%) infections in healthy blood donors in São Paulo state. Donors with asymptomatic Plasmodium infection could be
reservoirs of transfusion-transmitted malaria (TTM). As these
individuals have low parasitic infections with negative thick In contrast to asymptomatic Plasmodium infections that
can only be detected by PCR, the presence of anti-Plasmodium
antibodies can reveal recent or past infections27. In our study,
11.8% of specimens were reactive to any of the tested antigens,
with 3.5% reactive to P. falciparum, 7.7% to P. vivax, and
30.9% of a subsample of 42 individuals reactive to P. malariae. The frequency of reactivity was low in relation to the study of
Azevedo (1997, unpublished data) in which 47.8% of samples
from the outbreak of 1993 in the Rio Bonito district showed
reactivity of IgG antibodies and 17.4% of IgM antibodies
(indirect immunofluorescence). These studies were repeated in
the area in 1996 with a higher number of subjects and revealed
a frequency of 35.4% of IgG antibodies against the asexual
blood forms of P. vivax, while no IgM antibodies were found. These findings were corroborated by Mattos et al.6 Cerutti et
al.20 performed a cross-sectional study of 65 patients and all
specimens showed a positive reaction to all the variants of
antibodies against the circumsporozoite protein of P. vivax,
with positive reactions to P. Laboratory tests falciparum
Positive
0
1
1
0
1
1
0
1
1
0
1
1
Negative
24
287
311
11
301
312
13
28
41
35
278
313
Total
24
288
312
11
302
313
13
29
42
35
279
314
P. malariae
Positive
1
5
6
0
6
6
6
0
6
6
0
6
Negative
23
283
306
11
296
307
7
29
36
29
279
308
Total
24
288
312
11
302
313
13
29
42
35
279
314
Cohen’s kappa index (κ) for associations between molecular and serological results was 0.07 (range: -0.07-0.20, CI: 95%, p>0.05) for P. vivax,
-0.07 (range: -0.02-0.01, CI: 95%, p>0.05) for P. falciparum, and 0.542 (range: 0.27-0.82, CI: 95%, p<0.05, “moderate”) for P. malariae. The
same calculations were performed for all Plasmodium species and identified a kappa index of 0.29 (range: 0.11-0.46, CI: 95%, p<0.05, “fair”). IgG: Immunoglobulin G: PvMSP1-19 : 19 kDa C-terminal region of the Merozoite Surface Protein 1 de P. vivax. ship between PCR and serology results of individuals in the municipality of Guapimirim, Rio de Janeiro, Brazil. Cohen’s kappa index (κ) for associations between molecular and serological results was 0.07 (range: -0.07-0.20, CI: 95%, p>0.05) for P. vivax,
-0.07 (range: -0.02-0.01, CI: 95%, p>0.05) for P. falciparum, and 0.542 (range: 0.27-0.82, CI: 95%, p<0.05, “moderate”) for P. malariae. The
same calculations were performed for all Plasmodium species and identified a kappa index of 0.29 (range: 0.11-0.46, CI: 95%, p<0.05, “fair”). IgG: Immunoglobulin G: PvMSP1-19 : 19 kDa C-terminal region of the Merozoite Surface Protein 1 de P. vivax. occur throughout the year, with an annual average of 4 or fewer
cases across the state particularly during the warmer season7,23. Interestingly, malaria outbreaks associated with P. simium
occurred in areas close to the municipality of Guapimirim in the
summers of 2015 and 20169. In our study, 9 individuals (2.8%)
with asymptomatic Plasmodium infection were diagnosed using
PCR analysis, 2 of which were positive for P. vivax (0.6%),
1 (0.3%) for P. falciparum, and 6 for P. malariae (1.9%). None
of these individuals developed clinical malaria within 30 days
of follow-up. Although most of the infected individuals live
in the towns of Orindi and Paradise, there were no significant
differences between the assessed locations, probably due to
the small number of positive samples. Laboratory tests It is important to note
the presence of asymptomatic infection in individuals with no
previous history of malaria, travel to endemic areas, or contact
with a previous malaria-infected person. We speculated that
these subclinical infections were probably autochthonous cases. Only a few studies on asymptomatic infection by Plasmodium
species in the extra-Amazon region have been carried out to
date. Curado et al.24 demonstrated the presence of asymptomatic
infections with P. falciparum and mixed malaria caused by
P. falciparum and P. vivax in the Atlantic Forest region in São
Paulo state. Cerutti et al.20 conducted a population-based study
in the Espírito Santo state and obtained similar results. No
parasites were detected by microscopy in 1,777 blood samples
from residents of the area with reported malaria cases, but
asymptomatic infections with P. vivax (1.5%), P. malariae
(0.9%), and P. falciparum (0.5%) were diagnosed by PCR. De Alencar et al.25 in Espírito Santo state found a prevalence
of 3.4% of P. malariae and P. vivax infections. Maselli et al26
revealed a prevalence of P. falciparum (5.14%) and P. vivax
(2.26%) infections in healthy blood donors in São Paulo state. Donors with asymptomatic Plasmodium infection could be
reservoirs of transfusion-transmitted malaria (TTM). As these
individuals have low parasitic infections with negative thick blood smear results, detection can be difficult using routine
laboratory tests26. In our study, we identified 2.8% of participants
with asymptomatic infections, 6 of which were infected with
P. malaria, the most frequent parasite associated with TTM
in the Americas. Thus, further studies should be conducted
to identify risk factors in these asymptomatic individuals, to
establish diagnostic methods, and to prevent TTM. occur throughout the year, with an annual average of 4 or fewer
cases across the state particularly during the warmer season7,23. Interestingly, malaria outbreaks associated with P. simium
occurred in areas close to the municipality of Guapimirim in the
summers of 2015 and 20169. In our study, 9 individuals (2.8%)
with asymptomatic Plasmodium infection were diagnosed using
PCR analysis, 2 of which were positive for P. vivax (0.6%),
1 (0.3%) for P. falciparum, and 6 for P. malariae (1.9%). None
of these individuals developed clinical malaria within 30 days
of follow-up. Although most of the infected individuals live
in the towns of Orindi and Paradise, there were no significant
differences between the assessed locations, probably due to
the small number of positive samples. Laboratory tests malariae (n=42)1
Any species (n=314)
%Pv
uOR (CI: 95%)
aOR (CI: 95%)
%Pf
uOR (CI: 95%)
aOR (CI: 95%)
%Pm
uOR (CI: 95%)
aOR (CI: 95%)
%
uOR (CI: 95%)
aOR (CI: 95%)
Age
≥15 years
6.3
0.49(0.20-0.18)
0.36(0.14-.96)*
2.1
0.25(0.07-0.8)*
0.03(0.00-0.09)*
29.0
0.71(0.16-3.06)
0.93(0.73-1.16)
9.7
0.57(0.27-1.21)
0.52(0.23-1.19)
<15 years
12
-
-
7.9
-
-
36.9
-
-
15.8
-
-
Total
7.7
-
-
3.5
-
-
30.9
-
-
11.1
-
-
Sex
Male
7.5
0.96(0.41-.23)
-
3.8
1.13(0.34-3.8)
-
21.0
0.38(0.09-1.56)
-
11.2
1.01(0.49-2.06)
-
Female
7.8
-
-
3.3
-
-
39.1
-
-
11.1
-
-
Total
7.7
-
-
3.5
-
-
30.9
-
-
11.1
-
-
Entry to the forest for hunting, leisure, or collecting plants
Yes
14.3
2.71(1.15-6.4)*
2.73(1.01-7.38)*
4.3
1.31(0.34-5.2)
1.02(0.54-2.20)
44.4
0.98(0.24-4.07)
0.69(0.54-0.86)
17
1.99(0.93-4.23)
2.09(0.87-4.84)
No
5.8
-
-
3.3
-
-
45.0
-
-
9.4
-
-
Total
7.7
-
-
3.5
-
-
-
-
11.1
-
-
Housing location
Barrera
2.0
1
1
0
1
1
33.3
1
1
3.8
1
1
Monte Oliveti
13.3
1.29(0.41-9.1)
12.62(1.06-297)
0
**
-
100.0
**
-
13.3
1.24(0.27-5.74)
5.01(0.55-46.52)
Garrafão
3.7
0.41(0.09-1.8)
2.53(0.23-56.42)
0
**
-
20.0
0.52(0.05-5.20)
-
5.5
0.41(0.12-1.38)
1.55(0.24-12.25)
Caneca Fina
15.8
2.67(0.98-7.2)
8.71(1.31-173.74
7.9
2.86(0.72-11.3)
-
42.9
1.87(0.35-9.93)
-
18.4
2.00(0.81-4.21)
5.17(1.11-37.29)
Orindi
5.9
0.68(0.26-1.7)
3.48(0.55-67.97)
4
1.21(0.35-4.22)
-
23.1
0.57(0.127-2.55)
-
11.9
1.11(0.53-2.34)
3.69(0.93-24.73)
Paraiso
13.2
2.16(0.85-51)
10(1.62-94.85)*
7.5
2.95(0.83-10.5)
-
25.0
0.67(0.147-3.02)
-
17
1.83(0.81-4.21)
5.98(1.42- 41.21)*
Total
7.7
-
-
3.5
-
-
31.0
-
-
11.1
-
-
Symptoms in the month preceding the study
Yes
3.8
0.46(0.06-.53)
-
3.8
1.10(0.13-9.01)
0
**
-
3.8
0.29(0.04-2.27)
-
No
8
-
-
3.5
-
-
30.9
-
-
11.8
-
-
Total
7.7
-
-
3.5
-
-
30.9
-
-
11.1
-
-
Previous malaria
Yes
0
**
-
0
**
-
0
**
-
0
**
-
No
7.8
-
-
3.6
-
-
30.9
-
-
11.3
-
-
Total
7.7
-
-
3.5
-
-
30.9
-
-
11.1
-
-
*p<0.05, **Undefined. CI: confidence interval. aOR: adjusted odds ratio, uOR: unadjusted odds ratio. 1Only 42 samples. The main finding of our study is the
association between entering the forest
and positive serology for Plasmodium vivax. Laboratory tests vivax classic (VK210) in 25.4%,
to VK247 in 6.3%, to P. vivax-like in 10%, and to P. malariae
in 15.1% specimens. The same authors assessed healthy
individuals that had been in contact with malaria patients and
observed a high percentage of antibodies to P. vivax (37.7%
IgG and 6.2% IgM) and P. malariae (44.6% IgG and 15.8%
IgM) and a prevalence of P. falciparum reactivity of 13.5% for
IgM and 13% for IgG antibodies against asexual forms of the
parasite. With our results, we cannot conclude that serology
can predict infection. 6/9 Rev Soc Bras Med Trop Vol.:52:e-20180537, 2019 TABLE 4: Reactivity associated with IgG anti-PvMSP119, anti-Pf IgG, and anti-Pm IgG in specimens from residents of the Guapimirim municipality, Rio de Janeiro, Brazil. P. vivax (n=312)
P. falciparum (n=313)
P. Laboratory tests Rev Bras Malariol Doencas Trop. 1950;3:473-87. 4. Chagas C. Luta contra a malária-conferência proferida no Núcleo
Colonial S. Bento, abril 1933. In: Carlos Chagas. Discursos e
conferências. Rio de Janeiro: A Noite; 1935. p. 191-231. Enhancing the knowledge on malaria in residents of the
extra-Amazonian areas is an important aspect of disease
elimination. Although transmission in this area was interrupted
50 years ago5, 73% of actual residents have insufficient
knowledge on malaria transmission and prevention. Azevedo et
al.6 found a similar percentage in the Nova Friburgo region. It
is noteworthy that 79.3% of participants associated the summer
with an increase in mosquito density particularly at nightfall,
but a large proportion of the population did not use any control
measures for avoiding insect bites. Malaria diagnosis is a big
challenge in these areas because medical doctors often do not
consider the disease when a patient presents with fever22. 5. Moraes HF. SUCAM: sua origem sua história. 2 ed. Brasília:
SUCAM; 1990. 484p 6. Mattos M. S, Oliveira-Ferreira J, Castro M. G, Lourenço-de-
Oliveira R: Malária autóctone no município de Nova Friburgo -
Rio de Janeiro [Internet]. Proceedings of the IV Reunião Nacional
dos Pesquisadores em Malária, Summary 13. Manaus, Brasil. 1993 [cited Dec 12]. Avaliable from: http://www.scielo.br/scielo. php?script=sci_nlinks&pid=S00740276201400050061800077&lng
=en. 7. Miguel RB, Peiter PC, De Albuquerque H, Coura JR, Moza P. G,
Costa A DE P, Brasil P, Suárez-Mutis MC. Malaria in the state of
Rio de Janeiro, Brazil, an Atlantic Forest area: an assessment using
the health surveillance service. Mem Inst Oswaldo Cruz. 2014;
109(5):634-40. According to the WHO, residual malaria occurs sporadically
in places with interrupted transmission and remaining
determinants of transmission42. These areas require assessment
of favorable locations for Anopheles larvae habitats, food
sources, and invasion of Anopheles by Plasmodium. All these
conditions are present in the Atlantic Forest region, but on a
much smaller scale compared to the Amazon region36,37,38,39,40. Albuquerque et al. 43 recently constructed a model for evaluation
of territory receptivity in order to strengthen entomological
surveillance of Nyssorhynchus mosquitoes for imported
malaria cases. The study showed that pluviosity, temperature,
geomorphology, and vegetation variables could be used to
create a model of surveillance, but a specific model needs to be
generated for Kerteszia anophelines responsible for bromeliad
malaria in these areas. 8. Lupi O, Vidigal AC, Show CL, de Pina Costa Anielle, Saraiva RP,
Daniel-Ribeiro CD, Brasil P. Laboratory tests The chance to have a positive serology
was 2.7 times (range: 1.01-7.38, CI:
95%, p<0.05) higher for people who
entered the forest compared with
those that did not. These results differ
from those obtained by Maselli et al.26
who showed a positive association
between residing in the mountain
area of the Atlantic Forest region with
P. falciparum and P. vivax infections
and forest fragmentation. Nonetheless,
the study was carried out with samples
from blood donors in São Paulo state
and the origin of samples could explain
the divergences from our results. The presence of asymptomatic
P. falciparum infections detected in our
study is a cause of concern, but other
authors20,24,26 made the same observation
in the Espírito Santo and São Paulo
states. Recently, Laporta et al.28 showed
an unexpectedly high proportion of
P. falciparum on anophelines in the
São Paulo state. These results are
challenging the traditional “bromeliad-
malaria” paradigm that proposes a
sylvatic cycle in bromeliad areas with
an interaction between an An. kerteszia
vector (particularly An. cruzii), a non-
human primate reservoir (Alouatta
species, Brachyteles species, and
Cebinae subfamilies), and a P. simium
or P. vivax parasite9,30,31,32,33,34,35. As PCR
used in our study cannot differentiate
between a P. vivax and P. simium
infection, more sophistical analyses are
required. Humans could be accidentally
infected when they enter the forest for
recreation or work by invading the
transmission cycle of the parasite in
the wild36,37,38,39,40. On the other hand,
P. falciparum is a virulent Plasmodium
species in the Amazon region and any
infection by this parasite should result
in malaria disease if the individual is
not immune. The fact that individuals
do not develop symptoms in the extra-
Amazonian region may be due to
antigenic variability of P. falciparum,
but this question must be further
clarified in future studies involving
sequencing of parasite DNA29,41. Another important and unexpected
finding of our study was the higher 7/9 Miguel RB et al. - Plasmodium infection in Rio de Janeiro chance of obtaining positive serologies for P. falciparum and
P. vivax in individuals under 15 years of age compared to older
individuals. No previous study has reported a similar trend, and
our findings may present a novel epidemiological scenario in
this context that needs to be elucidated in future studies. 3. Carvalho FF, Rachou RG. Considerações sobre a malária no Estado
do Rio de Janeiro com especial referência ao Vale do Rio Piraí. Conflict of Interest: The authors declare that there are no conflicts of interest. 15. Sanchez MCA, Avila SLM, Quartier-Oliveira VP, Ferreira AW. Malaria serology: performance of six Plasmodium falciparum
antigen extracts and of three ways of determining serum titers in
IgG and IgM-ELISA. Rev Inst Med Trop Sao Paulo. 1993;35(6):495-
502. Financial Support: This research was funded by Fundação Carlos Chagas
Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, financial
support process 214879/E_06/2015) and Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES, financial support 001). 16. Ferreira AW, Sanchez MCA. Human malaria: standardization of
serologic tests for individual diagnostic and seroepidemiologic
surveys. Rev Inst Med Trop Sao Paulo. 1988;30(3):137-46. Laboratory tests Estudo dos casos suspeitos de malária
importada, um Centro de Referência na região extra-Amazônica. Research. 2014; 1: 10.13070/rs.pt. DOI: 10.13070/rs.pt.1.613 9. Brasil P, Zalis MG, de Pina-Costa A, Siqueira AM, Júnior CB, Silva
S, et al. Outbreak of human malaria caused by Plasmodium simium
in the Atlantic Forest in Rio de Janeiro: a molecular epidemiological
investigation. Lancet Glob Health. 2017; 5(10):e1038-e1046. 10. World Health Organization (WHO). Global technical strategy for
malaria 2016–2030. Geneva: WHO; 2016. 35p. 11. Ministério da Saúde (MS). Secretaria de Vigilância em Saúde. Manual de diagnóstico laboratorial da malária / Ministério da
Saúde, Secretaria de Vigilância em Saúde (Série A. Normas e
Manuais Técnicos). 2ª edição. Brasília: MS; 2009. 116 p. 12. Snounou G. Detection and identification of the four malaria parasite
species infecting humans by PCR amplification. Mol Biochem
Parasitol. 1996;50:263-91. Acknowledgments: We offer our deepest thanks to the institutions that
provided technical support for our study. We thank Arianni Rondelli Sanchez
and Eduardo Milton Ramos-Sanchez for preparing the P. vivax recombinant,
Fabiana Maria de Souza Leoratti for preparing the extract, Dr. Silvia Di Santi
for blade preparation of P. malariae and Dr. Daniel Câmara for statistical
analysis. Special thanks to the research participants in the Guapimirim
municipality. 13. Suárez-Mutis MC, Cuervo P, Leoratti FM, Moraes-Avila SL,
Ferreira AW, Fernandes O, Coura JR. Cross sectional study reveals
a high percentage of asymptomatic Plasmodium vivax infection in
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Brazil. Parasit vectors. 2018;11:256-62. 31. Gadelha PE. From forest malaria to bromeliad Malaria: a case-
study of scientific controversy and malaria control. Parassitologia. 1994;36:175-95. OPEN ACCESS
https://creativecommons.org/licenses/by/4.0/ 9/9 OPEN ACCESS
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Supplementary Figure 3 from <i>WWOX</i> Gene Expression Abolishes Ovarian Cancer Tumorigenicity <i>In vivo</i> and Decreases Attachment to Fibronectin via Integrin α<sub>3</sub>
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Interview with Peter Mertens and Wolfgang König: “From Reasonable Automation to (Sustainable) Autonomous Systems”
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Bus Inf Syst Eng
https://doi.org/10.1007/s12599-022-00753-z PROFILE PROFILE Interview with Peter Mertens and Wolfgang Ko¨nig: ‘‘From
Reasonable Automation to (Sustainable) Autonomous Systems’’ J. Dibbern (&)
Institut Fu¨r Wirtschaftsinformatik, Abteilung Information
Engineering, Muesmattstrasse 27, 3012 Bern, Switzerland
e-mail: jens.dibbern@iwi.unibe.ch R. Beck
IT University of Copenhagen, Rued Langgaards Vej 7,
2300 Copenhagen, Denmark
e-mail: romb@itu.dk M. Wiener
Chair of Business Information Systems, esp. Business
Engineering, TU Dresden, Helmholtzstr. 10, 01069 Dresden,
Germany
e-mail: martin.wiener@tu-dresden.de Interview with Peter Mertens and Wolfgang Ko¨nig: ‘‘From
Reasonable Automation to (Sustainable) Autonomous Systems’’ Roman Beck • Jens Dibbern • Martin Wiener The Author(s) 2022 Prof. Dr. Dr. h. c. mult. Peter Mertens
Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg
Chair of Informaon Systems – Innovaon & Value
Creaon (Wi1)
Lange Gasse 20
90403 Nuremberg
Germany
Prof. Dr. Wolfgang König
Goethe-Universität Frankfurt
Execuve Director of the House of Finance
Theodor-W.-Adorno-Platz 3
60323 Frankfurt am Main
Germany Prof. Dr. Dr. h. c. mult. Peter Mertens
Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg
Chair of Informaon Systems – Innovaon & Value
Creaon (Wi1)
Lange Gasse 20
90403 Nuremberg
Germany Prof. Dr. Dr. h. c. mult. Peter Mertens
Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg
Chair of Informaon Systems – Innovaon & Value
Creaon (Wi1)
Lange Gasse 20 Prof. Dr. Wolfgang König
Goethe-Universität Frankfurt
Execuve Director of the House of Finance
Theodor-W.-Adorno-Platz 3
60323 Frankfurt am Main
Germany Peter Mertens is Professor Emeritus of Wirtschaftsinfor-
matik
at
the
Friedrich-Alexander-Universita¨t
(FAU) M. Wiener
Chair of Business Information Systems, esp. Business
Engineering, TU Dresden, Helmholtzstr. 10, 01069 Dresden,
Germany
e-mail: martin.wiener@tu-dresden.de 12 123 12 R. Beck et al.: Interview with Peter Mertens and Wolfgang Ko¨nig, Bus Inf Syst Eng Erlangen-Nu¨rnberg. After studying industrial engineering,
he completed his doctoral studies and his habilitation at
the TH Darmstadt (1961) and the TU Mu¨nchen (1966),
respectively. From 1966 to 1968, he worked for a large
software and consulting firm in Switzerland, first as a
system designer and later as a managing director. In 1968,
Peter Mertens took over the first chaired professorship
specialized in business data processing at the University of
Linz. He is considered one of the founding fathers of
Wirtschaftsinformatik in the German-speaking world. Until
September 2005, Peter Mertens held the Chair of Business
Administration, especially Wirtschaftsinformatik I at the
Faculty of Business and Social Sciences of FAU. In par-
allel, he was head of the computer science research group
‘‘Business Applications’’ at FAU’s Faculty of Engineering. Since fall 2005, he works as an emeritus professor at his
former chair. Peter Mertens is the author of numerous
books, including 23 monographs. He has also been
involved in the editing of 26 collective works. The first
volume of his book ‘‘Integrated Information Processing’’
has been published in 18 editions. Some of his books have
been translated into English, Chinese, Italian, and Russian. Interview with Peter Mertens and Wolfgang Ko¨nig: ‘‘From
Reasonable Automation to (Sustainable) Autonomous Systems’’ Among other awards, he is a Fellow of the German
Informatics Society, an honorary doctor of five universities
in Germany, Austria, and Switzerland, and has been
awarded the Order of Merit of the Federal Republic of
Germany. From 1990 until 2000, Peter Mertens served as
Editor-in-Chief for WIRTSCHAFTSINFORMATIK (now:
BISE). BISE: In the context of the subject of our special issue,
you, Professor Mertens, proposed the goal of ‘‘reasonable
full automation’’ already back in 1995. (Note: Peter Mer-
tens formulated this goal for the first time during a keynote
speech
held
at
the
International
Conference
on
Wirtschaftsinformatik in Frankfurt/Main, organized by
Wolfgang
Ko¨nig.)
What
were
the
motivations
for
this proposal back then? Mertens: My motivation was to provide a fashion-inde-
pendent, long-term goal in the back and forth of fads,
constant relabeling, and actual advances. I like to use the
metaphor of the ship’s captain (or his compass) from ear-
lier times, who oriented himself to the polar star. He did not
want to reach the polar star, which would have been uto-
pian. As such, the notion of reasonable full automation is a
concrete utopia of philosophy (Ernst Bloch). In my opinion, extensive automation is essential, espe-
cially for Germany, given the catastrophic age structure of
the German population. However, it must be reasonable. A
counterexample: The use of an autonomous vehicle, such
as a self-driving car, does not bring any productivity ben-
efits for a manager who would like to study documents
while driving, as she or he can be called upon by the car to
take over steering and braking in confusing traffic situa-
tions. According to psychological studies, after the han-
dover from the car to the human, it will take 12 to
15 seconds for the latter to get an overview of the situation. By then, the car would have traveled a few hundred meters
and a serious crash may have already happened. For this
reason, the manager is not allowed to study documents
while driving her/his autonomous vehicle and thus does not
gain any working time. Therefore, automation is not rea-
sonable in this example. Until 2016, Wolfgang Ko¨nig was Professor of Business
Administration, especially Information Systems and Infor-
mation Management at the Faculty of Economics and
Business Administration of Goethe University Frankfurt a. BISE: What factors have influenced this development? BISE: What factors have influenced this development? Ko¨nig: Automation essentially depends on three determi-
nants: First, the performance/price ratio of ICT has been
both steadily and massively increasing for the last 50 years. In this regard, not much will change, at least not in the
foreseeable future. For example, on the hardware side, the
available computing power per US dollar has almost dou-
bled every year. Although ICT themselves absorbed some
of the technical improvements, this is an ‘‘insane’’ driving
force. Fig. 1 Development of automated systems over time Ko¨nig: Generally, the development of automation over
time can be illustrated in a pyramid. (Note: see Fig. 1
below.) The smallest pyramid (A) may symbolize the first
level of (partial) automation, so to speak. In the area of
business applications, this took place in the 1950s. These
were very simple administrative tasks (e.g., the documen-
tation of insurance contracts), which took advantage of the
then still quite small mass-processing capability of a
computer. An important basis of administration systems are
legal regulations, such as the posting of an invoice as soon
as the amount exceeds the legally binding threshold of a
company. These are mandatory regulations and there is in
fact no freedom of action. Over time, many of these
A-pyramids have developed side by side. Different appli-
cation
experiences were
bundled
through
knowledge
transfer; the theory of replacing simple administrative
processes with information and communication technolo-
gies (ICT) deepened and differentiated (as depicted in the
vertical dimension of the pyramid); and first approaches to
standardization emerged. The second determinant is the human controllability of
advances in the symbiotic human-machine system, which
have been made possible by the above-mentioned technical
improvements. In other words, both human designers and
users must spend significant time learning to implement
these rapidly increasing technical options in relevant
application contexts – and then also to control the system
outcomes. Those contexts have different characteristics
regarding the reproducibility of a specific result and the
endurability of any errors that may occur along the way. For example, from a scientific standpoint, controlling the
movements of a spaceship can be more easily achieved
than ‘‘reading’’ a person’s state of mind from a photo; and
an error in the former case is often fatal, whereas in the
latter case, one can often leave it at an apology. Interview with Peter Mertens and Wolfgang Ko¨nig: ‘‘From
Reasonable Automation to (Sustainable) Autonomous Systems’’ M., and until January 2022, he was Chairman of the
E-Finance Lab (since 2020: efl– the Data Science Insti-
tute) at Goethe University. Since 2008, he holds the posi-
tion of Executive Director of the House of Finance of
Goethe University, and since 2016, he serves as Senior
Professor at Goethe University. From 1998 until 2008,
Ko¨nig served as Editor-in-Chief for WIRTSCHAFTSI
NFORMATIK (now: BISE). It should also be noted that I never intended a short-term
realization of the full-automation goal; rather, this concrete
utopia was meant to give direction to system architects and
others, including legislators. For example, any legislation
and related reforms that impair the use of automation
technology or even prevent it – that is, are not automation-
friendly – are to be scrutinized particularly closely. Here is
a concrete example: in Austria, the registration of a new-
born child can be done immediately from the delivery
nurse’s computer screen. Consequently, the formalities at
the residents’ registration office, which issues the birth
certificate, and at the local office responsible for the
approval and payment of child benefits can be fully auto-
mated; that is, relevant documents are prepared by the
Austrian authorities in an automated fashion and then sent
to the parents for their files, and child benefits are paid out
to the parents automatically. In Germany, however, such
automation would not be possible in many communities, as
current legislation still requires parents to register their
newborn child in an ‘‘analog’’ manner. Both Peter Mertens and Wolfgang Ko¨nig are clearly
among the research pioneers when it comes to automated
systems, which can be seen as a precursor of the central
topic of this special issue: autonomous systems (AS). The
key difference between automated systems and AS is that,
in AS, machines or other technology actors have at least
some agency (i.e., they can act autonomously), whereas in
automated systems, the agency still lies with humans –
who, for example, define the relevant rule system – and
machines/technologies merely automate the execution of
these predefined rules. 12 123 123 123 R. Beck et al.: Interview with Peter Mertens and Wolfgang Ko¨nig, Bus Inf Syst Eng Fig. 1 Development of automated systems over time time and simultaneously an increase of their heights. Again, the pressure to do so often comes from the
administrative level. For instance, think of the ever-grow-
ing anti-money laundering regulations in the financial
world. Interview with Peter Mertens and Wolfgang Ko¨nig: ‘‘From
Reasonable Automation to (Sustainable) Autonomous Systems’’ All in all, this development can be characterized as
an extensive chain of careful palpations – always against
the backdrop that the consequences of an ‘‘extension error’’
need to be kept under control. BISE: What factors have influenced this development? BISE: What key challenges stood or stand in the way of
tapping the full potential of automated systems? Why has
the implementation of such systems often failed? BISE: What key challenges stood or stand in the way of
tapping the full potential of automated systems? Why has
the implementation of such systems often failed? Mertens: In the above-referenced case, where a technol-
ogy actor has made a wrong decision, a task may have been
automated based on a wrong prognosis. Such automation
was not reasonable then, at least not under the given cir-
cumstances. A prominent example in this context is the
infamous automation project ‘‘Halle 54’’ by Volkswagen
(VW) in the early 1980s. (Note: the name ‘‘Halle 54’’ refers
to a VW production facility designed for full automation.)
The project was based on a misjudgment regarding the
degree of maturity of the computer-integrated manufac-
turing (CIM) principle back then. Among other things, VW
underestimated the efforts of maintaining the automation
technology, while overestimating the motivation of the few
remaining production employees. Ko¨nig: With respect to Fig. 1, it is important to note that
different industries and individuals are likely to go through
different trajectories on their way from A to C, depending
for instance on the requirements of the business world and
depending on their own training and experience, as well as
their individual level of motivation. Consider the mega-
trend in economically developed countries that, over time,
the main share of their gross national product has moved
from ‘‘material industries,’’ where for example a machine
or a chemical reactor represents the core of a solution (as in
the automotive and chemical industry), to the high-tech
service sector – think, for example, of the engineering
sector of the chemical industry. Both aforementioned
technical devices are not readily modifiable during the
production
process,
which
restricts
the
respective
scheduling and planning processes. As such, users have
fewer degrees of freedom and must adapt to the given
machine structures, which cannot be changed on short
notice. In fact, parallel to the increase of the share of ser-
vices in the gross national product, the value-added share
of the ICT sector rises considerably. The engineering and
ICT sectors are more focused on user benefits – people and
their behavior are at the center of a solution. BISE: What factors have influenced this development? Industrial
services, for example, include more flexible ways of pro-
ducing and distributing goods, which places higher intel-
lectual demands on system developers, service providers,
and, of course, end users. Due to their comparatively
greater reliance on the human factor, services pose a par-
ticularly challenging automation problem – although here,
too, the classic administrative processes must be automated
first. Ko¨nig: Some additional examples: First, as with any new
technology, there are always excessive expectations at one
point in time. The Gartner Hype Cycle expresses this
explicitly. Second, too little attention has been paid to the
overall social consequences of automation. For example,
job holders (in the ‘‘manual’’ world) have almost always
protested and agitated against having their jobs, their work,
replaced by robots/technology. And third, if – despite all
the cautious probing of new ‘‘automation territory’’ – a
corresponding technology has made some serious wrong
decision on behalf of humans, there will be a host of critics
who have always known better, especially in Germany. Against this backdrop, the most important failure factor
has been and continues to be humans in their role as
individual stakeholders; that is, as system providers (e.g.,
when unrealistic user expectations are propagated), or as
developers, or as users, etc. In this regard, I still see the
machine as a programmed mechanism of action that is
made available to the world intentionally, or erroneously,
by humans. How can these challenges be overcome? A first impor-
tant answer is to improve education and experience. For
example, do we need more skilled programmers and sys-
tem designers? Apparently yes. Do we need more highly
skilled staff members to control the quality of the systems
we buy in the world? Apparently yes. BISE: To what extent are the challenges you mentioned
above also relevant in relation to the development of
(sustainable) AS? How can these challenges be overcome? Mertens: Broadly speaking, ICT-related advances are
naturally moving us forward along the time axis in many
areas of the economy and society (‘‘technology push’’). On
the other hand, as already noted above, a growing ‘‘demand
pull’’ results primarily from the current demographic situ-
ation in Germany. Both factors (i.e., technology push and
demand pull) influence the technical progress. In this
context, a fundamental problem – then as now – is that
many (new) systems are not sufficiently tested. BISE: What factors have influenced this development? Based on the experiences gained with the automation of
administrative tasks (A), attempts were made to automate
disposition systems (B), which offer some degree of free-
dom for relevant actors – be they humans or machines. In
logistics, freight scheduling is a good example. Here again,
the first task was and is to gain experience with (partial)
automation, to bundle different deployment experiences,
and
to
test
standardization
approaches. Clearly,
the
automation challenges at this level (B) are more complex
than the challenges at the lower level (A). However, the
increasingly complex challenges have been mitigated by
the fact that the general performance of machines has
increased noticeably over the years – and continues to do
so. The third determinant is the intellectual depth of the
replacement of human labor by computers. For decades,
Peter Mertens, and our discipline in general, has been
talking about automation in the context of administration,
disposition, and planning systems – in this order. This
means that with growing intellectual depth, there is often
also greater potential for the beneficial use of ICT (when
compared with the purely manual handling of relevant
processes
or
compared
with
earlier
versions
of
automation). Against this backdrop, Peter Mertens‘ notion of rea-
sonable full automation refers to the entire pyramid. (Note:
again, see Fig. 1.) Here, it must be ensured that an
investment in the degree of automation – and ‘‘full’’ means
100% after all – pays off at every level, including for
instance the repair and clean-up costs if the automation
technology ever takes a decision that human supervisors
consider to be ‘‘wrong’’ in retrospect. Lastly, based on the automation experiences at levels A
and B, automation experiments were carried out with
planning systems that are characterized by a comparatively
high degree of freedom for the individual actor. The dynamics of such a stacked hierarchy can be
depicted as a widening of the pyramids’ basis (A-C) over 12 3 3 R. Beck et al.: Interview with Peter Mertens and Wolfgang Ko¨nig, Bus Inf Syst Eng especially elderly bank customers tend to lose a great deal
of time familiarizing themselves with constantly changing
system versions. As such, it may well take bank customers
less time to pay a bill using a conventional (‘‘analog’’)
transfer slip on which their IBAN is already pre-printed
than to train themselves to use the latest software version. BISE: What factors have influenced this development? For
instance, online banking systems are often largely auto-
mated but also ‘‘sloppily’’ implemented, which implies that BISE: What do you think: where does the journey lead to
for AS in the future – also regarding the limits of techno-
logical autonomy as well as the tension between this
autonomy and human autonomy? Mertens: This cannot be answered in general terms. Would anyone argue today with the human right of self-
determination in order to ban automatic washing machines
in the basement? Would one classify the technical aids for 123 123 R. Beck et al.: Interview with Peter Mertens and Wolfgang Ko¨nig, Bus Inf Syst Eng flight captains as a restriction of the pilot’s responsibility? The question of when exactly an automated system should
hand over to the pilot (and vice versa) can only be
answered through very meticulous analysis of relevant
situations, but not in general. Ko¨nig: In successful symbiotic systems, the common
denominator between humans and machines are standards,
which are stacked in a multi-level hierarchy. At the lowest
level, we see elementary standards (like the letters of an
alphabet or the digits of a number system). We study, or
have studied, to learn and apply these stacked standards in
a subject area, and a dissertation aims to develop and apply
new methods to a particular problem, thereby probing and
potentially changing the existing stack of standards. For
humans, the application of standards limits the degree of
volatility and unpredictability of their actions. At the same
time, theory and experience teach us that reasonable stan-
dards (i.e., those that benefit many users) at lower levels
open up immense opportunities to individualize solutions
at higher levels of the stack by recombining standard ele-
ments. As such, we are challenged to invest into European
standards – also in the field of AS. This cannot simply be
‘‘outsourced’’ to the U.S. or China. Ko¨nig: AS are certainly an important step on the devel-
opment path outlined. Here, if the definition of ‘‘au-
tonomous’’ is taken seriously, the question of how
corresponding systems adapt to ever-changing circum-
stances must be at the center, such as the ability of
machines to reprogram themselves. In that case, the pyra-
mid introduced above may get another development level
on top; or it might be that the amplitude of the Gartner
Hype Cycle increases over time as automation develop-
ment progresses. BISE: What factors have influenced this development? In this case, the reprogrammability of
machines would be associated with (too?) high develop-
ment risks. To me, the dichotomy of human versus technology
autonomy seems to reach far into the future. But clearly: in
selected work environments, machines already carry out
independent planning. So, in principle, why shouldn’t they
be able to reprogram themselves in the future, especially if
certain operational prerequisites are given. This would
probably put another ‘‘rocket stage’’ on top of the currently
known complexity. BISE: Thank you very much for all the insights, as well as
for taking the time to participate in this interview, which is
greatly appreciated! Funding Open access funding provided by University of Bern. Funding Open access funding provided by University of Bern. Mertens: In any case, regarding the inherent tension
between human and technology autonomy, the develop-
ment of unethical AS must be avoided at all costs. For
example, an AS in which the death of humans is statisti-
cally accepted cannot be reasonable. 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/. BISE: Finally, what advice would you give young BISE
scholars who intend to write their dissertation, or the like,
in the field of AS? Mertens: Think of the higher-level goals that are relevant
for the survivability of our economy and society (‘‘demand
pull’’), and do not philosophize over many pages of your
dissertation about the nature of humans as such! 123 123 12
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3D flow field measurements outside nanopores
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RESEARCH ARTICLE | MAY 12 2022
3D flow field measurements outside nanopores Rev. Sci. Instrum. 93, 054106 (2022)
https://doi.org/10.1063/5.0083054 a)Authors to whom correspondence should be addressed: kandres a)Authors to whom correspondence should be addressed: kandrese@gettysburg.edu and ufk20@ ABSTRACT We demonstrate a non-stereoscopic, video-based particle tracking system with optical tweezers to study fluid flow in 3D in the vicinity
of glass nanopores. In particular, we used the quadrant interpolation algorithm to extend our video-based particle tracking to displace-
ments out of the trapping plane of the tweezers. This permitted the study of flow from nanopores oriented at an angle to the trapping
plane, enabling the mounting of nanopores on a micromanipulator with which it was then possible to automate the mapping procedure. Mapping of the voltage driven flow in 3D volumes outside nanopores revealed polarity dependent flow fields. This is in agreement with
the model of voltage driven flow in conical nanopores depending on the interaction of distinct flows within the nanopore and along the
outer walls. 24 October 2024 04:47:11 © 2022 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license
(http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0083054 I. INTRODUCTION principle of measuring nanopore electroosmotic flow using optical
tweezers. Specifically, a 2.16 μm diameter polystyrene bead (Kisker,
Germany) suspended in salt solution is held in an optical trap just
outside the pore entrance. Applying a voltage causes
fluid flow
around the nanopore that perturbs the trapped particle and displaces
it in the direction of the flow, thereby revealing information about
the field’s direction and strength. By changing the position of the
nanopore tip relative to the trapped bead while recording the forces,
the flow field is mapped. Solid-state nanopores underpin a simple but powerful molec-
ular sensing platform that has blossomed into an important tool
for biophysics and beyond. The basic operating principle of these
sensors involves using an electric field to drive analytes through
the nanopore with the translocation observed as a change in ionic
current. The operation of nanopore devices is, however, governed
by a subtle interplay of phenomena: target molecules are driven
electrophoretically,1–3 and this driving force is opposed by a viscous
drag force.4 This drag force arises not only from Stokes drag but
also from counterions on the target molecule driven in the opposite
direction by the electric field4 and from the voltage-driven, elec-
troosmotic flow (EOF) from the pore surface.3,5 Characterizing EOF
and broader nanopore flow behavior offers novel physical insights
into the hydrodynamics of nanochannel systems6 and has applica-
tions in molecular sensing,7 gel electrophoresis,4 and electrophysiol-
ogy.8 At these small length scales, however, the relevant forces have
pico-Newton magnitudes, leading to significant challenges in their
measurement. Articles You May Be Interested In Cation dependent electroosmotic flow in glass nanopores
Appl. Phys. Lett. (September 2019)
Note: Direct force and ionic-current measurements on DNA in a nanocapillary
Rev. Sci. Instrum. (August 2011)
Voltage-modulated polymer nanopore field-effect transistor for multi-sized nanoparticle detection
Nanotechnol. Precis. Eng. (May 2024) Cation dependent electroosmotic flow in glass nanopores Appl. Phys. Lett. (September 2019) 24 October 2024 04:47:11 Review of
Scientific Instruments ARTICLE scitation.org/journal/rsi AFFILIATIONS 1 Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, United Kingdom
2Department of Physics Gettysburg College Gettysburg Pennsylvania 17325 USA 1 Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, United Kingdom
2Department of Physics, Gettysburg College, Gettysburg, Pennsylvania 17325, USA Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, United Kingdom
2Department of Physics, Gettysburg College, Gettysburg, Pennsylvania 17325, USA III. PARTICLE TRACKING To initiate the in-plane tracking algorithm in LabVIEW, a
cursor is used to specify the approximate bead center. The algo-
rithm then follows the steps detailed in Figs. 2(a)–2(d). Initially, a FIG. 2. 3D particle tracking. Both in-plane (x, y) tracking and out-of-plane (z)
tracking require identification of the particle center (a)–(d), while (e)–(g) are specific
to z tracking. (a) A frame is cropped to a 20 × 20 px2 (x, y) or 80 × 80 px2 (z)
sub-array approximately centered on the trapped bead. (b) Intensity profiles in x
and y are obtained by averaging over 4 × 20 px2 rectangular bins, producing I(x)
and I(y). (c) The cross-correlation between each intensity profile and its reverse,
I⋆(x), I⋆(y), is found. (d) A quadratic fit to the cross-correlation determines the
particle center (x, y position) with sub-pixel accuracy. To obtain the z coordinate:
(e) The 80 × 80 px2 frame is quartered, and a radial intensity profile is calculated
per quadrant. (f) The four profiles are averaged, and this mean profile is compared
to each entry of a lookup table (LUT). (g) χ2 difference is used to compare the
profile to the LUT, and a quadratic fit around the minimum of the χ2 difference
enables sub-LUT resolution. (h) Typical intensity profile of a trapped particle (inset
80 × 80 px2) averaged from 20 radial profiles over one quadrant. (i) χ2 difference
LUT comparison for the shown radial profile, with the quadratic fit shown in red. FIG. 1. Schematic of the experimental setup showing relative positions of glass
nanopore, micromanipulator, and salt bath. An illustration of the movement of a
bead resulting from electroosmotic flow due to an applied voltage is shown in the
blue dashed box. The force on the bead can have components in the x, y, and z
directions depending on the position of the pore relative to the bead. making the changing of solution or addition of analytes possible
and also emulates electrophysiology experimental setups. Thus, this
platform offers an opportunity to study flows in electrophysiology
experiments with glass nanopores, such as the concentration gradi-
ent driven outflows believed to prevent nanopore clogging during
in vivo experiments.8,10 24 October 2024 04:47:11 Figure 1 shows a schematic of the setup used to measure
nanopore flow fields in 3D. III. PARTICLE TRACKING Nanopores have a nominal diameter
of 160 nm and are produced using a laser pipette puller (P2000/F,
Sutter Instruments, USA) from quartz glass capillaries. Nanopores
are assembled into an HL-U pipette holder (Molecular Devices,
USA). The holder is connected to a patch-clamp amplifier (Axon
Axopatch 200B, Molecular Devices, USA) via the head stage. The
head stage is itself mounted on a micromanipulator (Patchstar, Sci-
entifica, UK). The micromanipulator enables the positioning of the
nanopore in 3D space, with a resolution of 20 nm and a travel of
20 mm in x, y, and z. The head stage and nanopore are enclosed
by a Faraday cage during experiments to minimize both electrical
noise and mechanical noise from airflow. The amplifier outputs to a
control personal computer using a PCIe-6251 data acquisition card
(National Instruments, USA), capable of 1.25 × 106 samples per
second. This allows the amplifier to be controlled by and recorded
from using a custom written LabVIEW program (LabVIEW 2016,
National Instruments, USA). The micromanipulator is managed
with the same program, enabling the scanning of the nanopipette
through a range of x, y, and z positions automatically throughout an
experiment. The optical tweezers consists of a 1064 nm laser focused
through a 60× water-immersion objective with 1.2 NA in an inverted
microscope configuration. Further details of the optics can be found
in Ref. 11. FIG. 2. 3D particle tracking. Both in-plane (x, y) tracking and out-of-plane (z)
tracking require identification of the particle center (a)–(d), while (e)–(g) are specific
to z tracking. (a) A frame is cropped to a 20 × 20 px2 (x, y) or 80 × 80 px2 (z)
sub-array approximately centered on the trapped bead. (b) Intensity profiles in x
and y are obtained by averaging over 4 × 20 px2 rectangular bins, producing I(x)
and I(y). (c) The cross-correlation between each intensity profile and its reverse,
I⋆(x), I⋆(y), is found. (d) A quadratic fit to the cross-correlation determines the
particle center (x, y position) with sub-pixel accuracy. To obtain the z coordinate:
(e) The 80 × 80 px2 frame is quartered, and a radial intensity profile is calculated
per quadrant. (f) The four profiles are averaged, and this mean profile is compared
to each entry of a lookup table (LUT). II. EXPERIMENTAL BACKGROUND Previous studies considered flows through nanopores in the x,
y trapping plane of the optical tweezers by using in-plane tracking
of trapped particles to measure flow forces in the x, y plane. Full
characterization of flow profiles also requires mapping of the out-of-
plane z component of the flows, orthogonal to the tracking plane. As
such, here, we outline an extension of the flow mapping technique
to flow forces in 3D. l
To achieve this, glass capillary nanopores were mounted on
a micromanipulator at an angle to the trapping plane, enabling
straightforward positioning of the pore relative to a trapped bead
(Fig. 1). This arrangement permits automated mapping of flow
forces in the 3D space around the nanopore. Mounting the nanopore
on a micromanipulator allows for open bath nanopore experiments, Optical tweezers, with sensitivity to forces on the order of 1 pN,
can be used to measure the forces of these nanoscale fluid flows
and map flow fields,3 and have previously revealed salt dependent
flow behaviors around glass capillary nanopores.3,9 The graphic in
the blue dashed box below the schematic in Fig. 1 illustrates the Rev. Sci. Instrum. 93, 054106 (2022); doi: 10.1063/5.0083054
© Author(s) 2022 93, 054106-1 Review of
Scientific Instruments ARTICLE scitation.org/journal/rsi FIG. 1. Schematic of the experimental setup showing relative positions of glass
nanopore, micromanipulator, and salt bath. An illustration of the movement of a
bead resulting from electroosmotic flow due to an applied voltage is shown in the
blue dashed box. The force on the bead can have components in the x, y, and z
directions depending on the position of the pore relative to the bead. (x, y) and out-of-plane (z) tracking are performed separately with x,
y tracking performed live and z tracking performed post-acquisition. The x, y tracking of a trapped particle is performed using a CMOS
camera (MC1362, Mikrotron, Germany). The CMOS camera is
interfaced to the primary computer using a PCIe-1433 frame grabber
with a full configuration (two cables) Camera Link (National Instru-
ments, USA). To achieve adequate sampling for particle tracking, the
camera records at 1000 fps. Images are taken over a region of interest
(ROI) of 128 × 100 px2 centered on the optical trap. IV. RESULTS Combining 3D tracking with a micromanipulator mounted
nanopore, we recorded the flow fields generated by EOF in a vol-
ume outside a glass nanopore. As mentioned earlier, this recording
procedure is automated; at each position in the volume, a negative
voltage is applied, followed by a positive voltage, while the posi-
tion of the bead is recorded. After this, the micromanipulator moves
the nanopore to a new position in the volume and the procedure
is repeated. Figure 3 shows a typical map generated from a 160 nm
pore in 10 mM NaCl solution (2D views of the regions closest to the
nanopore tip are shown in Fig. S2). In Fig. 3(a), at −1 V, a stronger
central flow can be seen with the weaker flow in the spaces above
and below. In contrast, in Fig. 3(b), at +1 V, a more uniform flow
field is observed. That the two flow fields are not mirror images of
each other corroborates the inner/outer flow model of conical glass
nanopore EOF developed by Laohakunakorn et al.,3 whereby the
bulk flow is the result of the interplay of opposing flows through the
nanopore and along the outer walls. The vertical component of indi-
vidual flow forces is strongest and, thus, most visible close to the tip. Though the glass nanopore was inserted into the salt bath at an angle
of ∼15○, the flow forces are not directed at this angle, demonstrating
the non-ideal geometric reality at the tip of glass nanopores. To track the trapped particle’s movement out-of-plane, the par-
ticle is simultaneously imaged using a CCD camera (DMK31AF03,
Imaging Source, Germany) at 30 fps and 1024 × 768 px2 with syn-
chronized recording to the CMOS controlled with LabVIEW. The
routine is based on the Quadrant Interpolation algorithm developed
by van Loenhout et al.12 and is again illustrated in Fig. 2. Center
finding [Figs. 2(a)–2(d)] follows the same method as x, y tracking
with some differences arising from the lower bit depth of the CCD
camera noted here. In particular, the median intensity of the edges
of the ROI in each dimension is used for initial background subtrac-
tion to improve the signal-to-noise ratio. The pixel intensity in each
dimension is then summed and divided by the total number of pix-
els to estimate the particle center. III. PARTICLE TRACKING (g) χ2 difference is used to compare the
profile to the LUT, and a quadratic fit around the minimum of the χ2 difference
enables sub-LUT resolution. (h) Typical intensity profile of a trapped particle (inset
80 × 80 px2) averaged from 20 radial profiles over one quadrant. (i) χ2 difference
LUT comparison for the shown radial profile, with the quadratic fit shown in red. Forces are measured through video-based tracking of the
trapped particles. To track particle motion in 3D, both in-plane Rev. Sci. Instrum. 93, 054106 (2022); doi: 10.1063/5.0083054
© Author(s) 2022 93, 054106-2 93, 054106-2 Review of
Scientific Instruments Review of
Scientific Instruments ARTICLE scitation.org/journal/rsi scitation.org/journal/rsi sub-array of the ROI centered on the bead (20 × 20 px2 in size) is
defined [Fig. 2(a)]. Mean intensity profiles in x and y, I(x) and I(y),
are obtained by averaging over 4 × 20 px2 rectangular bins centered
to the sub-array in x and y, respectively [Fig. 2(b)]. These profiles
are reversed to create I⋆(x) and I⋆(y), and the cross-correlation
between each profile and its reverse is performed [Fig. 2(c)], e.g.,
I(x)☀I⋆(x). A quadratic fit to the seven central points of the cross-
correlation is used to determine the center position with sub-pixel
accuracy [Fig. 2(d)]. 20 profiles from one quadrant is shown in Fig. 2(h). The χ2 compari-
son of this profile to the LUT is shown in Fig. 2(i) with the quadratic
fit overlaid. We determined the axial force constant as a function of
laser power and in the process also determined the Brownian motion
limited resolution of our z-tracking implementation to be 20 nm
(supplementary material, Sec. I, Fig. S1). IV. RESULTS The ROI is cropped to a sub-array
of 80 × 80 px2 centered on that estimate and the cross correlation is
computed and fit with a quadratic as before to determine the center
with sub-pixel accuracy. Using this more accurate estimate of the center, the cropped
image is quartered and a radial coordinate system is imposed on
each quadrant. A radial intensity profile is calculated per quadrant,
I(r) [Fig. 2(e)], and the four I(r) are averaged together to obtain
a mean radial intensity profile for that frame. The mean profile is
compared to a series of intensity profiles in a lookup table (LUT),
where each profile corresponds to a specific z-position [Fig. 2(f)]. LUT profiles are created by applying the technique described so far
[Figs. 2(a)–2(e)] to a particle fixed on a coverslip. A piezo-stage is
used to move in controlled z steps with the step size dictating the
resolution of the LUT. By fitting a quadratic about the minimum of
the χ2 difference between the measured profile and LUT profiles, the
z position of the trapped bead is determined with sub-LUT accuracy
[Fig. 2(g)]. A typical mean intensity profile generated by averaging 24 October 2024 04:47:11 V. DISCUSSION AND CONCLUSIONS In this work, we have demonstrated how an optical tweezers
equipped for 2D particle tracking can be adapted to perform 3D
tracking, with two cameras for x, y, and z tracking, respectively. By
combining this with a micromanipulator mounted nanopore, we
have developed an automated 3D flow mapping platform. Measur-
ing flow forces at each point in 3D provides a means to observe the FIG. 3. 3D vector plots of the voltage induced flow fields in the vicinity of a nanopore in 10 mM NaCl solution. (a) Flow in the volume outside a nanopore when −1 V
is applied. (b) Flow in the same volume when +1 V is applied. Axes are labeled relative to the nanopore; axial is parallel to the long axis of the capillary, transverse is
perpendicular to that and the vertical axis is orthogonal to the transverse–axial (x, y) plane. Arrow length corresponds to the magnitude of the force experienced by the
trapped bead as a result of the flow at a given position. FIG. 3. 3D vector plots of the voltage induced flow fields in the vicinity of a nanopore in 10 mM NaCl solution. (a) Flow in the volume outside a nanopore when −1 V
is applied. (b) Flow in the same volume when +1 V is applied. Axes are labeled relative to the nanopore; axial is parallel to the long axis of the capillary, transverse is
perpendicular to that and the vertical axis is orthogonal to the transverse–axial (x, y) plane. Arrow length corresponds to the magnitude of the force experienced by the
trapped bead as a result of the flow at a given position. Rev. Sci. Instrum. 93, 054106 (2022); doi: 10.1063/5.0083054
© Author(s) 2022 93, 054106-3 Review of
Scientific Instruments ARTICLE scitation.org/journal/rsi U.F.K. was supported by the ERC Consolidator Grant (Designer-
Pores No. 647144). U.F.K. was supported by the ERC Consolidator Grant (Designer-
Pores No. 647144). true geometry of a glass nanopore aperture; we believe this offers a
route to quantify their geometry more precisely. The use of a 30 fps
camera makes our implementation of z-tracking inexpensive and
accessible. While the low sampling rate precludes the study of out-
of-plane forces using variance and power spectrum based analyses,
a higher frame-rate camera could be introduced if needed. As the
off-line implementation of z-tracking is hardware independent, it is
readily portable to other experiments needing 3D particle tracking. SUPPLEMENTARY MATERIAL 24 October 2024 04:47:11 7G. Huang, K. Willems, M. Soskine, C. Wloka, and G. Maglia, Nat. Commun. 8,
935 (2017). See the supplementary material for the measurement of axial
trap stiffness and z resolution along with presenting single plane flow
maps derived from the 3D volume shown in Fig. 3. 8J. Rabinowitz, M. A. Edwards, E. Whittier, K. Jayant, and K. L. Shepard, J. Phys. Chem. A 123, 8285 (2019). 9J. Mc Hugh, K. Andresen, and U. F. Keyser, Appl. Phys. Lett. 115, 113702 (2019). 10 t, M. Wenzel, Y. Bando, J. P. Hamm, N. Mandriota, J. H. Rabinow 10K. Jayant, M. Wenzel, Y. Bando, J. P. Hamm, N. Mandriota, J. H. Rabinowitz,
I. J.-L. Plante, J. S. Owen, O. Sahin, K. L. Shepard, and R. Yuste, Cell Rep. 26, 266
(2019) 10K. Jayant, M. Wenzel, Y. Bando, J. P. Hamm, N. Mandriota, J. H. Rabinowitz,
I. J.-L. Plante, J. S. Owen, O. Sahin, K. L. Shepard, and R. Yuste, Cell Rep. 26, 266
(2019). REFERENCES 1J. Mathé, H. Visram, V. Viasnoff, Y. Rabin, and A. Meller, Biophys. J. 87, 3205
(2004). 2U. F. Keyser, B. N. Koeleman, S. van Dorp, D. Krapf, R. M. M. Smeets, S. G. Lemay, N. H. Dekker, and C. Dekker, Nat. Phys. 2, 473 (2006). 3N. Laohakunakorn, V. V. Thacker, M. Muthukumar, and U. F. Keyser, Nano Lett. 15, 695 (2015). 4S. van Dorp, U. F. Keyser, N. H. Dekker, C. Dekker, and S. G. Lemay, Nat. Phys. 5, 347 (2009). 5S. Ghosal, J. D. Sherwood, and H.-C. Chang, Biomicrofluidics 13, 011301 (2019). 6R B S h
h J H
d P R
d R
M d Ph
80 839 (2008) 5S. Ghosal, J. D. Sherwood, and H.-C. Chang, Biomicrofluidics 13, 011301 (2019). 6R. B. Schoch, J. Han, and P. Renaud, Rev. Mod. Phys. 80, 839 (2008). DATA AVAILABILITY The data that support the findings of this study are available
from the corresponding authors upon reasonable request. The 3D
tracking code used in this work is available at https://github.com/
AndresenLab/otnp_3Dtrak. V. DISCUSSION AND CONCLUSIONS We note that in the event a particle being tracked is larger or smaller
than the particle that was used to generate the LUT, the particle’s
trapped Brownian motion will be centered around a z-position off-
set from the LUT center, providing a means of detecting this size
difference. Although changes in position will still be detected, this
may result in inaccuracies in measuring the magnitude of changes
in the z-position of different sized particles. To minimize the risk of
this occurring, care was taken to use only particles that were of the
same size as that used to generate the LUT, and no size differences
were observed. A more robust solution that can be implemented in a
future iteration of this tracking method would be to generate a series
of LUTs from a selection of particles that cover the range of expected
size variations, then compare the center of the trapped Brownian
motion of the particle to the central radial intensity profile of each
LUT to determine which is the best match and, thus, most accurately
maps to that particle size. Rev. Sci. Instrum. 93, 054106 (2022); doi: 10.1063/5.0083054
© Author(s) 2022 ACKNOWLEDGMENTS I. J.-L. Plante, J. S. Owen, O. Sahin, K. L. Shepard, and R. Yuste, Cell Rep. 26, 266
(2019). J.M.H. thanks Michael I. Walker for his assistance with pro-
gramming the micromanipulator and acknowledges funding from
AFOSR (Grant No. FA9550-17-1-0118). A.L.T. acknowledges sup-
port from the University of Cambridge Ernest Oppenheimer Fund. 11O. Otto, F. Czerwinski, J. L. Gornall, G. Stober, L. B. Oddershede, R. Seidel, and 11O. Otto, F. Czerwinski, J. L. Gornall, G. Stober, L. B. Oddershede, R. Seidel, and
U. F. Keyser, Opt. Express 18, 22722 (2010). U. F. Keyser, Opt. Express 18, 22722 (2010). 12 12M. T. van Loenhout, J. W. J. Kerssemakers, I. De Vlaminck, and C. Dekker,
Biophys. J. 102, 2362 (2012). Rev. Sci. Instrum. 93, 054106 (2022); doi: 10.1063/5.0083054
© Author(s) 2022 Rev. Sci. Instrum. 93, 054106 (2022); doi: 10.1063/5.0083054
© Author(s) 2022 Rev. Sci. Instrum. 93, 054106 (2022); doi: 10.1063/5.0083054
© Author(s) 2022 93, 054106-4 93, 054106-4
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English
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Pediatric Cardiac Intensive Care Society Statement: caring for children with critical cardiac disease across the globe
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Cardiology in the young
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cc-by
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Pediatric Cardiac Intensive Care Society Statement: caring for
children with critical cardiac disease across the globe Katherine L. Brown,1,2 Dorothy M. Beke,3 David S. Cooper,4 Graeme MacLaren5,6 1Charles West Division, Great Ormond Street Hospital; 2Institute of Cardiovascular Science, University College London,
London, United Kingdom; 3Cardiac Intensive Care Unit, Department of Nursing, Boston Children’s Hospital, Boston,
Massachusetts; 4Cardiac Intensive Care Unit, The Heart Institute, Cincinnati Children’s Hospital Medical Center,
Cincinnati, Ohio, United States of America; 5Paediatric Intensive Care Unit, Department of Paediatrics, The Royal
Children’s Hospital, University of Melbourne, Melbourne, Victoria, Australia; 6Cardiothoracic Intensive Care Unit,
National University Health System, Singapore, Singapore Received: 15 September 2017; Accepted: 15 September 2017 published in an open access format in order to
enhance the speed and efficiency with which the
information presented is shared. W
C
S
i W
E
ARE
BOTH
DELIGHTED
AND
PROUD
TO
introduce this ‘Global Statement’ spon-
sored by the Pediatric Cardiac Intensive
Care Society (PCICS), which contains nine articles
dedicated to a range of important topics related to
caring for children with critical cardiac disease
around the globe. This Global Statement was
developed on behalf of PCICS with the following
issues in mind: Second, paediatric cardiac disease is seen the world
over,1 and while paediatric cardiac critical care clin-
icians face common challenges no matter where they
practice – we all deal with patients who have infections,
late diagnoses of congenital heart disease, or failure to
thrive at one time or another – nonetheless, the pre-
valence and severity of individual specific clinical pro-
blems within this spectrum vary by geographical
location. For example, although all of us deal on a
regular basis with management of postoperative low
cardiac output syndrome, those practitioners based in
low- and middle-income settings have the greatest
experience in management of late repair of tetralogy of
Fallot in deeply cyanotic patients, or cardiac repairs in
severely malnourished patients and those affected by
tuberculosis. Doctors and nurses based in low- and
middle-income settings encounter a greater range and
severity of clinical scenarios within these categories, and
hence they have evolved skills and expertise in dealing
with these in the best possible way to ensure optimal
outcomes among their patients. Within this PCICS
Global Statement, state-of-the-art material on all of
these topics is presented directly by experts in the field
based in –amongst other places – Asia, Africa, and
South America.2–4 First, the world is getting smaller. Correspondence to: K. L. Brown, Charles West Division, Great Ormond Street
Hospital, London, WC1N 3JH, United Kingdom. Tel: +44 207 813 8180;
Fax: 44 207 829 8673; E-mail: Katherine.brown@gosh.nhs.uk Cardiology in the Young (2017), 27(Suppl. 6), S1–S2
© Cambridge University Press, 2017. This is an Open Access article,
distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
doi:10.1017/S1047951117002517 References 1. Musa NL, Hjortdal V, Zheleva B, et al. The global burden of
paediatric heart disease. Cardiol Young 2017; 27 (Suppl 6): S3–S8. 1. Musa NL, Hjortdal V, Zheleva B, et al. The global burden of
paediatric heart disease. Cardiol Young 2017; 27 (Suppl 6): S3–S8. 2. Murni IK, MacLaren G, Morrow D, Iyer P, Duke T. Perioperative
infections in children with congenital heart disease. Cardiol Young
2017; 27 (Suppl 6): S14–S21. 2. Murni IK, MacLaren G, Morrow D, Iyer P, Duke T. Perioperative
infections in children with congenital heart disease. Cardiol Young
2017; 27 (Suppl 6): S14–S21. 3. Argent AC, Balachandran R, Vaidyanathan B, Khan A, Krishna
Kumar R. Management of undernutrition and failure to thrive in
children with congenital heart disease in low and middle income
countries. Cardiol Young 2017; 27 (Suppl 6): S22–S30. 4. Iyer PU, Moreno GE, Fernando Caneo L, Faiz T, Shekerdemian LS,
Iyer KS. Management of late presentation congenital heart disease. Cardiol Young 2017; 27 (Suppl 6): S31–S39. 5. Bronicki RA, Pollack U, Argent AC, et al. Global perspective on
training and staffing for paediatric cardiac critical care. Cardiol
Young 2017; 27 (Suppl 6): S9–S13. 5. Bronicki RA, Pollack U, Argent AC, et al. Global perspective on
training and staffing for paediatric cardiac critical care. Cardiol
Young 2017; 27 (Suppl 6): S9–S13. Finally, a range of mechanisms have evolved to
measure, monitor, and ultimately improve patient
outcomes, this being a general principle that has very
wide support within the PCICS organisation. As
discussed in the article by Hickey et al,9 these include
the development of multi-institutional clinical data-
bases to which centres can add patient data in order
to compare their outcomes with those of others, the
use of common and consistent methodologies to
adjust for the severity of case mix, the identification of
quality metrics to benchmark individual centre 6. Bastero P, Staveski SL, Zheleva B, et al. Partnership models for
the establishment of sustainable paediatric cardiac surgical and
cardiac intensive care programs in low and middle income
countries. Cardiol Young 2017; 27 (Suppl 6): S55–S60. 7. Molloy FJ, Nguyen N, Mize M, et al. Medical missions for the
provision of paediatric cardiac surgery in low and middle income
countries. Cardiol Young 2017; 27 (Suppl 6): S47–S54. 8. Ramnarayam P, Intikhab Z, Spenceley N, Iliopoloulos I, Duff A,
Millar J. Inter-hospital transport of the child with critical cardiac
disease. Acknowledgements The authors thank the PCICS president Sandra
Staveski and the editor in chief of Cardiology in the
Young Jeff Jacobs for their tireless support for the
Global Statement. Fourth, it is important to remember that variations
in management for critically ill cardiac patients have
been developed to best fit the needs applicable across
a great range of settings, and that there is “more than
one way to peel an orange”. Human beings are
resourceful and imaginative, as is illustrated by the
solutions found by health professionals working
together to meet the inherent challenges that arise
in order to deliver the best care. Truly excellent
paediatric cardiac programmes have evolved over
time in all continents of the globe, finding their own
pathways to deliver outstanding care for children
with heart disease via a range of means. In addition,
paediatric cardiac care has been supported to develop
excellence by fostering inter-institutional partner-
ships, the examples of which – Jamaica, Mexico,
India, and Viet Nam – are discussed in the Global
Statement by Bastero et al6; with the input of “mis-
sions” or visiting paediatric cardiac surgical trips, a
detailed account of which is provided by Molloy
et al7; and in finding creative ways to care for criti-
cally ill children during transportation from one
place to another, as discussed by Ramnarayam et al.8 Pediatric Cardiac Intensive Care Society Statement: caring for
children with critical cardiac disease across the globe It is very unu-
sual for a major event in one part of the world to
remain unknown and unheard of on the other side of
the globe. Similarly, we can all read and learn from
our distant colleagues, as well as our nearest neigh-
bours, by logging onto a computer and checking for
the latest updates online. Just as this holds true for
new developments, it also applies to long established
lessons, especially if they convey something particu-
larly important or insightful. However, despite the
ubiquitous nature of news media and the internet,
there exist significant and important barriers to the
seamless sharing of knowledge with colleagues
around the world such as the cost of access to scien-
tific journals and travel to academic meetings, as well
as the challenges of finding time among the demands
of busy clinical jobs to undertake these activities. Consequently, this PCICS Global Statement is Third, all paediatric cardiac programmes have a
common goal of treating patients to the highest standard
and delivering optimal outcomes while working with https://doi.org/10.1017/S1047951117002517 Published online by Cambridge University Press Cardiology in the Young: Volume 27 Supplement 6 2017 S2 available resources, and this depends on the availability
of a suitably skilled workforce. PCICS as an organisation
holds a fierce commitment to education, training, and
guidance as to best practice. This was a motivation in
their commissioning of this Global Statement. In the
Global Statement, Bronicki et al5 present a range of
experiences and approaches to the training of doctors
and nurses around the world in the care of critically ill
children with heart disease. Those health professionals
reporting from low- and middle-income settings cited
the challenges created by a paucity of large training
centres and the material impact of a lack of personnel
and resources upon their training. Clinicians reporting
from higher-income settings celebrated the value of
experience gained through centralisation to form large,
well-resourced training centres and the adherence to
common standards across professional groups. performance, and the fostering of a commitment to
using improvement science methodologies. With widely varying resources, training, and
personnel, paediatric cardiac critical care clinicians
work towards one common goal: to alleviate the
suffering of, and provide the best possible outcomes
for, children with both congenital and acquired heart
disease. This Global Statement should be the first
of many initiatives that the PCICS will commission
to help them along their way. Financial Support This research received no specific grant from any
funding agency, commercial or not-for-profit sectors. Conflicts of Interest None. References Cardiol Young 2017; 27 (Suppl 6): S40–S46. 9. Hickey PA, Connor JA, Cherian KM, et al. International quality
improvement initiatives. Cardiol Young 2017; 27 (Suppl 6): S61–S68. https://doi.org/10.1017/S1047951117002517 Published online by Cambridge University Press
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German
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Die Behandlung der Schlaflosigkeit
|
Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift
| 1,912
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public-domain
| 3,321
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Die Behandlung der Schlaflosigkeit.
Klinischer Vortrag.
Von E. Meyer in Kinigsberg i. Pr. M. H. ! Vor eilliger Zeit wurde ich zur Beratung bei einem
7 jährigen Knaben zugezogen, dessen Krankengeschichte kurz
folgende war: Einziges Kind. Vater an Paralyse gestorben, Mutter nervös, mehr-
fach Fälle von Tuberkulose in der Familie. Immer schwächlich, ]eieht
eriegbar und ängstlich. von guter Intelligenz. Vor kurzem Masern
ohne besondere Komplikation, in der Zeit danach abends sehr ängstlich,
4ehreit, cinc böse Fee komme, auch nachmittags, wenn es, wie bisher,
schlafen soll. Will nicht mehr allein schlafen. Diese wenigen Fälle ausgenommen, pflegt die
Schlaf-
losigkeit stets eine Folge anderer Störungen zu sein oder als
ein S y m p t o m mit anderen sich zu einem Symptornenkomplex
oder Krankheitsbild zu vereinen. In diesen bekannten Fest-
stellung liegt ja der Schlüssel zur Bekämpfung der Schlaf-
losigkeit: die Erforschung und Beseitigung der Grund-
krankheit, deren Folge sie ist oder deren Symptom sie neben
anderen Erscheinungen bildet. Tn der Behandlung nimmt
sie abei deshalb eine gewisse Sonderstellung ein, weil sie oft
besonders in den Vordergrund tritt und jedenfalls die Be-
schwerde ist, unter der die körperlichen wie die geistigen
Funktionen am meisten zu leiden haben. Es ist der Typus eines nervösen Kindes von ausgesprochen
psychopathischer Konstitution, von dem wir hier hören ; der
Gegenstand steter Sorge der Angehörigen wie des Hausarztes. I n der Rekonvaleszenz von einer körperlichen Erkrankung ist
eine Steigerung der Nervosität eingetreten, die sich vor allem
in Beängstigungen zur Zeit des Einschlafens äußert. Wi hi
fi d
i b i Ki d
d E
h Wie hier, so finden wir bei Kindern und Erwachsenen
als Ausdruck nervöser Störung, angeborener oder erworbener
Art, ganz ungemein häufig Beeinträchtigungen des Schlafes,
oft als eine der Hauptbeschwerden, ja als die Beschwerde,
die den Kranken zum Arzte führt. Es verlohnt sich daher
wohl, bei der Schlaflosigkeit und ihrer Behandlung einmal
länger zu verweilen, auch deshalb, weil gegenüber der Legion
neuer Schlafmittel, deren Anpreisungen den Arzt tagtäglich
verwirrend überschwemmen, Sammlung und Umschau von Zeit
zu Zeit geboten ist. Bei nervösen Kindern ist Störung des Schlafes, vor alleni
Erschwerung des Einschlafens oder unruhige Unterbrechungen
des Schlafes, nichts Seltenes. In leichteren Fällen, wo es sich
um sensitive Kinder mit abnormer Erregbarkeit und labilern
Affekt handelt, finden wir nach jedem, oft auch wenig erheb-
lichen,
ungewohnten Ereignis, so Besuch bei Bekannten,
Kindergesellschaft, Geburtstagsfest u. dgl., abends eine unge-
wöhnliche Lebhaftigkeit und Unruhe, sodaß die Kinder nicht
zur Ruhe kommen können. Die Behandlung der Schlaflosigkeit.
Klinischer Vortrag.
Von E. Meyer in Kinigsberg i. Pr. Derartige Schlafstörungen finden
wir nicht selten nur in gewissen Zeiten körperlicher und psy-
chischer Angegniffenheit bei solchen leicht nervösen Kindern
auftreten. Man darf auch nicht vergessen, daLI Uebermüdung
und stärkere seelische Erregung auch bei sonst anscheinend
normalen Kindern das Einschlafen erschweren. h i i
li
di
i
i
ll
i d
i Wenn wir von Schlaflosigkeit
sprechen,
so
geschieht
das nur cum grano salis, insofern vollständiger Schlafmangel
über mehr als wenige Tage eigentlich nur bei Erregungs- und
Angstzuständen schwerer Psychosen sich findet, sonst handelt
es sich durchweg nur urn Verminderung des Schlafes in ver-
schiedenem Grade. So sind auch die Klagen unserer Patienten
über totálen Schlafmangel seit Wochen aufzufassen. Trotz
aller gegenteiligen Versicherungen liegt zumeist nur eine Be-
einträchtigung des Schlafes in Dauer und Wirkung vor; ins-
besondere in der Weise, daß morgens das Gefühl des Erfrischt-
seins fehlt und Ermüdung, eingenommener Kopf etc. eine
vollkommen durchwachte Nacht vortäuschen. Schwieriger liegen die Dinge in Fällen, wie dem eingangs
mitgeteilten, mit stark nervöser Konstitution, die sich
auch in nicht geringen anderweitigen nervös-psychischen Er-
scheinungen kundtut, ganz besonders, wenn noch andere Um-
stände hinzukommen, die, wie körperliche Schwächung, see-
lische Erschütterung,
geeignet sind,
das labile psychische
Gleichegwicht zu erschüttern. Es kommt bi solchen Kindern
zu Angstzuständen, die sich an eine besonders stark affekt-
betonte Vorstellung, zum Teil auf Halluzinationen oder Illu-
sionen beruhend, anschließen und das Einschlafen hindern. in anderen Fällen beobachten wir nach anfänglich ruhigem Schlaf
schreckhaftes Auffahren mit länger anhaltender ängstlicl1er
Erregung, oder Träume, die in starker motorischen Aktion
zum Ausdruck kommen und von einem Gefühl morgendlicher
Ermüdung gefolgt sind. Die Behandlung soll vor allem
die allgemeine nervöse Erregbarkeit verringern durch Kräf-
tigung des Nervensystems und Hygiene der gesamten Lebens-
führung, wozu als sehr wichìtiger Faktor hinzukommen muß
psychische Beeinflussung durch Eltern und Hausarzt, mit
ruhigem Eingehen und Verständnis, ebenso fern von Härte Bevor wir nun die Schlafstörungen näher ins Auge fassen,
müssen wir einen Augenblick bei dem ,,normalen" Schlaf
verweilen, weil wir ja danach unsere Fragen audi bei den Klagen
über Schlaflosigkeit einrichten müssen. Man pflegt zu unter-
scheiden einen
,,abendlichen"
und einen
, .morgendlichen"
Schlaftypus, je nachdem der tiefste Schlaf in die erste oder
zweite Hälfte der Schlafzeit fällt. Die erstere Schiafart scheint
bei gesunden Kindern die Regel, während die zweite bei Er-
wachsenen häufiger anzutreffen ist. BERLIN, DEN 12. SEPTEMBER 1912 38. JFIHRORNG. Nr. 37 Isolierte einfache Schlafstörung ohne sonstige krank-
hafte Erscheinungen zu gleicher Zeit kommt kaum vor. Man
findet sie am ehesten als letzte Folge überstandener körper-
licher Erkrankungen, insbesondere längere Zeit noch nach
einer Influenza. Auch babe ich sie in einzelnen Fällen peno-
disch auftreten sehen, wie eine Art Aequivalent periodischer
psychotischer Erscheinungen . Gerade diese
letztgenannte
Form ist einer Behandlung sehr schwer zugänglich. VERLAG: GEORG THIEME
LEI P116.RABENSTEINPLATZ Z VERLAG: GEORG THIEME
LEI P116.RABENSTEINPLATZ Z Die Behandlung der Schlaflosigkeit.
Klinischer Vortrag.
Von E. Meyer in Kinigsberg i. Pr. Eine scharfe Trennung
ist aber nicht angängig, es gibt sehr vielfache Uebergänge,
Gewohnheit und Lebensverhältnisse führen zu mannigfachen
Verschiebungen, auch kommen bei denselben Individuen die-
selben Typen vor. 216 DEUTSCHE MEDIZINISCHE WOCHENSCHRIFT. 1722 Nr. 37 Nr. 37 und Erregbarkeit vor allem Schlafstörungen in verschiedener
Form, oft als einfache Schlaflosigkeit, sich finden. und Zorn wie von übertriebener Milde. Nötigenfalls ist ein
Erfolg der Behandlung nur durch Entfernung aus der Familie
zu erreichen. Eigentliche Schlafmittel wird man höchstens
in den extremsten Fällen einmal benutzen. D ß
b i Ki d
h
B
i
h h i
d und Erregbarkeit vor allem Schlafstörungen in verschiedener
Form, oft als einfache Schlaflosigkeit, sich finden. und Erregbarkeit vor allem Schlafstörungen in verschiedener
Form, oft als einfache Schlaflosigkeit, sich finden. g
Auch der nicht seltenen Schlaflosigkeit im Klimakterium,
mit Unruhe, Beklemmung, Wallungen etc. sei hier noch gedacht. Daß man bei Kindern auch an Beeintthchtigung des
Schlafes durch körperliche Störungen, so z. B. durch Oxyuris,
denken muß, ist bekannt. Die Therapie der Schlaflosigkeit muß gerade bei der letzt-
genannten Form mit den einfachsten Dingen beginnen, i m me r
mitderVoraussetzung, daß in ersterLinie stets das Grund-
le id e n zu bekämpfen ist und die Schlaflosigkeit nur infolge
ihrer besonders unangenehmen und quälenden Eigenart Grund
zu besonderen Maßnahmen gibt. Die Therapie der Schlaflosigkeit muß gerade bei der letzt-
genannten Form mit den einfachsten Dingen beginnen, i m me r
mitderVoraussetzung, daß in ersterLinie stets das Grund-
le id e n zu bekämpfen ist und die Schlaflosigkeit nur infolge
ihrer besonders unangenehmen und quälenden Eigenart Grund
zu besonderen Maßnahmen gibt. Die Schlaflosigkeit der Erwachsenen kann auch
durch körperliche Störungen sekundär veranlaßt sein. Herz-
und Lungenleiden mit ihren Begleiterscheinungen, nicht selten
auch Hautaffektionen, geben besonders den Anlaß dazu. Ferner kommen hier eine Reihe sohmerzhafter Nerven-
krankheiten in Betracht, vor allem Neuralgien, Neuritiden
und Tabes, weiter u. a. Lues cerebrospinalis mit ihren gerade
nachts exazerbierenden Kopfschmerzen und Tumor cerebri. In allen diesen Fällen ist die Schlaflosigkeit kein Symptom,
sondern nur eine Folgeerscheinung ; sie ist es aber zuweilen,
die den Kranken zuerst zum Arzte und so zur Erkenntnis
des Leidens führt. Die Hygiene des Schlafes", insbesondere des Schlaf-
zimmers, darf mit allen ihren anscheinenden Kleinigkeiten
nicht gering geachtet werden. Die Behandlung der Schlaflosigkeit.
Klinischer Vortrag.
Von E. Meyer in Kinigsberg i. Pr. Sie bildet eine Hauptklage während der Ent-
ziehungskur, auch dann, wenn andere Beschwerden wenig
hervortreten, und sie kann noch längere Zeit nach Abschluß
der Entziehung in hartnäckiger Weise fortbestehen. Daß die tiefsten und am längsten anhaltenden Schlafstö-
rungen bei ausgesprochen psychotischen Zuständen verschie-
denster Art und Aetiologie auftreten, ist bekannt und bedarf
keines näheren Eingehens. Ich mache Sie auch darauf aufmerksam, daß die Elektro-
th e r ap ie in gewisser Form den Schlaf befördert. W
di
dik
Mi
l
h
i d Im Verlauf des Morphinismus beobachten wir Schlaf-
losigkeit oft. Sie bildet eine Hauptklage während der Ent-
ziehungskur, auch dann, wenn andere Beschwerden wenig
hervortreten, und sie kann noch längere Zeit nach Abschluß
der Entziehung in hartnäckiger Weise fortbestehen. D ß di
i f
d
l
h l
d
S hl f Was die medikamentösen Mittel angeht, so wird man
auch hier zuerst zu den einfachsten und harmiosesten greifen,
die als Schlafmittel nicht eigentlich gelten können. Das sind
die Baldrianpräparate in ihrer verschiedenen Form, die
häufig schon die Schlafstörungen vermindern. Ferner tun
in manchen Fällen Aspirin (0,5), Pyramidon (0,2-0,3)
gut, wie die Patienten zuweilen empirisch an sich erprobt
haben. Daß die tiefsten und am längsten anhaltenden Schlafstö-
rungen bei ausgesprochen psychotischen Zuständen verschie-
denster Art und Aetiologie auftreten, ist bekannt und bedarf
keines näheren Eingehens. Wie in der täglichen Praxis die funktionellen Nerven-
störungen bei weitem überwiegen, so ist auch die praktisch
wichtigste Form der symptomatischen
Schlaflosigkeit
die,
welche bei den funktione lien Neurosen auftritt. Wir rechnen
zu letzteren ja alles das, was wir als Hysterie, Neurasthenie etc. führen, zum Teil erworbener Art, zum großen Teil erwachsen
auf dem Boden psychopathischer Konstitution. In verschie-
dener Weise tritt da die Schlaflosigkeit in die Erscheinung:
Die Kranken befällt abends eine gewisse Unruhe, sich steigernd
bis zum Angstgefühl, körperliche Sensationen, Juckreiz, Schmer-
zen etc. treten auf, und so kommen sie erst spät und ermattet
zu kurzer Ruhe. Andere Kranke können nicht frei werden
von bestimmten Gedanken, die sich ihnen beinahe zwangs-
artig aufdrängen, gleichgültigen Inhaltes oder auch an wichtige
Entschlüsse der Vergangenheit oder Zukunft anknüpfend. Es ist geradezu ein Sturm von Gedanken, die auftauchen. In
anderen Fällen schlafen die Kranken ein, aber sehr lebhafte
Träume mit motorischen Entladungen lassen sie aufwachen
oder führen zu einer Art Erschöpfung, sodaß die Kranken
morgens nicht frisch sind. 1) Dornblüth, Die Schlaflosigkeit und ihre Behandlung (Leipzig
1912) bringt beherzigenswerte Ausführungen hierüber. Die Behandlung der Schlaflosigkeit.
Klinischer Vortrag.
Von E. Meyer in Kinigsberg i. Pr. Unter genauer Berücksichtigung
der gesamten Persönlichkeit des Kranken kann der Arzt da-
durch zuweilen Wunder verrichten, leichtere nervöse Schlaf-
losigkeit völlig beseitigen.1)
Er wird den Kranken auch auf
allerlei kleine Hiffsmittel, Zählen u. dgl. hinweisen, die alle die
Loslösung von der Gedankenarbeit des Tages, den Ausschluß
äußerer Eindrücke bezwecken. Eine solche innerlich wie äußer-
lich
möglichst
günstige
psychische Konstellation für den
Sch]af zu erzielen, dazu bedarf es nicht selten einer Art Er-
z je u ng , bei jugendlichen, aber auch älteren Nervösen, be-
sonders bei stark psychopathischer Veranlagung. H d
h
i
h
M ß
h Dieses Dokument wurde zum persönlichen Gebrauch heruntergeladen. Vervielfältigung nur mit Zustimmung des Verlages. Anders liegt es bei dem Gros der Fälle von Schlaflosigkeit. Ei
l ä ß
i h i h
i
i
h E k Einmal äußern sich nicht wenige organische Erkran-
kungen des Nervensyste nis u. a. in Schlafmangel, und zwar
ist dabei bedeutungsvoll, daß dieser oft geradezu die initiale
Beschwerde ist. So kommen Paralytiker manches Mal nur
mit der Klage über absolute Schlaflosigkeit zum Arzt, ähnliches
finden wir bei Hirntumoren und Lues cerebri. Beeinträchtigung
des Schlafes, speziell frühes Erwachen, beobachten wir auch
vielfach bei der Arteriosklerose des Gehirns. Hydrotherapeutische
Maßnah men:
Umschläge,
,nasse Strümpfe' ', Einpackungen, partielle Bäder verschiedener
Art, etwas prolongierte, nicht zu warme Vollbäder etc. sind bei
vielen Fällen leichter Schlaflosigkeit, aber auch bei manchen
etwas schwereren Grades günstig. Empfohlen ist die Behandlung
mit Bädern etc. auch gerade bei den klimakteriellen Be-
schwerden, insbesondere bei den Schlafstörungen jener Zeit. Hervorzuheben ist schließlich allgemein, daß gerade hydro-
therapeutische Eingriffe individuell sehr verschieden wirken
und daher sorgfältig ausprobiert werden müssen. i
h l
ill i h hi
d ß di
i
ff Eine zweite Kategorie bilden die Vergiftungen, so der
chronische Alkoholismus, der Nikotinismus, auch die Intoxi-
kationen mit Kaffee und Tee, bei denen ebenfalls die Schlaf-
losigkeit lange Zeit so ausschließlich in den Vordergrund
treten kann, daß sie den Arzt über das eigentliche Leiden täuscht. Häufig sind freilich allgemein nervöse Erscheinungen, auch
Präkordialangst, lJnruhe, Erregung etc. mit der Schlaf losig-
keit vergeselischaftet. g
g
p
Einschalten will ich hier, daß die Hypnose ein vortreff-
liches ,,Schlafmittel" sein kann, doch bedarf sie besonderer
Erfahrung, die dem Praktiker naturgemäß meist fehlt. h
h Si
h d
f
f
k
d ß di
l k g
Im Verlauf des Morphinismus beobachten wir Schlaf-
losigkeit oft. Die Behandlung der Schlaflosigkeit.
Klinischer Vortrag.
Von E. Meyer in Kinigsberg i. Pr. Sicherlich wird dabei das eine oder andere Hypnoticum,
das Ihnen aus eigener Erfahrung oder nach Berichten be-
sonders gut dünkt, fehlen, doch scheinen mir eben clic Haupt-
sache die allgemeinen Gesichtspunkte zu sein. Es scheint auch, worauf Bürgi hingewiesen, der Versuch
geboten, die Dosis nicht auf einmal, sondern in zwei Teilen
etwa, in Abständen von V4 1/2 Stunde zu geben. Die Wirkung
soll dann größer sein. g
Eine ältere, wohl empirisch gewonnene Erfahrung ist es,
daß die Vereinigung zweier Schlafmittel oft besonders
guten Erfolg hat. So hat man schon lange die Kombination
von Chloralhydrat mit Morphium bei starken Erregungen als
sehr wirksam kennen gelernt, vielfach werden Rezepte von Vero-
na! und Codein etc. verordnet, auch macht sich dieses Be-
streben, kombinierte Wirkungen zu erreichen, bei der Dar-
stellung neuer Schlafmittel geltend. Diese praktisch wichtige
Tatsache hat B ürg j in experimentellen Untersuchungen auf
sichere Basis zu stellen sich bemüht.') Das früher viel gebrauchte Morphiu m, vor allem sub-
kutan, ist als Schlafmittel durchaus zu vermeiden, wegen der
Gefahr der Angewöhnung. Auch das Chioraihydrat, das an sich sehr wirksam,
ist wegen seiner ungünstigen Wirkung auf das Herz besser
durch andere Hypnotica zu ersetzen, ganz besonders sollte
es deshalb bei Arteriosklerose, bei symptomatischen Psychosen,
beim Delirium tremens und ähnlichem vermieden werden. Gerade bei solchen sind das Paraldehyd in Dosen von 3,0
bis 5,0 (evtl. in Himbeerlimonade oder dgl. wegen des scharfen
Geruchs oder Geschmacks), Amylenhydrat (2,0-3,0), evtl. Dormiol in Kapseln (2-3 zu 0,5), auch Isopral, zu bevorzugen. Vor wenigen Jahren waren Trional und Sulfonal (1,0-2,0
pro Dosis) besonders viel im Gebrauch. Ihre Wirkung war
gut, doch führten sie zuweilen zu Hämatoporphinurie, was
besonders bei Morphinismus und bei bestehendem Nieren-
leiden beobachtet wurde. Die Behandlung der Schlaflosigkeit.
Klinischer Vortrag.
Von E. Meyer in Kinigsberg i. Pr. Er kommt zu dem Schluß, daß ein Gemisch von zwei Narcotica
(für Hypnotica gilt das gleiche) dann einen Effekt, der über dem Addi-
tionsergebnis liegt, (also eine Potenzierung') verursacht, wenn seine
zwei Glieder verschiedene pharmakologische Angriff spunkte haben;
bei gleichem Angriffspunkt der zwei Komponenten erzielt die Kombi-
nation nur ein Additionsergebnis.")
Seine Untersuchungen in dieser
Richtung führten auch zu dem speziellen Ergebnis, daß auch die allcr-
kleinsten Mengen starke pharmakologische Effekte auslösen können,
wenn sie einer größeren Menge einer anderen Arznei beigegeben worden
sind."3) Diese beiden Mittel sind verdrängt durch das Veronal,
das solcher unangenehmen Nebenwirkung entbehrt und in
Dosen von 0,5-1,0 auch bei stärkerer Schlaflosigkeit in der
Regel sehr gut und schnell (nach etwa 1 Stunde) wirkt; es hat
auch den Vorteil, daß es sich in warmer Flüssigkeit löst. In
manchen Fällen wird über ein gewisses Eingenommensein im
Kopf morgens geklagt, bei längerem Gebrauch hören wir von
Schläfrigkeit, auch Erschwerung der Sprache; besonders bei
alten Leuten ist zuweilen eine stärkere Benommenheit auf-
fallend. Im ganzen muß das Veronal aber als ausgezeichnetes
Schlafmittel angesehen werden. M. H.! Ich glaube, daß die Berücksichtigung dieser Fest-
stellungen B ii rg is, wenn sie sich weiterhin bestätigen sollten,
auch in der Praxis zu einem klareren Urteil und besserer
Auswahl gegenüber dem Ansturm der Hypnotica beitragen wird. Noch günstiger wirkt vielleicht das Veronalnatrium
oder Medinal, das noch besser löslich ist, etwas schneller
vielleicht wirkt und noch weniger zu Nachwirkungen Anlaß
gibt. Doch scheint das Verona! nach meinen und anderer
Autoren Erfahrungen stärker zu sein. Manche Kranke be-
merken auch eine schnelle Abnahme der Wirkung beim Medinal. g
Von einigen Seiten werden ferner das He d on al, besonders
auch das Code onal gelobt. In neuerer Zeit ist von vielen Untersuchern besonders
auf das Adalin aufmerksam gemacht, das zwar schwächer
als Veronal und Medina! ist, aber bei der Schlaflosigkeit der
funktionellen Neurosen und anderen leichten Schlafstörungen
sehr gute Erfolge aufweist. Es steht dem Bro mural nahe,
ist aber in der Wirkung sicherer und frei von wesentlichen
Nebenerscheinungen. Genannt seien hier noch das Hypnal, Hypnoval, Ma-
lonal, Veronazetin, das Apoual, aus allerjüngster Zeit das
Lu minal. Letzteres wird in den vorliegenden Mitteilungen
sehr warm empfohlen,
es
scheint
auch subkutan und in
Suppositorienform gut anwendbar. Die Behandlung der Schlaflosigkeit.
Klinischer Vortrag.
Von E. Meyer in Kinigsberg i. Pr. Sehr viele Nervöse sind abends
sehr mude, schlafbedürftig, können aber nachher nicht ein-
schlafen, fühlen sich sehr schwach, ohne jede Ursache. Schließ-
lieu haben solche Kranke abends direkt Angst vor dein Versuch
des Einschafens und schlafen schon deshalb nicht. In anderen
Fällen erwachen die Kranken schon nach drei oder vier Stunden
ohne jeden Grund und können nicht wieder einschlafen. Ailes das führt uns hinüber zu der so häufigen Schlaf Zur Bekämpfung der Schlaflosigkeit genügen
vielfach
auch die Bro msalze, die ich zu den ausgesprochenen Schlaf-
mitteln noch nicht rechnen möchte. Sie verdienen wegen
ihrer allgemein die Erregbarkeit des Nervensystems herab-
setzenden Wirkung durchaus ihren alten, guten Ruf. B i d
V
d
i
li h
H
i
i d Bei der Verordnung eigentlicher Hypnotica wird man
sich ganz besonders wieder gegenwärtig halten, daß die Schlaf-
losigkeit in der Regel nur ein Symptom it, daß immer die
Grundkrankheit zu beseitigen bleibt. Das soll auch den Pa-
tienten stets bewußt sein, die sonst schließlich nur einfach
die Verschreibung eines Schlafmittels wünschen. Wi d
k
d
h i
i Wird man, was vorkommt, durch intensivere
Schlaf-
störungen schon bald gezwungen, zu einem Schlafmittel zu
greifen, um einmal eine oder ein paar gute Nächte herbei-
zuführen, so wird alles das zu beherzigen sein. Ohne weiteres
ist ja auch klar, daß der regelmäßige Gebrauch von Schlaf-
mitteln auf die Dauer nicht ohne unangenehme Folgen bleibt
und schon dadurch in psychischer Hinsicht ungünstig wirkt,
daß er zu einer Gewohnheit, zu einer Art Leidenschaft wird. M. H.! Eine lückenlose Aufzählung und Besprechung aller
gebräuchlichen Schlafmittel zu geben, ist bei ihrer ungeheuren
Zahil nicht möglich. - Brachte doch z. B. Rabow 1911
in
den Neuesten Arzneimitteln" nicht
weniger
als
21,
ungerechnet die Narcotica, die doch mehr oder weniger, wie
z. B. das Pantopon, auch als Schlafmittel benutzt werden. - Es hätte das auch keinen Wert für Sie, würde eher ver- Ailes das führt uns hinüber zu der so häufigen Schlaf-
losigkeit bei der, ich möchte sagen, Nervosität des täg-
ichen Le hens, wo neben gewisser Abspannung, Ermüdung DEUTSCHE MEDIZINISCHE WOCHENSOERIFT. 1723 12. September 1912. wöhnung zu vermeiden und recht bald wieder ohne Schlaf-
mittel auszukommen. wirrend wirken ; ich beschränke mich daher darauf, Ihnen
über eine Anzahl von Schlafmitteln, soweit sie besonders viel
in Gebrauch sind und empfehlenswert scheinen, kurz zu be-
richten. Die Behandlung der Schlaflosigkeit.
Klinischer Vortrag.
Von E. Meyer in Kinigsberg i. Pr. Mehr Mittel Ihnen aufzuzählen, wäre zwecklos, es wird
Ihnen wie auch mir immer einmal vorkommen, daß ein Patient
Sie nach einem Schlafmittel fragt, das Ihnen, wenn Sie ganz
aufrichtig sein wollen, noch nicht vorgekommen Ist. M. H.! Wir haben uns, schon der knappen Zeit wegen,
auf die rein praktische Seite unseres Themas beschränkt und
von chemischen etc. Auseinandersetzungen, so wichtig sie sind,
abgesehen. Nur einige allge meine Punkte bleiben mir zu
erledigen noch übrig. Einmal ist es ein alter Erfahrungssatz, daß es in Fällen,
wo man gezwungen ist, längere Zeit Hypnotica zu geben,
von Vorteil Ist, zu variieren, z. B. den einen Abend Veronal,
den anderen Dormiol, den dritten Codeonal etc. zu geben. Die Wirkung scheint besser zu sein, die Schädigungen ge-
ringer. Ferner empfiehlt es sich, wenn eine bestimmte Dosis
eines Mittels gut gewirkt hat, möglichst bald auf ein geringeres
Quantum herabzugehen, auch einmal auszusetzen, um die gerade
genügende Dosis festzustellen, vor allem auch, um die Ge- 216 *
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https://openalex.org/W4280605595
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https://www.frontiersin.org/articles/10.3389/fpubh.2022.886262/pdf
|
English
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Prenatal Detection of Congenital Heart Diseases Using Echocardiography: 12-Year Results of an Improving Program With 9782 Cases
|
Frontiers in public health
| 2,022
|
cc-by
| 7,569
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ORIGINAL RESEARCH
published: 13 May 2022
doi: 10.3389/fpubh.2022.886262 ORIGINAL RESEARCH
published: 13 May 2022
doi: 10.3389/fpubh.2022.886262 Prenatal Detection of Congenital
Heart Diseases Using
Echocardiography: 12-Year Results
of an Improving Program With 9782
Cases Yanqiu Ou 1, Michael S. Bloom 2, Jinzhuang Mai 1, Melissa Francois 3, Wei Pan 4,
Xiaohua Xiao 5, Ximeng Wang 1, Zhiqiang Nie 1, Yanji Qu 1, Xiangmin Gao 1, Yong Wu 1,
Xiaoqing Liu 1, Jian Zhuang 1 and Jimei Chen 1* Yanqiu Ou 1, Michael S. Bloom 2, Jinzhuang Mai 1, Melissa Francois 3, Wei Pan 4,
Xiaohua Xiao 5, Ximeng Wang 1, Zhiqiang Nie 1, Yanji Qu 1, Xiangmin Gao 1, Yong Wu 1,
Xiaoqing Liu 1, Jian Zhuang 1 and Jimei Chen 1* 1 Department of Cardiac Surgery, Guangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong
Academy of Medical Sciences, Guangzhou, China, 2 Department of Global and Community Health, George Mason University,
Fairfax, VA, United States, 3 Department of Biomedical Sciences, University at Albany, State University of New York, Albany,
NY, United States, 4 Department of Pediatric Cardiology, Guangdong Cardiovascular Institute, Guangdong Provincial
People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China, 5 Department of Cardiology, Boai Hospital
of Zhongshan, Zhongshan, China Edited by:
Corrado Romano,
University of Catania, Italy Edited by:
Corrado Romano,
University of Catania, Italy
Reviewed by:
Chao-Yu Guo,
National Yang Ming Chiao Tung
University, Taiwan
Ivana I. Kavecan,
University of Novi Sad, Serbia
*Correspondence:
Jimei Chen
jimei@hotmail.com Background: A provincial program combining the effect of a government investment in
prenatal screening and a specialized cardiac center was introduced in 2004, to improve
prenatal diagnosis by echocardiography for congenital heart diseases (CHDs) in the
Guangdong Registry of Congenital Heart Disease, China. Reviewed by:
Chao-Yu Guo,
National Yang Ming Chiao Tung
University, Taiwan
Ivana I. Kavecan,
University of Novi Sad, Serbia Objectives: To evaluate the effects of this program on the prenatal diagnosis rate (PDR)
by echocardiography and termination of pregnancy (TOP). *Correspondence:
Jimei Chen
jimei@hotmail.com Methods: A retrospective study from 2004-2015 included 9782 fetuses and infants
diagnosed with CHDs. The PDR was calculated for major and minor CHDs during
pre-, mid- and post-program time-intervals. Multivariable logistic regression was utilized
to analyze the associations between program implementation and the timing of CHD
diagnosis (prenatal vs. postnatal) by different hospital levels. The rate for TOP were
also evaluated. Specialty section:
This article was submitted to
Children and Health,
a section of the journal
Frontiers in Public Health Results:
The PDR increased by 44% for major CHDs in the post-program interval
relative to the pre-program interval. The three most frequently diagnosed subtypes
prenatally were hypoplastic left heart syndrome (84%), double outlet right ventricle
(83%) and severe pulmonary stenosis (82%). Participants with a high school education
experienced a greater increase in PDR than those without a high school education. The odds for a prenatal vs. a postnatal diagnosis for major CHD were greater after
introduction of the program than before (adjusted odd ratio= 20.95, 95% CI:2.47,
178.06 in secondary hospitals; and adjusted odd ratio=11.65, 95% CI:6.52, 20.81
in tertiary hospitals). The TOP rate decreased from 52.3% pre-program to 19.6%
post-program among minor CHD fetuses with a prenatal diagnosis (P for trend
=0.041). A lower proportion of TOP were attributed to minor CHDs after the program. Received: 28 February 2022
Accepted: 19 April 2022
Published: 13 May 2022 Professional and Place The specialized cardiac center, Guangdong Cardiovascular
Institute, established and maintained a network of GRCHD
across the province with the support of the Guangdong
Department of Health, since 2004, aiming to unify the diagnosis
and treatment of CHD. The network included the largest
local hospitals and maternal and child care centers from
various geographic areas across Guangdong province. Beginning
with 19 surveillance sites at inception, the network gradually
expanded to 39 sites in 20 cities at present, including primary,
secondary, and tertiary hospitals (10, 11). The surveillance sites
screen women and diagnose CHDs, which are reported to
Guangdong Cardiovascular Institute in a standardized fashion. Guangdong Cardiovascular Institute, a specialized cardiac center
in Guangzhou city, the provincial capital, is the coordinating and
technical support center for the network. The coordinating center
oversees the GRCHD, provides technical training, support, and
quality control, and reports data to the Guangdong Department
of Health. In addition, a CHD referral network ensures priority
treatment for complicated and uncertain cases. Suspected
CHDs are referred to Guangdong Cardiovascular Institute,
where pediatric cardiologists validate the prenatal diagnosis;
karyotyping is routinely recommended. After birth, newborns
are evaluated by an obstetrician, pediatrician, or cardiologist
before discharge, or within 72 h, to determine whether further
investigation for potential CHD was required. All CHD cases are
confirmed by B-mode echocardiography, cardiac catheterization,
surgery, autopsy, or necropsy in the case of stillbirth. A 1-year
followed up for the cases were conducted. Guangdong Cardiovascular Institute in Guangdong, China,
initiated a program for improving the early diagnosis of
CHDs with the support of the provincial government in
2004. Government policies promoted screening for congenital
abnormalities, provincial networking, and professional training
to improve detection, with the aim to improve CHD detection. Before 2006, sparse resources and few skilled ultrasonographers
limited ultrasound screening for fetal abnormalities to evaluate
obstetric complications or other high-risk pregnancies (8). However, many CHDs occur among “low risk” populations,
mothers without known risk factors (9). Therefore, population-
based
CHD
screening
strategies
have
grown
worldwide. Therefore, with the support of the provincial government,
Guangdong Cardiovascular Institute established the Guangdong
Registry of CHD (GRCHD) in 2004. The GRCHD provides
professional education and training in early CHD diagnosis, and
to offer medical priority for diagnosis to major cases, also to
streamline CHD reporting across a network of 40 hospitals in
Guangdong province. Abbreviations: AVSD, atrioventricular septal defect; ASD, atrial septal defect;
CHDs, congenital heart diseases; DORV, double outlet right ventricle; GRCHD,
Guangdong Registry of CHD; HLHS, hypoplastic left heart syndrome; ICD-10,
International Classification of Diseases, 10th Revision; PA, pulmonary atresia;
PDR, prenatal detection rate; TA, tricuspid atresia; TAPVR, total anomalous
pulmonary venous return; TGA, dextro-transposition of the great arteries; ToF,
tetralogy of Fallot; TOP, termination of pregnancy; VSD, ventricular septal defect. Citation: Ou Y, Bloom MS, Mai J, Francois M,
Pan W, Xiao X, Wang X, Nie Z, Qu Y,
Gao X, Wu Y, Liu X, Zhuang J and
Chen J (2022) Prenatal Detection of
Congenital Heart Diseases Using
Echocardiography: 12-Year Results of
an Improving Program With 9782
Cases. Front. Public Health 10:886262. doi: 10.3389/fpubh.2022.886262 Front. Public Health 10:886262. doi: 10.3389/fpubh.2022.886262 Front. Public Health 10:886262. doi: 10.3389/fpubh.2022.886262 May 2022 | Volume 10 | Article 886262 Frontiers in Public Health | www.frontiersin.org Ou et al. Ou et al. Improving Program for CHD’s Prenatal Diagnosis Conclusions: The program combining the advantages of government investment and a
specialized cardiac center appeared to increase the PDR by echocardiography for CHDs
in an unselected population. The TOP rate among minor cases with prenatal diagnosis
declined significantly after implementation of the program. Conclusions: The program combining the advantages of government investment and a
specialized cardiac center appeared to increase the PDR by echocardiography for CHDs
in an unselected population. The TOP rate among minor cases with prenatal diagnosis
declined significantly after implementation of the program. Keywords: congenital disease, birth defects, echocardiography, prenatal diagnosis, program, termination
of pregnancy Professional and Place In this report, we primarily aimed to assess the impact of
the GRCHD program on the prenatal detection rate (PDR) and
timing of diagnosis for CHDs, in an unselected population. We
also examined the influence of the program on the termination
of pregnancy (TOP) in the GRCHD. Provincial Improving Program for Early
Diagnosis of CHD Congenital heart diseases (CHDs) remain the most common
congenital malformations, with a global incidence of 6-12/1000
live births (1). CHDs account for 46% of all deaths due to
congenital malformations and 3% of all infant deaths (2). ∼20–
30% of CHDs are “major”, defined as potentially life threatening
and requiring surgery within the first year of life. Timing of
diagnosis is a crucial factor for the prognosis of major CHD
(3–5). Prenatal diagnosis of CHD can lead to timely delivery
of medical treatment that may improve clinical outcomes and
decrease neonatal morbidity and mortality (6). Over the past two
decades, a great deal of emphasis has been placed on prenatal
echocardiographic screening for CHDs, increased provider
training, and establishing a national screening program (7). The provincial improving program included three major
components
and
was
summarized
into
“4P”
Mode
(Supplementary Figure 1): Policy Government
recommended
the
population
to
congenital
abnormality screening. In 2006, the Guangdong Province Health
Department formally recommended that all pregnant women
receive routine ultrasound examination, including screening for
fetal congenital anomalies, between 18 and 24 weeks of gestation
(8). The recommendations for a basic cardiac examination
included a 4-chamber view during prenatal ultrasound and,
when technically feasible, views of the outflow tracts. Updated
guidelines for prenatal ultrasound examinations by the Chinese
Doctor
Association,
Council
on
Ultrasound
(2012)
also
included views of three vessels when technically feasible (12). According to the updated recommendations, examination via
fetal echocardiography is warranted for abnormal obstetrical
ultrasound, the family history of CHD, maternal diabetes, or use
of in vitro fertilization, in accordance with the practice guidelines
for the performance of fetal echocardiography (13). Afterwards,
some cities where the network sites located in across the
province have initiated government-funded prenatal screening
for congenital abnormalities beginning in 2008. Besides, Practice
Guidelines for the Performance of Fetal Echocardiography in
Guangdong Province was issued by the Guangdong Eugenics
Association Council on Congenital Heart Diseases to guide the
prenatal couseling on CHDs (14). Data Analysis y
The 12-year period was divided into three time-intervals, 1)
2004-2005, was defined as the “pre-program” interval, as there
were no provincial CHD diagnosis data available before 2004;
2) 2006-2010, was defined as the “mid-program” interval; and
3) 2011-2015, was defined as the “post-program” interval. Characteristics of the participants, PDR, and timing of diagnosis
were described for each CHD subtype by different intervals. The PDR was calculated as number of prenatal diagnosis
cases divided by the total cases, for different CHD group (i.e.,
major CHDs or minor CHDs), CHD subtypes, and different
program time intervals. P for trend was calculated by Cochran-
Armitage trend test to estimate changing PDR trends during
the different program time intervals. Multivariable logistic
regression models were used to estimate the odds of a prenatal
vs. postnatal diagnosis for major CHDs in the pre-program
and mid-program/post-program intervals, stratified by different
hospital levels. Factors adjusted in the multivariable regression
was those highly related to the prenatal detection of CHDs,
such as maternal age (>35 /≤35 years), domestic migrant
population (people moving from rural or undeveloped areas to
Guangdong areas for work without permanent city residence;
yes/no), maternal education (more than high school/completion
of high school/less than high school), total previous live births
(0/1/≥2), family history of CHD (yes/no), maternal diabetes
(including pregestational and gestational diabetes, yes/no),
multiple gestation (yes/no), infant sex (female/male), extra-
cardiac defects or chromosomal abnormality (yes/no), and
fetus with suspected cardiac abnormality on basic obstetric
ultrasound (yes/no). The multiplicative scale of interaction
was calculated by evaluating the product term for time-
intervals or other factors and hospital levels. The statistical
significance of the product term was determined by using the
Wald statistic. Data Collection All CHDs in newborns and stillbirths are reported to the
national registry system for all birth defect. More detailed
information obtained from hospital medical records is provided
to the GRCHD. The GRCHD also obtains information from
mothers through face-to-face interviews and a structured,
standardized questionnaire on CHD risk factors as previously
reported (10). Promotion
d Guangdong Cardiovascular Institute offers annual technical and
standardization training on CHDs for stafffrom all network
sites, including echocardiography knowledge and skills, fetal
and infant cardiac anatomy, prenatal counseling, clinical
management of CHDs, and intervention strategies. Completion
of a 3-month intensive pediatric echocardiography in-service
training
program,
offered
by
Guangdong
Cardiovascular
Institute, is required for the key echocardiographer from each
network site. Guangdong Cardiovascular Institute also offers May 2022 | Volume 10 | Article 886262 Frontiers in Public Health | www.frontiersin.org 2 Improving Program for CHD’s Prenatal Diagnosis Ou et al. anomalous pulmonary venous return (TAPVR); (6) tricuspid
atresia (TA); (7) truncus arteriosus; (8) aorta defects, such
as aortic coarctation, atresia/hypoplasia/interruption of the
aortic arch, and severe aortic valve stenosis; (9) double outlet
right ventricle (DORV), (10) Ebstein anomaly; (11) severe
pulmonic stenosis; (12) single ventricle complex; and (13)
complete and partial atrioventricular septal defect (AVSD). Minor CHDs included: (1) isolated septal defects, such as
isolated VSD, isolated atrial septal defect (ASD), and a
combination of VSD and ASD; and (2) all other specific
defects. Those with only prenatal diagnosis of ASD as an
isolated heart disease, preterm infants with isolated patent
ductus arteriosus, cases with isolated patent foramen ovale
younger than 1 year, and cases without specific diagnosis
or only with mild valve lesions were not considered to
be CHDs. on-site and off-site technical consultation to network sites,
provides timely feedback on referral cases, and helps improve
diagnostic skills at lower-level facilities. Study Design and Participants This is a multicenter population-based descriptive study of
livebirths and stillbirths from the GRCHD diagnosed with CHDs
with ultrasound, with estimated dates of delivery from 2004-
2015. The study was approved by the Ethics Committee of
Guangdong Provincial People’s Hospital (Reference number:
GDREC2011135H). Patient consent were obtained. Case Subclassification All CHD cases in the GRCHD were coded by two clinical
epidemiologists, with more than 20 years of experience in
pediatric and adult cardiology, based on the International
Classification of Diseases, 10th Revision (ICD-10) (Q20.000-
Q28.000). CHD was classified into major CHDs and minor
CHDs based on previous reports with slight modification
(15). Major CHDs included: (1) dextro-transposition of the
great arteries (TGA); (2) hypoplastic left heart syndrome
(HLHS); (3) pulmonary atresia (PA), with or without ventricular
septal defect (VSD); (4) tetralogy of Fallot (ToF); (5) total Missing data for maternal age (n = 486), domestic migration
status (n = 112), maternal education (n = 46) or maternal
previous live births (n = 43) were not imputed during analysis. Data were analyzed using SAS (v.9.3; SAS Institute, Inc., Raleigh,
NC USA). Statistical tests were two sided with a significance level
of P <0.05. May 2022 | Volume 10 | Article 886262 Frontiers in Public Health | www.frontiersin.org 3 Improving Program for CHD’s Prenatal Diagnosis Ou et al. TABLE 1 | Characteristics of congenital heart disease cases, 2004–2015. TABLE 1 | Characteristics of congenital heart disease cases, 2004–2015. Total (N = 9782)
Pre-program (N = 673)
Mid-program(N = 3331)
Post-program (N = 5778
Maternal age (years)*
<35
8316 (85)
504 (75)
2888 (87)
4924 (85)
≥35
980 (10)
57 (9)
294 (9)
629 (11)
Maternal ethnicity
Minorities
221 (2)
22 (3)
73 (2)
126 (2)
Han
9561 (98)
651 (97)
3258 (98)
5652 (98)
Domestic migrant population†
Yes
2552 (26)
223 (33)
1011 (30)
1318 (23)
No
7118 (74)
450 (67)
2318 (70)
4350 (77)
Maternal education‡
More than high school
1521 (16)
79 (12)
497 (15)
945 (17)
Completion of high school
3174 (33)
202 (30)
939 (28)
2033 (36)
Less than high school
5041 (52)
392 (58)
1895 (57)
2754 (48)
Maternal previous parity**
0
6495 (67)
466 (69)
2311 (69)
3718 (65)
1
2638 (27)
174 (26)
865 (26)
1599 (28)
2
606 (6.2)
33 (4.9)
154 (4.6)
419 (7.3)
Infant sex
Male
4783 (49)
356 (53)
1646 (49)
2781 (48)
Female
4632 (47)
313 (47)
1679 (50)
2640 (46)
Hospital level
Tertiary hospital
4798 (49)
469 (70)
1702 (51)
2627 (46)
Secondary hospital
4639 (47)
192 (29)
1488 (45)
2959 (51)
Primary hospital
345 (4)
12 (2)
141 (4)
192 (3)
Missing data, *486 cases; †112 cases; ‡46 cases; **43 cases. Case Subclassification Total (N = 9782)
Pre-program (N = 673)
Mid-program(N = 3331)
Post-program (N = 5778) Missing data, *486 cases; †112 cases; ‡46 cases; **43 cases. Characteristics of CHD Cases Characteristics of CHD Cases A total of 9782 cases were diagnosed with CHD, among 1046456
fetuses and infants during the study period (Table 1). Compared
to the pre-program interval, CHD cases in the mid-program and
post-program intervals were more likely to be a maternal age
≥35 years and nulliparous, and less likely to be domestic migrant
population and education less than high school (P<0.05). During
the study period, an increasing number of diagnoses were made
at secondary compared to tertiary hospitals. The proportion of
CHD cases diagnosed by tertiary hospitals significantly decreased
(P for trend <0.001) from 70% pre-program to 46% post-
program, while the proportion of CHD cases diagnosed by
secondary hospitals significantly increased (P for trend <0.001),
from 29% pre-program to 51% post-program. RESULTS 4 percentage points per year. The minor CHD PDR increased
9 percentage points from pre-program (4%) to post-program
(13%) (P for trend <0.001). Prenatal Diagnosis by CHD Subtypes g
y
yp
Except for severe PS, all CHD subtypes experience dramatic
increases in the PDRs (Table 2). The most significant PDR
improvements were made for TA and aortic defects, with
difference of more than 70% from the pre-program to the post-
program intervals, followed by the ToF (60%), DORV (53%), PA
(49%) and TGA (39%). Even with TAPVR, PDR increased by 32%
throughout from the pre- program to the post-program intervals. The highest PDRs post-program were found for HLHS (84%),
DORV (83%) and severe PS (82%). Almost half (49%) of TAPVR
were detected prenatally. When stratified by the education level,
we observed that the PDR improvement for major CHDs was
most significant in participants completing education of high
school, and then less than high school, and then higher than high
school (Supplementary Figure 2). Time Trends of PDR by Echocardiography
Of 9782 CHD cases diagnosed from 2004-2016, 2184 (22%)
were diagnosed prenatally. Of note, the PDRs for total CHDs
and CHD subgroups increased over time (Figure 1). Overall,
CHD PDR increased 17 percentage points from 9% pre-program
to 26% post-program (P for trend <0.001). The major CHD
PDR increased 44 percentage points (from 26% pre-program to
70% post-program, P for trend <0.001) an average increase of The times of prenatal and postnatal diagnosis also greatly
advanced as shown in Figure 2. The median gestational week at
prenatal diagnosis was 30 weeks pre-program and 25 weeks post-
program for each specific subtype, with statistically significant
differences for TGA, HLHS, ToF and SV (P for trend <0.001). May 2022 | Volume 10 | Article 886262 Frontiers in Public Health | www.frontiersin.org 4 Ou et al. Ou et al. Improving Program for CHD’s Prenatal Diagnosis FIGURE 1 | Trend for prenatal diagnosis rates for congenital heart diseases during pre-, mid- and post-program time intervals, by major and minor severity. CHD,
congenital heart disease. FIGURE 1 | Trend for prenatal diagnosis rates for congenital heart diseases during pre-, mid- and post-program time intervals, by major and minor severity. CHD,
congenital heart disease. TABLE 2 | Prenatal congenital heart disease diagnoses, by subtype, 2004–2015. Prenatal Diagnosis by CHD Subtypes Total
Pre-program
Mid-program
Post-program
n (PDR)
n (PDR)
n (PDR)
n (PDR)
P for trend
Major CHD
2155 (61)
152 (26)
693 (51)
1310 (70)
<0.001
TGA
485 (58)
40 (28)
167 (50)
278 (67)
<0.001
HLHS
144 (80)
4 (50)
39 (72)
101 (84)
0.028
PA
126 (56)
13 (15)
40 (55)
73 (64)
0.003
TAPVR
86 (48)
6 (17)
29 (52)
51 (49)
0.378
ToF
407 (50)
32 (9)
129 (26)
246 (69)
<0.001
TA
35 (60)
2 (0)
21 (57)
12 (75)
0.067
Truncus arteriosus
108 (51)
12 (33)
33 (49)
63 (56)
0.158
Aortic defects*
174 (71)
3 (0)
45 (60)
126 (76)
0.002
DORV
212 (81)
10 (30)
70 (84)
132 (83)
0.023
Ebstein anomaly
53 (76)
4 (75)
21 (71)
28 (79)
0.654
Severe PS
38 (84)
2 (100)
14 (86)
22 (82)
0.529
Single ventricle
62 (60)
6 (50)
18 (50)
38 (66)
0.259
AVSD
†
225 (54)
18 (33)
67 (36)
140 (66)
<0.001
Minor CHD
7627 (11)
521 (4)
2638 (9)
4468 (13)
<0.001
Isolated septal defects‡
3279 (12)
240 (5)
1034 (11)
2005 (14)
<0.001
Other specified defects
4348 (10)
281 (3)
1604 (8)
2463 (13)
<0.001
Total CHD
9782 (22)
673 (9)
3331 (18)
5778 (26)
<0.001
ASD, atrial septal defect; AVSD, atioventricular septal defect; CHD, congenital heart disease; DORV, double outlet right ventricle; HLHS, hypoplastic left heart syndrome; PA, pulmonary
atresia; PDR, prenatal detection rate; PS, pulmonary stenosis; TA, tricuspid atresia; TAPVR, total anomalous pulmonary venous return; TGA D-, Transposition of the great arteries; ToF,
tetralogy of fallot; VSD, ventricular septal defect. *Aorta defects includes aortic coartation, atresia/hypoplasia/interruption of the aortic arch and severe aortic valve stenosis. † AVSD includes complete and partial subtypes. ‡Isolated septal defects include isolated VSD, isolated, and combination of VSD and ASD. The bold values indicates the trend test for PDR in different time intervals is with
statistical significance. TABLE 2 | Prenatal congenital heart disease diagnoses, by subtype, 2004–2015. Odds of Prenatal Diagnosis for CHD in
Different Time-Intervals and Population Postnatal CHD diagnosis also advanced significantly (all P for
trend <0.001), with most major post-program CHDs detected
within three (TGA, PA, TAPVR, TA, PTA, aortic defect, and
Ebstein anomaly) to seven days (HLHS, ToF and DORV)
after delivery. Postnatal CHD diagnosis also advanced significantly (all P for
trend <0.001), with most major post-program CHDs detected
within three (TGA, PA, TAPVR, TA, PTA, aortic defect, and
Ebstein anomaly) to seven days (HLHS, ToF and DORV)
after delivery. To further elucidate the contribution of the provincial improving
program to the advancing major CHD PDR, multivariable
logistic regression was used to analyzed the odds of receiving a May 2022 | Volume 10 | Article 886262 Frontiers in Public Health | www.frontiersin.org 5 Improving Program for CHD’s Prenatal Diagnosis t al. Improving Program for CHD’s Prenatal Diagnosis
GURE 2 | Timing of prenatal and postnatal diagnosis for major congenital heart defects. DORV Double Outlet Right Ventricle, HLHS Hypoplastic Left Heart
ndrome, PA Pulmonary Atresia, PDR Prenatal Detection Rate, PS Pulmonary Stenosis, TA Tricuspid Atresia, TAPVR Total Anomalous Pulmonary Venous Return,
A D-Transposition of the Great Arteries, ToF Tetralogy of Fallot. natal vs. postnatal diagnosis for major CHD in the different
period; and aOR=20.95, 95% CI:2.47, 178.06 in post-program Ou et al. FIGURE 2 | Timing of prenatal and postnatal diagnosis for major congenital heart defects. DORV Double Outlet Right Ventricle, HLHS Hypoplastic Left Heart
Syndrome, PA Pulmonary Atresia, PDR Prenatal Detection Rate, PS Pulmonary Stenosis, TA Tricuspid Atresia, TAPVR Total Anomalous Pulmonary Venous Return,
TGA D-Transposition of the Great Arteries, ToF Tetralogy of Fallot. FIGURE 2 | Timing of prenatal and postnatal diagnosis for major congenital heart defects. DORV Double Outlet Right Ventricle, HLHS Hypoplastic Left Heart
Syndrome, PA Pulmonary Atresia, PDR Prenatal Detection Rate, PS Pulmonary Stenosis, TA Tricuspid Atresia, TAPVR Total Anomalous Pulmonary Venous Return,
TGA D-Transposition of the Great Arteries, ToF Tetralogy of Fallot. period; and aOR=20.95, 95% CI:2.47, 178.06 in post-program
period, respectively), and then the tertiary hospitals (aOR=4.51,
95% CI: 2.54, 8.03 in mid-program period; and aOR=11.65,
95% CI:6.52, 20.81 in post-program period, respectively). The interaction between time-intervals and hospital levels
was statistically significant (P<0.001). In addition, different
level centers had particular factors associated with increased prenatal vs. postnatal diagnosis for major CHD in the different
program intervals stratified by different hospitals. Frontiers in Public Health | www.frontiersin.org Odds of Prenatal Diagnosis for CHD in
Different Time-Intervals and Population As shown in
Figure 3, compared to cases in the pre-program period, major
CHD cases in mid-program and post-program periods had
higher odds of prenatal diagnosis. The increment of adjusted
OR (aOR) by time-interval was strongest in the secondary
hospitals (aOR=9.53, 95% CI: 1.11, 81.48 in mid-program May 2022 | Volume 10 | Article 886262 Frontiers in Public Health | www.frontiersin.org 6 Ou et al. Ou et al. Improving Program for CHD’s Prenatal Diagnosis FIGURE 3 | Multivariate logistic regression analysis for different time-interval associated with the prenatal diagnosis for major congenital heart diseases stratified by
different hospital levels, 2004–2015. The multivariable models in different hospital stratification was adjusted by maternal age (>35 yrs and≤35 yrs), domestic migrant
population (yes/no), maternal education (more than high school/completion of high school/less than high school), total previous live births (0/1/≥2), family history of
CHD (yes/no), maternal diabetes (yes/no), maternal hypertension (yes/no), infant sex (female/male), extra-cardiac/aneuploidy/genetic syndrome (yes/no), and fetus
with suspected cardiac abnormality on basic obstetric ultrasound (yes/no). OR, Odds Ratio; CI, confidence interval. FIGURE 3 | Multivariate logistic regression analysis for different time-interval associated with the prenatal diagnosis for major congenital heart diseases stratified by
different hospital levels, 2004–2015. The multivariable models in different hospital stratification was adjusted by maternal age (>35 yrs and≤35 yrs), domestic migrant
population (yes/no), maternal education (more than high school/completion of high school/less than high school), total previous live births (0/1/≥2), family history of
CHD (yes/no), maternal diabetes (yes/no), maternal hypertension (yes/no), infant sex (female/male), extra-cardiac/aneuploidy/genetic syndrome (yes/no), and fetus
with suspected cardiac abnormality on basic obstetric ultrasound (yes/no). OR, Odds Ratio; CI, confidence interval. PDR (Supplementary Table 1). Factors interacted with hospital
level included domestic migrant population, primiparous,
female infant, and fetus with extra-cardiac/aneuploidy/genetic
syndrome (P for multiplicative interaction <0.01). According to
the performance guideline in the GRCHD (13), indicators for
fetal heart screening include the presence of abnormal obstetrical
ultrasound results, the presence of a family history of CHD,
maternal diabetes, or use of in vitro fertilization, and so on. Except
for the major CHDs, total CHDs and minor CHDs with these
indicators were more easily to be detected prenatally (P<0.001,
Table 3). However, only about 8% of the cases had these factors,
like the presence of abnormal obstetrical ultrasound results, the
presence of a family history of CHD, maternal diabetes, or use of
in vitro fertilization, and so on. Interpretation
PDR Over Time and Subtype Detection The largest increase in PDRs with the progress of the program,
2 years after the start of the improving program, which
verified the effect of the program after a learning period for
the ultrosonographers. Our findings were was consistent with
previous studies of Netherlands, which reported the PDRs for
severe CHDs (n = 1912) increased from 35.8% in 2002 to
59.7% in 2012 after the 2007 introduction of a national prenatal
screening program (7). While the pre- and mid-program PDRs
of major CHDs were lower in the current study than reported
for developed countries, post-program PDRs were similar or
exceeded rates from developed countries. For example, the post-
program prenatal HLHS PDR in the current study (84%) was
similar to the 87.9% reported in Massachusetts, and higher than
the 50% reported for the multisite U.S. National Birth Defects
Prevention Study (4, 16). In our study, the detection rate for
TAPVR was higher than some studies from developed nations
reporting no prenatally detected cases (15). Termination of Pregnancy During the study period, the rate for TOP deceased from 40%
pre-program to 30% post-program among total CHD fetuses
with prenatal diagnosis (P for trend = 0.060), while the TOP rate
dropped from 52.3% to 19.6% among minor CHD fetuses with
prenatal diagnosis (P for trend = 0.041). The TOP attributed
largely to major CHDs, progressively lower proportion of TOP
were due to minor CHDs after the program (from 38% pre-
program to 22% post-program, P for trend = 0.028, Figure 4). Odds of Prenatal Diagnosis for CHD in
Different Time-Intervals and Population and fewer pregnancy terminations resulted from minor CHD. Following program implementation, PDRs increased almost 50%
for major CHDs, with 70% of major defects detected prenatally. Participants with a high school education experienced a greatest
increase in PDR than those without a high school education. While major CHD subtypes continued to be terminated,
termination of minor CHD subtypes diminished over time. To
the best of our knowledge, this is the first large population-based
study to investigate the effect of a regional program on CHD
in China. Main Findings In
this
large
population-based
study,
we
found
that
implementation of a provincial improving program for the
early diagnosis of CHDs was associated with greater CHD PDRs, May 2022 | Volume 10 | Article 886262 Frontiers in Public Health | www.frontiersin.org 7 Improving Program for CHD’s Prenatal Diagnosis Ou et al. TABLE 3 | Prenatal CHD diagnosis rates by presence of additional CHD risk factors. Risk Factor* Not Present, N (%)
PDR
Risk Factor* Present, N (%)
PDR
P-value for PDR
Total CHD
1991, 92%
22%
177, 8%
29%
<0.001
Major CHD
1214,92%
61%
100, 8%
60%
0.763
Minor CHD
777, 91%
11%
77, 9%
18%
<0.001
*Risk factors includes the presence of abnormal obstetrical ultrasound results, the presence of a family history of CHD, maternal diabetes, or use of in vitro fertilization, and so on. TABLE 3 | Prenatal CHD diagnosis rates by presence of additional CHD risk factors. CHD diagnosis rates by presence of additional CHD risk factors. FIGURE 4 | The distribution of the indicators for termination of a pregnancy
with a prenatal congenital heart disease diagnosis. BD, birth defect; CHD,
congenital heart disease; PDR, prenatal detection rate; TOP, termination of
pregnancy. specialized tertiary hospitals. And the multivariable logistic
regression stratified by the hospital level further convinced
that the improving program benefit the secondary and tertiary
hospitals, and had significant public health implication. Another important contributor was the inclusion of outflow
tracts view and 3-vessels views, in addition to 4-chamber
view, during prenatal ultrasound diagnosis. Theoretically, the 4-
chamber view detects >50% of serious cardiac malformations
when performed in mid-gestation. The addition of the outflow
tracts and 3-vessel with trachea view increases sensitivity
to as much as 90% (20–22). This can give explanation
to our finding that, both PDRs with lesions visible and
invisible on the 4-chamber view has significantly increased
(Supplementary Figure 3). The gap between the two groups
shortened, which implicated the detection of CHDs prenatally
does not rely on the 4-chamber view. FIGURE 4 | The distribution of the indicators for termination of a pregnancy
with a prenatal congenital heart disease diagnosis. BD, birth defect; CHD,
congenital heart disease; PDR, prenatal detection rate; TOP, termination of
pregnancy. Governmental recommendations are required to help allocate
medical investment in congenital abnormalities screening, and
standardized the clinicians daily work process. Termination of Pregnancy The TOP rate in our study population was much lower than
that in Norther China (40% vs. 85%) (23). And notably, we
were able to observe the TOP rate for minor case reducing
to 19.6%, as well as fewer TOP attributing to minor CHDs
post-program. We attribute these finding to the promotion of
the improving program, which provided training not only on
diagnosis skills, but also on standardized prenatal consultation. According to the Practice Guidelines for the Performance of Fetal
Echocardiography in Guangdong Province (14), we strongly
encouraged the family to give birth to the fetus with minor CHDs
and defects that will have promising prognosis postnatally. Main Findings In addition,
some cities across the region have initiated government-funded
prenatal screening for congenital abnormalities beginning in
2008, which has likely boosted the number of low-risk pregnant
mothers who have access to screening for cardiac defects. These
not only benefited participants with high level education, and to
a greater degree, also benefited those with high school education
or less than high school. We also found a noteworthy advancement of gestational age
diagnosis for prenatal CHDs after program implementation. The
∼5 weeks of earlier prenatal diagnosis for major CHDs allows
more time for the family to plan the delivery at a higher or
specialized medical center, or to receive intervention in utero
to prevent deterioration. For postnatal major CHDs diagnosed
after program implementation, we also found a median diagnosis
time 3 to 7 days earlier than pre-program which may allow for
earlier and perhaps more effective treatment of very critical and
severe lesions. Frontiers in Public Health | www.frontiersin.org Role of The Provincial Improving Program on PDR
Increase Technical training and education is vital to improve the PDR. Fetal echocardiography, in experienced hands, has been reported
to detect up to 90% of CHDs in low-risk population (9). However,
given the spectrum and complexity of CHD pathologies,
obstetrical ultrasound screening has a very low diagnostic yield
(10%−26%) (17–19). Therefore, the standardized on-site and
off-site training, that pediatric cardiologists provide to network
hospital physicians and ultrasonographers, is important to
improve the overall level of CHD diagnosis using ultrasound. The effectiveness of this technical training provided to network
hospitals is supported by our results, which suggests a greater
frequency of CHD cases reported by secondary hospitals in in
the post-program interval, even exceeding the PDR of more We also found that the overall and minor TOP rate for CHDs
with prenatal detection in the current study was lower than
our previously report about the specialized prenatal consultation
service in our specialized center (40% vs. 58.1% for overall, 19.6%
vs. 30.1% for minor CHDs) (24). The discrepancies were mainly
due to different study population and design. The current study
was population-based and demonstrate the “real world” data,
validating the influence of the specialized prenatal consultation
service in the generalized population. May 2022 | Volume 10 | Article 886262 8 Improving Program for CHD’s Prenatal Diagnosis Ou et al. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fpubh. 2022.886262/full#supplementary-material Supplementary Figure 1 | The diagrammatic drawing for the “4P” Mode to
improve the diagnosis capability across the region. Supplementary Figure 1 | The diagrammatic drawing for the “4P” Mode to
improve the diagnosis capability across the region. Supplementary Figure 1 | The diagrammatic drawing for the “4P” Mode to
improve the diagnosis capability across the region. Supplementary Figure 2 | Prenatal diagnosis rate for congenital heart disease by
maternal education, 2004-2015. Supplementary Figure 2 | Prenatal diagnosis rate for congenital heart disease by
maternal education, 2004-2015. CONCLUSIONS The results of this large multicenter study indicate that a
program combining the advantage of government investment,
and a specialized cardiac center is an effective means to
improve the PDR of CHDs in an unselected population. The TOP rate among minor cases with prenatal diagnosis
declined significantly with the program. We presume that
similar programs will be effective for improving prenatal CHD
diagnoses in other large Chinese provinces and in Asian
countries with different levels of hospitals. Future studies will
be necessary to evaluate the effectiveness of this approach in
different populations. Strengths and Limitations GDCHD network. We were not authorized to share the data
outside the network. Requests to access the datasets should be
directed to ouyanqiu@gdph.org.cn. This study is one of the few and the largest studies to report
a provincial program to improve the PDR across the region. The long study period enabled assessment of the program by
different time intervals, and the large number of cases size
enabled evaluation of PDR by different CHD subtypes. Also,
we evaluated the change for TOP rate by program progress. In addition, each CHD case was individually reviewed by two
clinical epidemiologists trained in pediatric and adult cardiology. Similar to several previous studies (25, 26), our definition
of major CHD required documentation of clinical severity in
addition to ICD code. ACKNOWLEDGMENTS We appreciate the efforts of all participating centers from the
Guangdong Registry of CHD that collected data for this study. We thank Srila Sen and Youlian Liao from the Centers for Disease
Control and Prevention, Atlanta, GA, USA for their dedicated
editorial assistance. FUNDING This work was supported by the National Key Research and
Development Program [No. 2018YFC1002600], the National
Natural Science Foundation of China [No. 81903287], the
Natural Science Foundation of Guangdong Province [Nos. 2021A1515011445
and
2020A1515010743],
Guangdong
Provincial
Key
Laboratory
of
South
China
Structural
Heart
Disease
[No. 2012A061400008]
and
Guangdong
Provincial
Clinical
Research
Center
for
Cardiovascular
Disease [No. 2020B1111170011]. This work was supported by the National Key Research and
Development Program [No. 2018YFC1002600], the National
Natural Science Foundation of China [No. 81903287], the
Natural Science Foundation of Guangdong Province [Nos. 2021A1515011445
and
2020A1515010743],
Guangdong
Provincial
Key
Laboratory
of
South
China
Structural
Heart
Disease
[No. 2012A061400008]
and
Guangdong
Provincial
Clinical
Research
Center
for
Cardiovascular
Disease [No. 2020B1111170011]. AUTHOR CONTRIBUTIONS YO and JC conceived and designed the study and designed the
protocol. YO, JM, and MF conducted the statistical analysis. MB
critically reviewed the manuscript. All authors contributed to the
article and approved the submitted version. ETHICS STATEMENT The studies involving human participants were reviewed and
approved by the Ethics Committee of Guangdong Provincial
People’s
Hospital
(Reference
number:
GDREC2011135H). Written
informed
consent
to
participate
in
this
study
was
provided
by
the
participants’
legal
guardian/next
of kin. However, there were several potential limitations of the study. First, the GRCHD is not a province-wide registry, only covering
about 12% newborns from the whole province. Therefore, it
could not capture all CHD cases from the province. However, the
GRCHD included the largest local hospitals and maternal and
child care centers from each city across Guangdong province,
which have the capability of detecting CHD prenatally or in
newborns. We think the GRCHD could represent the overall level
for diagnosing CHD across the whole province. Moreover, there
was no control group without any program implementation, or
a parallel data set for comparison. An ongoing project using
two parallel population-based cohorts with and without program
implementation, is conducted in Zhongshan City, Guangdong
Province, which is expected to shorten this limitation. Several
other factors may contribute to improved PDR, including
affordability, social insurance schemes, and access to healthcare
in general. It was a limitation that we did collect these data to
evaluate their influence. Furthermore, the influence of differences
in diagnosis time on later mortality, morbidity, and quality of life
of the affected babies is not currently available. The long-term
follow-up for these CHD patients and prognosis evaluation is
required, to validate the effect of prenatal diagnosis. REFERENCES Marek
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chamber view plus three-vessel and trachea view for a complete evaluation
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JP, et al. Hospitalizations, costs, and mortality among infants with critical
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screening for cardiac anomalies: the value of routine addition of the aortic
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diagnosis and treatment planning of congenital heart defects-possibilities and
limits. World J Pediatr. (2009) 5:18–22. doi: 10.1007/s12519-009-0003-8 22. DATA AVAILABILITY STATEMENT Supplementary Figure 3 | Trend for prenatal diagnosis rates for congenital heart
disease during pre-, mid- and post-program time intervals, by invisible or visible
on the 4-chamber view of fetal ultrasound screening. ∗P<0.05 compared to the
pre-program PDR in lesion invisible on 4-chamber view. Supplementary Figure 3 | Trend for prenatal diagnosis rates for congenital heart
disease during pre-, mid- and post-program time intervals, by invisible or visible
on the 4-chamber view of fetal ultrasound screening. ∗P<0.05 compared to the
pre-program PDR in lesion invisible on 4-chamber view. The datasets presented in this article are not readily available,
because the data was generated from different hospitals of the May 2022 | Volume 10 | Article 886262 Frontiers in Public Health | www.frontiersin.org Improving Program for CHD’s Prenatal Diagnosis Ou et al. Copyright © 2022 Ou, Bloom, Mai, Francois, Pan, Xiao, Wang, Nie, Qu, Gao, Wu,
Liu, Zhuang and Chen. 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) and the
copyright owner(s) 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. Frontiers in Public Health | www.frontiersin.org REFERENCES Practice guidelines for the performance of fetal echocardiography
in Guangdong province. Int Med Health Guid News. (2015) 21:739–40.. Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. 14. Guangdong
Provincial
People’s
Hospital,
Guangdong
Cardiovascular
Institute, Guangdong Eugenics Association Council on Congenital Heart
Diseases. Practice Guidelines for the Performance of Prenatal Counseling
on Fetal Congential Heart Diseases in Guangdong Province. Int Med Health
Guid News. (2015) 21:1033–36. Copyright © 2022 Ou, Bloom, Mai, Francois, Pan, Xiao, Wang, Nie, Qu, Gao, Wu,
Liu, Zhuang and Chen. 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) and the
copyright owner(s) 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. 15. Olney RS, Ailes EC, Sontag MK. Detection of critical congenital heart
defects: review of contributions from prenatal and newborn screening. Semin
Perinatol. (2015) 39:230–7. doi: 10.1053/j.semperi.2015.03.007 16. Ailes EC, Gilboa SM, Riehle-Colarusso T, Johnson CY, Hobbs CA, Correa A,
et al. Prenatal diagnosis of nonsyndromic congenital heart defects. Prenatal
Diag. (2014) 34:214–22. doi: 10.1002/pd.4282 May 2022 | Volume 10 | Article 886262 Frontiers in Public Health | www.frontiersin.org 10
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Angiogenesis Dysregulation in Term Asphyxiated Newborns Treated with Hypothermia
|
PloS one
| 2,015
|
cc-by
| 8,410
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Objective This study aimed to evaluate the expression of angiogenesis-related protein markers in as-
phyxiated newborns developing and not developing brain injury compared to healthy control
newborns. Published: May 21, 2015 Copyright: © 2015 Shaikh 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. OPEN ACCESS OPEN ACCESS
Citation: Shaikh H, Boudes E, Khoja Z, Shevell M,
Wintermark P (2015) Angiogenesis Dysregulation in
Term Asphyxiated Newborns Treated with
Hypothermia. PLoS ONE 10(5): e0128028. doi:10.1371/journal.pone.0128028
Academic Editor: Masaki Mogi, Ehime University
Graduate School of Medicine, JAPAN
Received: November 20, 2014
Accepted: April 21, 2015
Published: May 21, 2015
Copyright: © 2015 Shaikh 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. OPEN ACCESS
Citation: Shaikh H, Boudes E, Khoja Z, Shevell M,
Wintermark P (2015) Angiogenesis Dysregulation in
Term Asphyxiated Newborns Treated with
Hypothermia. PLoS ONE 10(5): e0128028. doi:10.1371/journal.pone.0128028
Academic Editor: Masaki Mogi, Ehime University
Graduate School of Medicine, JAPAN
Received: November 20, 2014
Accepted: April 21, 2015
Published: May 21, 2015
Copyright: © 2015 Shaikh 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. Citation: Shaikh H, Boudes E, Khoja Z, Shevell M,
Wintermark P (2015) Angiogenesis Dysregulation in
Term Asphyxiated Newborns Treated with
Hypothermia. PLoS ONE 10(5): e0128028. doi:10.1371/journal.pone.0128028 Academic Editor: Masaki Mogi, Ehime University
Graduate School of Medicine, JAPAN
Received: November 20, 2014
Accepted: April 21, 2015
Published: May 21, 2015 Academic Editor: Masaki Mogi, Ehime University
Graduate School of Medicine, JAPAN Angiogenesis Dysregulation in Term
Asphyxiated Newborns Treated with
Hypothermia Henna Shaikh1, Elodie Boudes1, Zehra Khoja1, Michael Shevell2, Pia Wintermark1*
1 Division of Newborn Medicine, Department of Pediatrics, McGill University, Montreal, Quebec, Canada,
2 Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada 1 Division of Newborn Medicine, Department of Pediatrics, McGill University, Montreal, Quebec, Canada,
2 Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada * pia.wintermark@bluemail.ch a11111 Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Funding: Pia Wintermark received research grant
funding from the Fonds de la Recherche en Santé
Québec (FRQS) Clinical Research Scholar Career
Award Junior 1, NeuroDevNet Networks of Centres of
Excellence (NCE) Cerebral Palsy Demonstration
Project, Canadian Institutes of Health Research
(CIHR) Operating Grant, and New Investigator
Research Grant from the SickKids Foundation and
the CIHR Institute of Human Development, Child and
Youth Health (IHDCYH). RESEARCH ARTICLE Background Neonatal encephalopathy following birth asphyxia is a major predictor of long-term neuro-
logical impairment. Therapeutic hypothermia is currently the standard of care to prevent
brain injury in asphyxiated newborns but is not protective in all cases. More robust and ver-
satile treatment options are needed. Angiogenesis is a demonstrated therapeutic target in
adult stroke. However, no systematic study examines the expression of angiogenesis-
related markers following birth asphyxia in human newborns. Design/Methods Twelve asphyxiated newborns treated with hypothermia were prospectively enrolled; six
developed eventual brain injury and six did not. Four healthy control newborns were also in-
cluded. We used Rules-Based Medicine multi-analyte profiling and protein array technolo-
gies to study the plasma concentration of 49 angiogenesis-related proteins. Mean protein
concentrations were compared between each group of newborns. Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Angiogenesis Dysregulation following Birth Asphyxia anti-angiogenic proteins, including insulin-growth factor binding proteins -1, -4, and -6, com-
pared to healthy newborns. anti-angiogenic proteins, including insulin-growth factor binding proteins -1, -4, and -6, com-
pared to healthy newborns. Competing Interests: This manuscript has been
contributed to, seen and approved by all the authors. The authors declare that they have no conflict of
interest. All the authors fulfill the authorship credit
requirements. No honorarium, grant or other form of
payment was received for the preparation of
this manuscript. Conclusions These findings suggest that angiogenesis pathways are dysregulated following birth as-
phyxia and are putatively involved in brain injury pathology and recovery. Results Compared to healthy newborns, asphyxiated newborns not developing brain injury showed
up-regulation of pro-angiogenic proteins, including fatty acid binding protein-4, glucose-6-
phosphate isomerase, neuropilin-1, and receptor tyrosine-protein kinase erbB-3; this up-
regulation was not evident in asphyxiated newborns eventually developing brain injury. Also, asphyxiated newborns developing brain injury showed a decreased expression of 1 / 15 PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Introduction In angiogenesis, a coordinated orchestra of proteins causes new blood vessels to sprout and
mature from existing vessels. This process begins with the degradation of the local extracellular
matrix and the weakening of tight junctions and interactions between endothelial cells and
pericytes. Vascular destabilization permits endothelial cell proliferation and migration to a site
of nascent tube formation. Angiogenesis is completed as intercellular interactions and the ex-
tracellular matrix are re-established to stabilize the new blood vessels [1]. Recent studies of
adult stroke highlight angiogenesis as a potential therapeutic target. Enhanced angiogenesis is
accompanied by enhanced neurogenesis and improved neurological recovery in animal models
of adult stroke [2–4] and is correlated with improved outcomes in human adults [5]. Neonatal encephalopathy is a major predictor of neonatal death and long-term neurological
deficits, including cerebral palsy, intellectual disability, and epilepsy [6]. Currently, neonatal
encephalopathy attributed to asphyxia is treated with mild hypothermia, which has been
shown to reduce mortality and morbidity in clinical trials [7]. However, hypothermia must be
initiated within 6 hours of life, requires cumbersome equipment, and shows decreased benefits
for severely asphyxiated newborns [8]; thus more robust and versatile treatment options are
needed. While hypothermia protects the brain from acute damage due to hypoxia-ischemia,
enhancing angiogenesis may help to restore the neurovascular niche, facilitating optimal neu-
ronal and glial re-growth in the sub-acute and chronic phases of hypoxia-ischemia [9, 10]. A
few angiogenic markers have been studied in asphyxiated newborns and in rodent models of
neonatal encephalopathy; these studies have demonstrated increased expression of VEGF, its
receptors, and other angiogenic proteins following the injury [11–15]. However, to date, no
systematic study examines the broader expression of angiogenesis-related markers following
asphyxia injury in human newborns. We hypothesized that angiogenesis is activated following birth asphyxia, and that this acti-
vation may differ between asphyxiated newborns developing and not developing brain injury. Thus, the objective of this study was to evaluate the expression of angiogenesis-related protein
markers in asphyxiated newborns treated with hypothermia developing and not developing
brain injury compared to healthy control newborns. Competing Interests: This manuscript has been
contributed to, seen and approved by all the authors.
The authors declare that they have no conflict of
interest. All the authors fulfill the authorship credit
requirements. No honorarium, grant or other form of
payment was received for the preparation of
this manuscript. Imaging The presence or absence of brain injury in asphyxiated and healthy control newborns was de-
fined by magnetic resonance imaging obtained around day 10 of life (range: day 9–13 of life),
as they have previously been reported to define precisely the extent of the brain injury in these
newborns [19–21]. Neuroradiologists blinded to the newborns’ clinical condition reviewed the
images and scored them using a previously described system for evaluating brain injury in as-
phyxiated newborns [22]. For the purpose of this study, asphyxiated newborns treated with hy-
pothermia were then classified as “with” or “without injury” depending on whether or not any
brain injury was observed on the magnetic resonance imaging. Methods
Patients We conducted a cohort study of term asphyxiated newborns admitted to our neonatal intensive
care unit who met the criteria for induced hypothermia [16–18]: (1) gestational age 36 weeks
and birth weight 2000 g; (2) evidence of fetal distress, e.g. history of an acute perinatal event,
cord pH 7.0 or base deficit 16 mEq/L; (3) evidence of neonatal distress, such as an Apgar
score 5 at 10 minutes, postnatal blood gas pH obtained within the first hour of life 7.0 or 2 / 15 PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Angiogenesis Dysregulation following Birth Asphyxia base deficit 16 mEq/L, or a need for ventilation initiated at birth and continued for at least
10 minutes; and (4) evidence of moderate to severe neonatal encephalopathy as evident by an
abnormal standard neurological exam and/or amplitude-integrated electroencephalogram. Eli-
gible patients received whole-body cooling to an esophageal temperature of 33.5°C initiated
within the first 6 hours of life, continued for 72 hours, and followed by slow rewarming. Clinical data, including gestational age, birth weight, sex, Apgar score at 10 minutes, use of
intubation and chest compression at birth, arterial cord pH, and initial infant blood gas pH
were collected prospectively for each asphyxiated newborn. The hour of life at which hypother-
mia was initiated was also calculated and recorded. Healthy term newborns with normal brain MRI findings were included as healthy controls. The research protocol was approved by the research ethics board from the Montreal Children’s
Hospital, McGill University Health Centre, and informed written consent was obtained from
the parents on behalf of their newborns in all cases. PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Angiogenesis-related proteins Nurses collected blood samples in tubes containing potassium EDTA at 24 hours of life for
healthy control newborns and at 6, 24, 48, 72, and 96 hours of life for asphyxiated newborns
treated with hypothermia. Blood samples were immediately centrifuged at 3600 rpm for 6 min-
utes at room temperature. Following centrifugation, plasma was removed, aliquotted, and
stored at -80°C until tested. Plasma samples were coded, and protein markers were measured
blindly with respect to patient identity and disease. The samples were thawed at room temperature, vortexed, spun at 13,000 x g for 5 minutes
for clarification and 40 mcL were transferred to a master microtiter plate for multi-analyte pro-
filing (MAP) antigen analysis. Using automated pipetting, an aliquot of each sample was intro-
duced into one of the capture microsphere multiplexes of the Rules-Based Medicine (Myriad
RBM) Custom Human multi-analyte profile (MAP) (Myriad, Austin, TX) (http://www.rules-
basedmedicine.com). These mixtures of sample and capture microspheres were thoroughly
mixed and incubated at room temperature for 1 hour. Multiplexed cocktails of biotinylated, re-
porter antibodies for each multiplex were then added robotically and, after thorough mixing,
were incubated for an additional hour at room temperature. Multiplexes were developed using
an excess of streptavidin-phycoerythrin solution, which was thoroughly mixed into each multi-
plex and incubated for 1 hour at room temperature. The volume of each multiplexed reaction
was reduced by vacuum filtration and the volume increased by dilution into matrix buffer for
analysis. Analysis was performed in a Luminex 100 instrument and the resulting data stream
was interpreted using proprietary data analysis software developed at Rules-Based Medicine
(Myriad RBM). For each multiplex, both calibrators and controls were included on each micro-
titer plate. 8-point calibrators were run in the first and last column of each plate and 3-level
controls were included in duplicate. Testing results were determined first for the high, medium 3 / 15 PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Angiogenesis Dysregulation following Birth Asphyxia and low controls for each multiplex to ensure proper assay performance. Unknown values for
each of the analytes localized in a specific multiplex were determined using 4- and 5- parameter
weighted and non-weighted curve fitting algorithms included in the data analysis package. Ex-
pression of 49 angiogenesis-related proteins was thus analyzed. Patients Twelve asphyxiated newborns treated with hypothermia and four healthy control newborns
were included in this study. Six of the asphyxiated newborns developed brain injury; four de-
veloped a basal ganglia injury pattern (“Fig 1”) and two a watershed injury pattern. The re-
maining six newborns did not develop any brain injury. Findings on brain MRI of the healthy
control newborns were normal. Clinical characteristics of all asphyxiated newborns treated
with hypothermia are shown in “Table 2”. Gestational age, birth weight, sex, Apgar score at 10
minutes, use of intubation and chest compression at birth, arterial cord pH, and initial blood
gas pH were not different between the asphyxiated newborns developing and not developing
brain injury. Angiogenesis-related proteins Analyzed proteins, listed in
“Table 1”, were selected based on angiogenic involvement and assay availability; the different
pathways in which they act are schematically represented in “S1 Fig”. Plasma concentrations
were reported for each sample unless the sample quantity was not sufficient (QNS), the con-
centration was below the lower limit of quantification (LLOQ), or the concentration was above
the highest quantifiable value. Data analysis Protein expression data were represented as mean concentration ± standard deviation for each
group of newborns. To test differences in protein expression between groups, we used Mann-
Whitney U tests. A p value < 0.05 was used to highlight protein expression differences between
groups. All analyses were performed with SPSS Version 20.0 for Windows (SPSS Inc., Chicago,
IL, USA). For graphical representation, ratios of group means were calculated and log trans-
formed to obtain fold change data for each protein marker. In addition, to improve data inter-
pretation, a DAVID functional clustering of Gene Ontology Terms was performed [23–24]. PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Expression of angiogenesis-related proteins at 24 hours of life Mean protein concentration values were compared between asphyxiated newborns developing
and not developing brain injury and healthy newborns at 24 hours of life (“Fig 2”). p
g
j
y
y
g
Asphyxiated newborns treated with hypothermia showed altered expression in 10 of the 49
angiogenesis-related proteins compared to healthy newborns (“Fig 2A”). Matrix metalloprotei-
nase-9 (MMP-9), which is important for degrading the extracellular matrix, was decreased. Fatty acid binding protein-4 (FABP-4) and galectin-3 (Gal-3), which enhance endothelial cell
survival, proliferation and migration, were up-regulated among asphyxiated newborns, while
kallikrein-5 (KLK-5) was down-regulated. VEGF-C, which promote endothelial cell permeabil-
ity along with survival and proliferation, was decreased. Brain derived neurotrophic factor
(BDNF), which has a dual impact on neurogenesis and angiogenesis, was down-regulated. The
anti-angiogenic proteins fibulin-1C (Fib-1C) and insulin-like growth factor-binding protein-6
(IGF-BP-6) were down-regulated in asphyxiated newborns. Finally, while the apoptotic protein
Fas ligand (FasL) showed decreased expression, its receptor (FasR) showed increased expres-
sion. When performing a DAVID functional clustering of Gene Ontology Terms using the 49
angiogenesis-related proteins as background and the abovementioned 10 dysregulated proteins PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 4 / 15 Angiogenesis Dysregulation following Birth Asphyxia Table 1. Angiogenesis-related protein markers analyzed in the study. Vascular
Destabilization
Endothelial cells
survival,
proliferation and
migration
Neurogenesis and
angiogenesis
Anti-angiogenesis
Apoptosis
Angiopoietin-2
(Ang-2)
Angiogenin (Ang)
Kallikrein 5 (KLK-5)
Brain-Derived
Neurotrophic Factor
(BDNF)
Endostatin
Fas Ligand (FasL)
Cathepsin D
AXL Receptor
Tyrosine Kinase
(AXL)
Macrophage-
Stimulating Protein
(MSP)
Neuron-Specific
Enolase (NSE)
Fibulin-1C (Fib-1C)
FasL Receptor (FasR)
Hepsin
Endoglin
Neuropilin-1 (NP-1)
Neuronal Cell Adhesion
Molecule (Nr-CAM)
Human Epididymis
Protein 4 (HE4)
Sortilin
Matrix
Metalloproteinase-3
(MMP-3)
Epidermal Growth
Factor Receptor
(EGFR)
Receptor tyrosine-
protein kinase erbB-3
(ErbB3)
Insulin-like Growth Factor-
Binding Protein 1
(IGFBP-1)
TNF-Related
Apoptosis-Inducing
Ligand Receptor 3
(TRAIL-R3)
Matrix
Metalloproteinase-7
(MMP-7)
Fatty Acid-Binding
Protein 4, Adipocyte
(FABP-4)
Stem Cell Factor
(SCF)
Insulin-like Growth Factor
Binding Protein 4
(IGFBP4)
Matrix
Metalloproteinase-9
(MMP-9)
Galectin-3
Superoxide
Dismutase 1, soluble
(SOD-1)
Vascular stabilization
Insulin-like Growth Factor
Binding Protein 5
(IGFBP5)
Matrix
Metalloproteinase-10
(MMP-10)
Glucose-6-phosphate
Isomerase (G6PI)
Tenascin-C (TN-C)
Cellular Fibronectin
(cFib)
Insulin-like Growth Factor
Binding Protein 6
(IGFBP6)
Heparin-Binding
EGF-Like Growth
Factor (HB-EGF)
Tumor necrosis factor
receptor 2 (TNFR2)
Collagen IV
Tissue Inhibitor of
Metalloproteinases 1
(TIMP-1)
Human Epidermal
Growth Factor
Receptor 2 (HER-2)
Vascular Endothelial
Growth Factor C
(VEGF-C)
Tyrosine kinase with Ig
and EGF homology
domains 2 (TIE-2)
Insulin-like Growth
Factor-Binding
Protein 2 (IGFBP-2)
Vascular Endothelial
Growth Factor
Receptor 2 (VEGFR-
2)
Insulin-like Growth
Factor-Binding
Protein 3 (IGFBP-3)
Vascular endothelial
growth factor receptor
3 (VEGFR-3)
Intercellular Adhesion
Molecule 1 (ICAM-1)
YKL-40
The 49 proteins analyzed in this study are categorized according to their predominant role in angiogenesis. Obviously, many proteins serve multiple,
context-dependent functions and may fit into more than one category. Table 1. Angiogenesis-related protein markers analyzed in the study. The 49 proteins analyzed in this study are categorized according to their predominant role in angiogenesis. Obviously, many proteins serve multiple,
context-dependent functions and may fit into more than one category. The 49 proteins analyzed in this study are categorized according to their predominant role in angiogenesis. Obviously, many proteins serve multiple,
context-dependent functions and may fit into more than one category. doi:10.1371/journal.pone.0128028.t001 as the Gene List, one cluster of proteins stood out all related to extracellular signaling activities
(MMP-9, KLK-5, Fib-1C and IGF-BP6); six proteins could not be grouped. When comparing asphyxiated newborns developing and not developing brain injury at 24
hours of life, the expression of 10 angiogenesis-related proteins was decreased in the prior
group compared to the latter (“Fig 2B”). PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Two of these proteins, angiopoietin-2 (Ang-2) and
hepsin destabilize the vasculature to permit new sprouting. Expression of heparin-binding epi-
dermal growth factor-like growth factor (HB-EGF), which is important for endothelial cell sur-
vival, proliferation, and migration, was decreased. Neuropilin-1 (NP-1), receptor tyrosine-
protein kinase erbB-3 (Erb-B3), and YKL-40, which promote similar endothelial cell effects,
also showed diminished expression. Expression of the angiogenic inhibitors IGF-BP-1, PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 5 / 15 Angiogenesis Dysregulation following Birth Asphyxia Fig 1. Brain MRIs of a term asphyxiated newborn treated with hypothermia, performed on day 9 of life. (A) The T2-weighted imaging shows the injury within the thalami and lentiform nuclei (arrows). doi:10 1371/journal pone 0128028 g001 Fig 1. Brain MRIs of a term asphyxiated newborn treated with hypothermia, performed on day 9 of life. (A) The T2-weighted imaging shows the injury within the thalami and lentiform nuclei (arrows). doi:10 1371/journal pone 0128028 g001 Fig 1. Brain MRIs of a term asphyxiated newborn treated with hypothermia, performed on day 9 of life. (A) The T2-weighted imaging shows the injury within the thalami and lentiform nuclei (arrows). Table 2. Clinical characteristics of the asphyxiated newborns treated with hypothermia. Variables
Healthy
newborns
(n = 4)
All asphyxiated
newborns (n = 12)
Asphyxiated newborns treated
with hypothermia not developing
brain injury (n = 6)
Asphyxiated newborns treated
with hypothermia developing
brain injury (n = 6)
p value
Clinical
characteristics
Gestational age
(weeks), mean ± SD
39.39 ± 0.85
39.08 ± 1.89
38.86 ± 184
39.31 ± 2.09
0.63
Birth weight (g),
mean ± SD
3412 ± 525
3131 ± 608
3183 ± 276
3080 ± 855
0.87
Sex
0.51
Male, n(%)
1 (25)
9 (75)
5 (83)
4 (67)
Female, n(%)
3 (75)
3 (25)
1 (17)
2 (33)
Apgar score 5 at 10
minutes, n(%)
0 (0)
9 (75)
5 (83)
4 (67)
0.51
Intubation at birth, n(%)
0 (0)
11 (92)
6 (100)
5 (83)
0.30
Chest compression at
birth, n(%)
0 (0)
7 (58)
4 (67)
3 (50)
0.56
Arterial cord pH,
mean ± SD
-
6.96 ± 0.18
7.03 ± 0.13
6.87 ± 0.21
0.33
Initial postnatal blood
gas pH, mean ± SD
-
7.06 ± 0.19
7.11 ± 0.15
7.03 ± 0.21
0.61
Initiation of hypothermia
(hours), mean ± SD
-
5.17 ± 0.86
5.29 ± 0.88
5.06 ± 0.92
0.69
doi:10.1371/journal.pone.0128028.t002 Table 2. Clinical characteristics of the asphyxiated newborns treated with hypothermia. PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 6 / 15 Angiogenesis Dysregulation following Birth Asphyxia Fig 2. Expression of angiogenesis-related protein markers at 24 hours of life. Mean concentrations in
each group were compared. p values were calculated to highlight significant protein expression differences
between the groups (* p < 0.05). For graphical representation, ratios between group mean concentrations
were calculated and log transformed to obtain fold change data for each marker. (A) Fold changes in protein
expression between asphyxiated newborns treated with hypothermia and healthy newborns. (B) Fold
changes in protein expression between asphyxiated newborns developing and not developing brain injury. doi:10.1371/journal.pone.0128028.g002 Fig 2. Expression of angiogenesis-related protein markers at 24 hours of life. Mean concentrations in
each group were compared. p values were calculated to highlight significant protein expression differences
between the groups (* p < 0.05). For graphical representation, ratios between group mean concentrations
were calculated and log transformed to obtain fold change data for each marker. (A) Fold changes in protein
expression between asphyxiated newborns treated with hypothermia and healthy newborns. (B) Fold
changes in protein expression between asphyxiated newborns developing and not developing brain injury. doi:10.1371/journal.pone.0128028.g002 IGF-BP-4, and IGF-BP-5 and the apoptotic protein sortilin was also decreased. When perform-
ing a DAVID functional clustering of Gene Ontology Terms using the 49 angiogenesis-related
proteins as background and the abovementioned 10 dysregulated proteins as the Gene List,
one cluster of proteins stood out all related to extracellular signaling activities (Ang-2, Erb-B3,
HB-EGF, IGF-BP-1, IGF-BP-4, IGF-BP-5, NP-1, YKL-40); one protein could not be grouped. IGF-BP-4, and IGF-BP-5 and the apoptotic protein sortilin was also decreased. When perform-
ing a DAVID functional clustering of Gene Ontology Terms using the 49 angiogenesis-related
proteins as background and the abovementioned 10 dysregulated proteins as the Gene List,
one cluster of proteins stood out all related to extracellular signaling activities (Ang-2, Erb-B3,
HB-EGF, IGF-BP-1, IGF-BP-4, IGF-BP-5, NP-1, YKL-40); one protein could not be grouped. Comparisons in protein expression between asphyxiated newborns not developing brain in-
jury and healthy newborns, as well as between asphyxiated newborns developing brain injury
and healthy newborns were included in “S2 Fig”. Comparisons in protein expression between asphyxiated newborns not developing brain in-
jury and healthy newborns, as well as between asphyxiated newborns developing brain injury
and healthy newborns were included in “S2 Fig”. PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Evolution of angiogenesis-related protein expression in the first days of
life Mean protein concentration values were also compared between asphyxiated newborns devel-
oping and not developing brain injury throughout the first days of life, i.e., at 6, 24, 48, 72, and
96 hours of life (“Fig 3”). Among the vessel destabilizing proteins, hepsin expression was de-
creased early after asphyxia at 6 and 24 hours of life in newborns developing brain injury. Ca-
thepsin-D (Cat-D) and MMP-3 expression decreased later at 72 hours. Angiopoietin-2 showed
an early and late decrease at 24 and 96 hours. Among other proteins like hepsin, involved in
endothelial cell survival, proliferation, and migration, macrophage-stimulating protein (MSP)
showed an early decrease at 6 hours of life. HB-EGF, NP-1, Erb-B3, and YKL-40 were de-
creased at 24 and 48 hours of life, and NP-1 was also decreased at 96 hours of life. Tumor ne-
crosis factor receptor-2 (TNFR-2) was decreased at 48 and 72 hours of life. Later, at 96 hours of 7 / 15 PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Angiogenesis Dysregulation following Birth Asphyxia Fig 3. Evolution of angiogenesis-related protein expression over time in asphyxiated newborns
developing brain HI injury compared to asphyxiated newborns not developing brain HI injury. The
heat map depicts the fold changes in protein concentration between the two groups. Fold change ranges are
denoted by the color coding in the legend at the right. ** p < 0.05. doi:10.1371/journal.pone.0128028.g003 Fig 3. Evolution of angiogenesis-related protein expression over time in asphyxiated newborns
developing brain HI injury compared to asphyxiated newborns not developing brain HI injury. The
heat map depicts the fold changes in protein concentration between the two groups. Fold change ranges are
denoted by the color coding in the legend at the right. ** p < 0.05. doi:10.1371/journal.pone.0128028.g003 doi:10.1371/journal.pone.0128028.g003 life, endoglin, intercellular adhesion molecule-1 (ICAM-1) and neuronal cell adhesion mole-
cule (NrCAM) were decreased. Among vessel stabilizing proteins, cellular fibronectin (cFib)
expression was decreased at 48 and 72 hours of life. The anti-angiogenic protein IGF-BP-5
showed an early decrease at 6 hours of life, which was maintained at 24 and 72 hours of life. IGF-BP-1 and IGF-BP-4 expression decreased at 24 and 48 hours of life. IGF-BP-6 showed a
later decrease in expression at 72 and 96 hours of life. Another anti-angiogenic protein, tissue
inhibitor of metalloproteinases-1 (TIMP-1), was also decreased at 48, 72, and 96 hours of life. Evolution of angiogenesis-related protein expression in the first days of
life Finally, among apoptotic proteins, tumor necrosis factor-related apoptosis-inducing ligand re-
ceptor-3 (TRAIL-R3) showed an early increase at 6 hours of life, sortilin was decreased at 24
and 48 hours of life and FasL expression was decreased at 72 hours of life. Angiogenesis Dysregulation following Birth Asphyxia endothelial cell survival, proliferation, and migration. This suggests that up-regulation of pro-
angiogenic proteins may be neuroprotective or neurorestorative in asphyxiated newborns. Pre-
vious studies examining the individual roles of these proteins support their isolated effects on
neuroprotection and neurorestoration. For example, animal models of adult stroke show that
NP-1 facilitates VEGF signaling to promote neurorestoration [25]. HB-EGF-related neuropro-
tection has also been demonstrated in adult rodents subjected to experimental stroke [26–28]. Additionally, increased Gal3 expression has been found in rodents following experimental hyp-
oxia-ischemia [29] and in the cerebrospinal fluid of asphyxiated newborns, where greater ex-
pression was correlated with a worse outcome [30]. Intriguingly, asphyxiated newborns developing brain injury showed decreased expression of
four anti-angiogenic proteins (i.e., Fib-1C, IGF-BP-1, IGF-BP-4, and IGF-BP-6) compared to
healthy newborns, while only two of these proteins (i.e., Fib-1C and IGF-BP-6) were down-
regulated in asphyxiated newborns not developing brain injury compared to healthy newborns. In addition, IGF-BP-1, -4, and -5 were less expressed in asphyxiated newborns developing
brain injury compared to those not developing brain injury. Evidence suggests that IGF-BP-1,
-4, -5 and -6, which bind IGF-1, inhibit angiogenesis [31–33]. While no previous studies have
examined the expression of the IGF-BPs after neonatal encephalopathy, animal models of neo-
natal hypoxia-ischemia demonstrate conflicting findings on IGF-1 expression after the insult
[34–37], and decreased serum IGF-1 has been reported in asphyxiated newborns [38]. The de-
creased expression of IGF-BPs and other anti-angiogenic proteins observed in the present
study may represent a mechanism for compensating for a failure to adequately up-regulate
pro-angiogenic proteins in asphyxiated newborns developing brain injury. In this study, protein expression was also altered in other steps of the angiogenesis pathway,
beginning with vascular destabilization. MMP-9 expression was decreased among asphyxiated
newborns not developing brain injury. Previous studies have found that MMP-9, which degrades
the extracellular matrix to facilitate vascular sprouting [39], is increased following hypoxia-ische-
mia in rodent models [40–42] and human newborns [42–43]. However, in another study, no
change was found in cerebrospinal fluid MMP-9 concentration following birth asphyxia in
human newborns [30]. Beyond facilitating angiogenesis, this increase in MMP-9 may contribute
to brain injury via blood brain barrier (BBB) disruption and neuroinflammation [40]. Therefore,
the decrease in MMP-9 expression presently observed among asphyxiated newborns not devel-
oping brain injury may reflect an endogenous neuroprotective or neurorestorative mechanism
or an effect of the hypothermia treatment. Discussion This is the first systematic study of the expression of angiogenesis-related protein markers fol-
lowing birth asphyxia in human newborns. The expression of angiogenesis-related proteins
was clearly dysregulated in asphyxiated newborns treated with hypothermia compared to
healthy newborns, suggesting that angiogenesis plays an important but under-recognized role
following birth asphyxia. Interestingly, asphyxiated newborns who did not develop brain injury showed increased ex-
pression of four proteins involved in endothelial cell survival, proliferation, and migration (i.e.,
FABP4, G6PI, NP-1 and Erb-B3) compared to healthy newborns, while only one such protein
(i.e., Gal3) was up-regulated in asphyxiated newborns developing brain injury. In comparison
to asphyxiated newborns not developing brain injury, those who did develop an injury showed
decreased expression of HB-EGF, NP-1, Erb-B3 and YKL-40, four proteins that promote 8 / 15 PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Angiogenesis Dysregulation following Birth Asphyxia This study also demonstrated decreased BDNF expression in all asphyxiated newborns. Pre-
viously, in studies of asphyxiated human newborns, increased BDNF levels have been found in
cord blood at birth [57], blood serum 24 hours after birth [58], and cerebrospinal fluid [59], This study also demonstrated decreased BDNF expression in all asphyxiated newborns. Pre-
viously, in studies of asphyxiated human newborns, increased BDNF levels have been found in
cord blood at birth [57], blood serum 24 hours after birth [58], and cerebrospinal fluid [59],
with the degree of increased expression correlated to brain injury severity [58]. Increased brain
BDNF expression was also found in a rat model of neonatal hypoxia-ischemia [60]. Decreased
BDNF expression may thus again reflect a neuroprotective or neurorestorative response or an
effect of the hypothermia treatment. with the degree of increased expression correlated to brain injury severity [58]. Increased brain
BDNF expression was also found in a rat model of neonatal hypoxia-ischemia [60]. Decreased
BDNF expression may thus again reflect a neuroprotective or neurorestorative response or an
effect of the hypothermia treatment. Among the proteins important for vascular stabilization, collagen IV expression was de-
creased in asphyxiated newborns developing brain injury compared to healthy newborns. No
previous studies of collagen IV expression following birth asphyxia exist; however, this protein
is an essential component of the extracellular matrix, and its breakdown is necessary for new
vessel sprouting. Therefore, decreased collagen IV expression may again indicate some com-
pensatory mechanism for a failure to adequately up-regulate pro-angiogenic proteins in as-
phyxiated newborns developing brain injury. Among apoptotic proteins, FasR was up-regulated in all asphyxiated newborns while FasL
was down-regulated in asphyxiated newborns developing brain injury compared to healthy
newborns. Additionally, sortilin expression was decreased in asphyxiated newborns developing
brain injury compared to those not developing brain injury. No previous studies of serum con-
centrations of these proteins in asphyxiated newborns exist; however, Fas signaling has been
shown to contribute to brain injury following neonatal hypoxia-ischemia [61]. This study was intended to highlight whether angiogenesis pathways are preferentially acti-
vated following birth asphyxia. As per the DAVID functional clustering of Gene Ontology
Terms, the dysregulated angiogenesis-related proteins were mostly involved in extracellular
signaling activities, but also in neurogenesis and programmed cell death. Given that this study
demonstrates a significant role for angiogenesis after birth asphyxia, further investigation with
larger sample sizes and targeted analysis of angiogenesis-related protein expression is defini-
tively warranted. An important next step also includes validation of the dysregulated proteins
using a distinct technique (e.g. Western blot). Such investigation may lead to the discovery of
potential novel therapeutic targets. It would have also been ideal to study a group of asphyxiat-
ed newborns not treated with hypothermia so to distinguish how the hypothermia treatment
rather than the asphyxia influenced some of the observed changes. However, as cooling is now
the standard of care, it is no longer ethically possible to not receive hypothermia to assess the
evolution over time of these angiogenesis-related proteins. In this study, systemic plasma samples were analyzed rather than brain-specific samples. While studying cerebrospinal fluid would have provided a more direct view of the brain’s an-
giogenic response, ethical and practical concerns make it difficult to collect serial cerebrospinal
fluid samples in sick newborns. Birth asphyxia has global effects on the newborn body, so al-
tered plasma protein expression reflects peripheral as well as brain injuries [42]. Nonetheless,
birth asphyxia does appear to disrupt the blood-brain barrier [62], which may release proteins
from the brain into the peripheral systemic circulation. A final limitation of any study of neo-
natal encephalopathy in human newborns is the difficulty of accurately determining the time
and duration of the asphyxial insult, which somewhat compromises the reliability of compari-
sons between newborns. Despite this, the current study demonstrates that angiogenic pathways
appear to be activated at a similar time in the different newborns. Ang-2 and hepsin are also involved in extracellular
matrix degradation and promote neovascular sprouting in angiogenesis [44–45]. Both Ang-2
and hepsin were expressed at lower levels in asphyxiated newborns developing brain injury, sug-
gesting that decreased expression of these proteins may contribute to injury pathology. VEGF-C expression was decreased in all asphyxiated newborns, which may represent a
mechanism for protecting against vascular permeability-related edema [46–48]. This is in con-
trast with data in rodent models demonstrating increased VEGF expression following hypoxia-
ischemia [12, 13, 40, 49] and a protective effect of VEGF administration following the insult
[50–51]. However, VEGF findings in human newborns vary more widely. While a few studies
have found increased VEGF expression in cord blood [52] and cerebrospinal fluid [53], others
have found either no difference in cerebrospinal fluid VEGF levels between asphyxiated and
healthy newborns [54] or lower serum VEGF expression in severely asphyxiated compared to
healthy newborns [55]. In the present study, KLK-5 expression was reduced in all asphyxiated
newborns; KLK-5 promotes extracellular matrix degradation and neovascular sprouting by in-
creasing MMP-9 activity, which, as previously noted, may contribute to brain injury [40, 56]. Decreased KLK-5 expression may therefore represent another possible mechanism of neuro-
protection or neurorestoration. 9 / 15 PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Supporting Information S1 Fig. Schematic representation of the different studied angiogenesis-related pathways. Expression of 49 angiogenesis-related proteins, which were selected based on angiogenic in-
volvement and assay availability, was analyzed. (TIF) S2 Fig. Expression of angiogenesis-related protein markers at 24 hours of life. Mean con-
centrations in each group were compared. p values were calculated to highlight significant pro-
tein expression differences between the groups ( p < 0.05). For graphical representation,
ratios between group mean concentrations were calculated and log transformed to obtain fold
change data for each marker. (A) Fold changes in protein expression between asphyxiated
newborns not developing brain injury and healthy newborns. Eleven angiogenesis-related
proteins showed altered expression. MMP-9, KLK-5, VEGF-C and BDNF levels remained re-
duced; however no change was evident in the levels of Gal-3. FABP-4 expression remained
elevated. In addition, a greater number of proteins enhancing endothelial cell survival, prolifer-
ation and migration were up-regulated among asphyxiated newborns not developing brain in-
jury. Expression of neuropilin-1 (NP-1), which is a VEGF receptor-2 (VEGFR-2) co-receptor,
was increased. The expression of other proteins important for endothelial cell survival, prolifer-
ation, and migration, including glucose-6-phosphate isomerase (G6PI) and receptor tyrosine-
protein kinase erbB-3 (Erb-B3), was also increased. The anti-angiogenic proteins Fib-1C and
IGF-BP-6 remained decreased. Among the apoptotic proteins, no change was apparent in FasL
expression; however FasR expression remained increased. When performing a DAVID func-
tional clustering of Gene Ontology Terms using the 49 angiogenesis-related proteins as back-
ground and the abovementioned 11 dysregulated proteins as the Gene List, two clusters of
proteins stood out, one related to neurogenesis (BDNF, NP-1, Erb-B3, VEGFC) and one relat-
ed to extracellular signaling activities (Fib-1C, IGF-BP-6, KLK-5, MMP-9); three proteins
could not be grouped. (B) Fold changes in protein expression between asphyxiated new-
borns developing brain injury and healthy newborns. Thirteen angiogenesis-related proteins
showed altered levels. While MMP-9 expression was not different between the two groups,
KLK-5, VEGF-C and BDNF still showed reduced expression. Among the proteins enhancing
endothelial cell survival, proliferation and migration, the expression of Gal3 and PlGF was still
increased; but the expression of NP-1, G6PI, Erb-B3 and FABP-4 was not different, and the ex-
pression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was de-
creased. Collagen-IV, which is important for vascular stabilization, showed reduced
expression. Among the anti-angiogenic proteins, Fib-1C and IGF-BP-6 expression remained
decreased. Conclusions In conclusion, this study highlights that angiogenesis pathways are dysregulated following
birth asphyxia and are putatively involved in brain injury pathology and recovery. Our results 10 / 15 PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 Angiogenesis Dysregulation following Birth Asphyxia suggest that a failure to up-regulate pro-angiogenic proteins may leave these newborns more
susceptible to eventual brain injury. Furthermore, those newborns who fail to up-regulate pro-
angiogenic proteins may attempt to compensate by down-regulating anti-angiogenic proteins. Overall, it seems that angiogenesis enhancement may be an important determinant of injury
outcome following birth asphyxia. These findings may have future therapeutic applications to
optimize eventual recovery following asphyxia and should be further investigated. PLOS ONE | DOI:10.1371/journal.pone.0128028
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sion of a greater number of anti-angiogenic proteins in these newborns compared to newborns
not developing brain injury. Finally, the apoptotic proteins sortilin and FasL remained de-
creased, while FasR remained increased. When performing a DAVID functional clustering of
Gene Ontology Terms using the 49 angiogenesis-related proteins as background and the
abovementioned 13 dysregulated proteins as the Gene List, two clusters of proteins stood out,
one related to programmed cell death (Fas, FasL, Sortilin) and one related to extracellular PLOS ONE | DOI:10.1371/journal.pone.0128028
May 21, 2015 11 / 15 Angiogenesis Dysregulation following Birth Asphyxia signaling activities (Collagen-IV, Fib-1C, KLK-5, IGF-BP-1, IGF-BP-4, IGF-BP-6); four pro-
teins could not be grouped. (TIF) signaling activities (Collagen-IV, Fib-1C, KLK-5, IGF-BP-1, IGF-BP-4, IGF-BP-6); four pro-
teins could not be grouped. (TIF) Acknowledgments The authors thank the families and their newborns for participating in this study. Author Contributions Conceived and designed the experiments: HS PW. Performed the experiments: HS EB ZK PW. Analyzed the data: HS PW. Contributed reagents/materials/analysis tools: HS EB ZK PW. Wrote the paper: HS EB ZK MS PW. PLOS ONE | DOI:10.1371/journal.pone.0128028
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Phospholipase A2 activity is required for immune defense of European (Apis mellifera) and Asian (Apis cerana) honeybees against American foulbrood pathogen, Paenibacillus larvae
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PLOS ONE PLOS ONE RESEARCH ARTICLE Gahyeon Jin, Md Tafim Hossain Hrithik, Eeshita Mandal, Eui-Joon Kil, Chuleui Jung,
Yonggyun KimID* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Department of Plant Medicals, Andong National University, Andong, Korea Department of Plant Medicals, Andong National University, Andong, Korea * hosanna@anu.ac.kr * hosanna@anu.ac.kr * hosanna@anu.ac.kr Abstract Honeybees require an efficient immune system to defend against microbial pathogens. The
American foulbrood pathogen, Paenibacillus larvae, is lethal to honeybees and one of the
main causes of colony collapse. This study investigated the immune responses of Apis mel-
lifera and Apis cerana honeybees against the bacterial pathogen P. larvae. Both species of
honeybee larvae exhibited significant mortality even at 102 103 cfu/mL of P. larvae by diet-
feeding, although A. mellifera appeared to be more tolerant to the bacterial pathogen than
A. cerana. Upon bacterial infection, the two honeybee species expressed both cellular and
humoral immune responses. Hemocytes of both species exhibited characteristic spreading
behaviors, accompanied by cytoskeletal extension along with F-actin growth, and formed
nodules. Larvae of both species also expressed an antimicrobial peptide called apolipo-
phorin III (ApoLpIII) in response to bacterial infection. However, these immune responses
were significantly suppressed by a specific inhibitor to phospholipase A2 (PLA2). Each hon-
eybee genome encodes four PLA2 genes (PLA2A ~ PLA2D), representing four orthologous
combinations between the two species. In response to P. larvae infection, both species sig-
nificantly up-regulated PLA2 enzyme activities and the expression of all four PLA2 genes. To
determine the roles of the four PLA2s in the immune responses, RNA interference (RNAi)
was performed by injecting gene-specific double stranded RNAs (dsRNAs). All four RNAi
treatments significantly suppressed the immune responses, and specific inhibition of the
two secretory PLA2s (PLA2A and PLA2B) potently suppressed nodule formation and ApoL-
pIII expression. These results demonstrate the cellular and humoral immune responses of
A. mellifera and A. cerana against P. larvae. This study suggests that eicosanoids play a
crucial role in mediating common immune responses in two closely related honeybees. OPEN ACCESS Citation: Jin G, Hrithik MTH, Mandal E, Kil E-J,
Jung C, Kim Y (2024) Phospholipase A2 activity is
required for immune defense of European (Apis
mellifera) and Asian (Apis cerana) honeybees
against American foulbrood pathogen,
Paenibacillus larvae. PLoS ONE 19(2): e0290929. https://doi.org/10.1371/journal.pone.0290929 Editor: Olav Rueppell, University of Alberta,
CANADA Editor: Olav Rueppell, University of Alberta,
CANADA Phospholipase A2 activity is required for
immune defense of European (Apis mellifera)
and Asian (Apis cerana) honeybees against
American foulbrood pathogen, Paenibacillus
larvae Gahyeon Jin, Md Tafim Hossain Hrithik, Eeshita Mandal, Eui-Joon Kil, Chuleui Jung,
Yonggyun KimID* 1. Introduction Insect pollinators sustain plant species and increase crop yields [1–3]. Indeed, a third of all
crops are pollinator-dependent, and in particular are dependent on bees such as the honeybees
[4]. For example, the economic value of pollination by honeybees was estimated to be over
18-fold greater than that of their honey production in Korea [5]. Honeybees are social insects
that build nests in which different generations live together with divided labors among the
workers, drones, and the queen, and in which immatures are five instar larvae and pupae [6]. The adult lifespan varies from a few weeks to several months or years depending on the caste
differentiation and even season. The Asian honeybee, Apis cerana, is also an important pollina-
tor that provides essential pollination services to agricultural plant communities in Asian
countries [7]. However, in recent decades, the A. cerana populations have declined in many
countries and subsequent reductions in pollination services greatly affect plant communities
[8,9]. Among factors leading to declines in bee populations, the use of pesticides is considered
to be the main cause [10]. However, in addition to the direct toxic actions of insecticides, sub-
lethal doses have significant adverse effects on honeybee immunity [11]. Competing interests: The authors have declared
that no competing interests exist. American foulbrood is the most destructive bacterial disease of the honeybee larvae, in
which only ten spores of the bacteria are sufficient to trigger a lethal infection [12,13]. Infection
typically occurs through feeding to the larvae by worker bees and roughly 12 h after ingestion,
the spores germinate in the larval gut epithelium to proliferate and kill the infected larva [14]. The dead larvae are then decomposed to form scales containing millions of the bacterial
spores, which are spread around the hive by worker bees, leading to massive colony collapse
[15]. Similar to other social insects, the honeybees defend against pathogenic microbes or
eukaryotic parasites with communal defenses and individual immunity [16]. Communal
defense represents hygienic behaviors like grooming and hive fever exhibited by young work-
ers [17–19]. Individual immunity includes cellular and humoral responses in combination
with a frontier barrier like a cuticle [16,20]. Suppression of these immune responses can lead
to colony collapse [21]. For example, an exposure of queens to neonicotinoid pesticides
reduced their total hemocyte number and impaired wound healing and antimicrobial peptide
(AMP) production, leading to an immunosuppressed state [22,23]. PLOS ONE PLOS ONE Honeybee immunity against the American founbrood disease Funding: National Research Foundation (NRF)
(2022R1A2B5B03001792) to Yonggyun Kim
National Research Foundation (NRF)
(2018R1A6A1A03024862) to Chuleui Jung. Funding: National Research Foundation (NRF)
(2022R1A2B5B03001792) to Yonggyun Kim
National Research Foundation (NRF)
(2018R1A6A1A03024862) to Chuleui Jung. Editor: Olav Rueppell, University of Alberta,
CANADA Received: August 16, 2023
Accepted: December 31, 2023
Published: February 6, 2024 Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0290929 Copyright: © 2024 Jin 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 paper and its Supporting Information
files. 1 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 1. Introduction The immunosuppression
induced by insecticides then increases the susceptibility to the pathogens as was demonstrated
in the larvae of A. mellifera exhibiting high mortality to P. larvae, after exposure to insecticides
[24]. Insect immunity is innate and its recognition against pathogens is programmed in the
genome by specific pattern recognition receptors [25]. Recent discovery of the infection-
induced damage signal via dorsal switch protein 1 (DSP1) is additional to the innate insect rec-
ognition system [26,27]. The recognition signal is propagated to nearby effector tissues such as
hemocytes and fat bodies [28]. A number of immune mediators have been identified and these
include nitric oxide, cytokines, biogenic monoamines, and eicosanoids. Each plays a crucial
role in activating immune effectors for various immune responses in insects [29]. Cross-talk
between immune mediators occurs upon various pathogen infections, and eicosanoids play a
central role in mediating immune signals with their chemical diversity [30]. These eicosanoids
are likely to mediate the immune responses of the honeybees because nodule formation in
response to bacterial infection was shown to be dependent on phospholipase A2 (PLA2) activ-
ity in A. mellifera [31]. PLA2 catalyzes the committed step for eicosanoid biosynthesis and releases arachidonic
acid (AA) from phospholipids [32]. Since the first PLA2 was isolated from snake venom, a
number of venomous or non-venomous PLA2s have been identified and classified into at least 2 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 PLOS ONE Honeybee immunity against the American founbrood disease 16 Groups (I-XVI) [33]. They are also classified into five major types: secretory PLA2s
(sPLA2s: Groups I-III, V, IX, X, XI, XII, XIII, XIV, and XV), calcium-dependent intracellular
PLA2 (cPLA2: Group IV), calcium-independent intracellular PLA2 (iPLA2: Group VI), lipo-
protein-associated PLA2 (LpPLA2: Groups VII and VIII), and adipose phospholipase A2
(AdPLA2: Group XVI) [34]. However, PLA2s encoded in honeybee genomes have not been
analyzed in terms of either their identities or functions. 16 Groups (I-XVI) [33]. They are also classified into five major types: secretory PLA2s
(sPLA2s: Groups I-III, V, IX, X, XI, XII, XIII, XIV, and XV), calcium-dependent intracellular
PLA2 (cPLA2: Group IV), calcium-independent intracellular PLA2 (iPLA2: Group VI), lipo-
protein-associated PLA2 (LpPLA2: Groups VII and VIII), and adipose phospholipase A2
(AdPLA2: Group XVI) [34]. However, PLA2s encoded in honeybee genomes have not been
analyzed in terms of either their identities or functions. This study identified the PLA2s encoded in two honeybee species, A. 2.1. Honeybee larvae collection and diet preparation The experiment was carried out on two species, A. mellifera and A. cerana, which were reared
in the experimental apiary of Andong National University. The honeybees were raised in non-
cultivated areas where exposure to agricultural chemicals such as pesticides including neonico-
tinoids was minimized. The experimental larvae were collected from beehives at the experi-
mental apiary. To isolate the queen and a few workers, a cage was used to separate them on a
new frame for 24 h. Afterward, the queens were released, and the frame containing newly laid
eggs was returned to the hive with a protective cage for an additional 70 h. Under these rearing
conditions, larvae underwent five instars (L1-L5). Larval stage used 2 days-old L5 individuals
for immunological assays. Bioassays against the bacterial pathogen used L3 larvae. 2.3. Chemicals Arachidonic acid (AA, 5,8,11,14-eicosatetraenoic acid) and dexamethasone (DEX, (11β, 16α)-
9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3) were obtained from Sigma-
Aldrich Korea (Seoul, Korea). They were dissolved in dimethyl sulfoxide (DMSO) to prepare
test solutions. To prevent any melanization of hemolymph, an anticoagulant buffer (ACB) was
prepared with 186 mM NaCl, 17 mM Na2EDTA, and 41 mM citric acid and adjusted to pH 4.5
with acetic acid. Phosphate-buffered saline (PBS) was prepared with 100 mM phosphate plus
0.75% NaCl and pH adjusted to 7.4 with NaOH. 1. Introduction mellifera and A. cer-
ana. The physiological functions of the PLA2s were assessed in mediating cellular and humoral
immune responses in the two species. Using the full genomes, this study proposed an immune
signal pathway associated with eicosanoid biosynthesis in honeybees. 2.2. Bacterial culture The bacterial pathogen, P. larvae, used in this study was obtained from Korean Agricultural
Colony Collection (KACC, RDA, Wanju, Korea) with an accession number of
NZ_CP019687.1. The bacterium was grown in brain heart infusion medium (BHI: Millipore,
Burlington, MA, USA) for 18 h at 30˚C with shaking at 180 rpm. 2.4. Hemocyte counts Ninety μL of ACB was mixed with the hemolymph samples collected from 25 individuals of L5
instar larvae. The collected hemolymph suspension was then centrifuged at 1,000 × g for 3 min
to obtain the cell pellet which was then resuspended in 40 μL of TC-100 insect tissue culture
medium (Hyclone, Daegu, Korea). Total hemocyte count (THC) and differential hemocyte
count (DHC) were determined with a hemocytometer. The classification of hemocyte types
was based on the morphological characteristics described by Lavine and Strand [35]. Each
treatment was replicated independently three times. 3 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 PLOS ONE Honeybee immunity against the American founbrood disease 2.7. Bioinformatics to predict PLA2 genes Eight PLA2 genes were retrieved with accession numbers XM_016916293.2 (Am-PLA2A),
XM_393116.7 (Am-PLA2B), XM_624469.6 (Am-PLA2C), JQ_900376.1 (Am-PLA2D),
XM_017064190.2 (Ac-PLA2A), XM_017059900.2 (Ac-PLA2B), XM_017051624.2 (Ac-PLA2C),
XM_ 017066061.2 (Ac-PLA2D). MEGA10 was used to construct a phylogenetic tree using the
Neighbor-joining method. Bootstrap values at each branch were calculated with 1,000 repeats. Interpro (http://www.ebi.ac.uk/interpro/) and Expasy (WWW.expasy.com) were used to pre-
dict domain and signal peptides. 2.5. Hemocyte-spreading assay L5 instar larvae of both honeybee species were used for the hemocyte-spreading behavior. Hemocytes were collected using ACB as described above and incubated on ice for 20 min. The
diluted hemolymph was then centrifuged at 1,000 × g for 3 min at 4˚C to get the pellet, which
was re-suspended in 300 μL of filter-sterilized TC-100 insect cell culture medium. Ten μL of
hemocyte suspension was laid on a glass coverslip. After removing supernatant, hemocytes
were fixed with 4% paraformaldehyde for 10 min at 25˚C. After washing three times with fil-
ter-sterilized PBS, hemocytes were then permeabilized with 0.2% Triton-X in PBS for 2 min at
25˚C. After washing three times, hemocytes were incubated with 5% skim milk for 10 min at
25˚C and subsequently with fluorescein isothiocyanate (FITC)-tagged phalloidin in PBS for 60
min. After washing three times, the cells were incubated with 4´,6-diamidino-2-phenylindole
(DAPI, 1 mg/mL). Finally, after washing twice in PBS, the cells were observed under a fluores-
cence microscope (DM2500, Leica, Wetzlar, Germany) at 400 × magnification. Hemocyte-
spreading was determined based on the extension of F-actin growth beyond the original cell
boundary. Scoring the spread cells was performed by counting the cells exhibiting F-actin
growth among 100 randomly chosen cells. Each treatment was replicated three times with
independent hemocyte preparations. 2.6. Nodulation assay Hemocyte nodule formation was evaluated in L5 larvae of A. mellifera and A. cerana. Each
larva was injected with 1 μL of overnight-cultured P. larvae (5 × 107 cells/mL) and 1 μL of a
test chemical (DEX or AA, 1 μg per larva), into the hemocoel through the proleg using a
micro-syringe (Hamilton, Reno, NV, USA). For controls, DMSO was injected along
with the bacteria. The injected larvae were then incubated at 25˚C for 8 h to reduce any vari-
ation in the nodule formation depending on varying ambient temperatures. After incuba-
tion, the larvae were dissected to count the melanized nodules under a microscope (Stemi
SV11, Zeiss, Jena, Germany) at 50× magnification. In each treatment, nine larvae were
assessed. 2.8. RNA extraction, cDNA construction, and qPCR L5 instar larvae, 2–3 day old pupae, and young worker bees less than 1 week old after emer-
gence were used for total RNA extraction after removing their intestines to avoid any contami-
nation derived from non-target organisms using Trizol reagent (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer’s instructions. Extracted RNA was used for synthesizing
complementary DNA (cDNA) using RT-premix (Intron Biotechnology, Seoul, Korea) con-
taining an oligo-dT primer. Synthesized cDNA was quantified with a spectrophotometer
(NanoDrop, Thermo Fisher Scientific, Wilmington, DE, USA). Synthesized cDNA (80 ng
per μL) was used as a template for quantitative PCR (qPCR) using gene-specific primers (S1 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 4 / 19 PLOS ONE Honeybee immunity against the American founbrood disease Table). qPCR was performed using SYBR Green real-time PCR master mixture (Toyobo,
Osaka, Japan) according to the guidelines of Bustin et al. [36] on a real-time PCR system (Step
One Plus Real-Time PCR System, Applied Biosystems, Singapore). The reaction mixture
(20 μL) contained 10 μL of Power SYBR Green PCR Master Mix, 1 μL of cDNA template (80
ng), and 1 μL each of forward and reverse primers, and 7 μL of deionized distilled water. The
temperature program for qPCR began with 95˚C heat treatment for 10 min followed by 40
cycles of denaturation at 94˚C for 30 s, annealing at 52˚C for 30 s, and extension at 72˚C for 30
s. The ribosomal protein gene, RL32, was used as an endogenous control. Each treatment was
replicated three times with independent samples. Expression analysis of qPCR was calculated
by comparative CT method [37]. 2.9. Virulence assay of P. larvae against the honeybee larvae Test L3 larvae were fed with an artificial diet containing royal jelly (44.3%), glucose (5.3%),
fructose (5.3%), yeast (0.9%), and sterilized water (44.3%) [38]. Diet was placed into wells of 48
well-plates containing individual larva. For the virulence test treatment, the artificial diet
included P. larvae at the pre-determined bacterial concentrations in colony-forming unit
(CFU). Ten larvae were used in each concentration treatment and replicated three times. 2.10. dsRNA preparation and RNAi treatment T7 promoter sequence was linked to gene-specific primers at the 5’ end. Using these primers, a
partial PLA2 gene was amplified. The PCR product was then used to generate double-stranded
RNA (dsRNA) using the Megascript RNAi Kit (Ambion, Austin, TX, USA). The dsRNA was
mixed with a transfection reagent (Metafectene Pro, Biontex, Planegg, Germany) in 1:1 ratio. Late L4 larvae were used for RNAi treatment. To administer the dsRNA, a microsyringe
(Hamilton, Reno, NV, USA) was employed to inject 1 μg of dsRNA per larva. A green fluores-
cence protein (GFP) was used as a control to prepare dsRNA. Each treatment was replicated
three times using independent RNA preparations. 2.11. Statistical analysis All experiments in this study were conducted in three individual replications. The results were
plotted using Sigma plot 10.0. Statistical analysis was performed using PROC GLM of the SAS
program [39] with a one-way analysis of variance. Significant differences among the means
were determined using the LSD test at a Type I error of 0.05, indicated by different letters. 3.1. Comparative analysis of P. larvae virulence against honeybees Bacterial administration of P. larvae to larvae used a feeding method, in which live bacterial
cells were incorporated in the artificial diet. The fed larvae suffered from bacterial pathogenic-
ity and some died with a blackened cadaver (Fig 1A). Virulence was dependent on the incuba-
tion time and A. cerana appeared to be more susceptible than A. mellifera in the median lethal
time (LT50): 3.65 days for A. mellifera and 2.48 days for A. cerana (Fig 1B) at the bacterial con-
centration (105 cfu/mL) by feeding. This differential susceptibility between two honey bee spe-
cies was also appeared in the median lethal dose (LC50): 1.1 × 105 cfu for A. mellifera and
2.4 × 104 cfu for A. cerana (Fig 1C) at 5 days after bacterial treatment. However, these two
median values were not statistically different at Type error = 0.05. 5 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 PLOS ONE Honeybee immunity against the American founbrood disease Fig 1. Relative virulence of P. larvae against honeybee larvae of A. mellifera (‘Am’) and A. cerana (‘Ac’). (A)
Pathogenic symptom of the infected larvae. (B) Time-mortality curves of two honeybee species infected with P. larvae. L2 larvae were treated with bacteria (105 cfu/mL) by feeding. (C) Dose-mortality curves at 5 days after bacterial
treatment (‘DAT’). An experimental unit consisted of 10 larvae. Each dose was replicated three times. Different letters
above standard deviation bars indicate significant differences among means at Type I error = 0.05. Fig 1. Relative virulence of P. larvae against honeybee larvae of A. mellifera (‘Am’) and A. cerana (‘Ac’). (A)
Pathogenic symptom of the infected larvae. (B) Time-mortality curves of two honeybee species infected with P. larvae. L2 larvae were treated with bacteria (105 cfu/mL) by feeding. (C) Dose-mortality curves at 5 days after bacterial
treatment (‘DAT’). An experimental unit consisted of 10 larvae. Each dose was replicated three times. Different letters
above standard deviation bars indicate significant differences among means at Type I error = 0.05. PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 3.2. Comparative analysis of immune responses of two honeybees Hemocytes were classified on the basis of cell morphology (Fig 2A). Total hemocyte counts
were not significantly different between the two honeybees (Fig 2B). At least three different
types of hemocytes were discriminated, most of which were granulocytes (> 80%) in both spe-
cies (Fig 2C). Granulocytes and plasmatocytes in particular, showed spreading on the plastic
surface. Specifically, granulocytes were spread in all directions around the entire cell contour
while plasmatocytes were spread unequally in specific directions. Oenocytoid cells were
unspread and had a small nucleus compared to cytoplasm. The bacterial treatment signifi-
cantly increased the hemocyte-spreading behavior in both honeybee species (Fig 2D). Hemocoelic injection of the bacteria into larvae stimulated nodule formation. The nodula-
tion was dependent on the incubation time and reached maximal levels 8 h after the bacterial
infection (Fig 3A). Most nodules were detected near the trachea and on the fat body (see inset
photos). The kinetics of nodule formation was not much different in the two species in terms
of the time to form the maximal number of nodules and the number of nodules. Moreover, PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 6 / 19 PLOS ONE Honeybee immunity against the American founbrood disease Fig 2. Hemocytes and their behavior (A. mellifera (‘Am’) and A. cerana (‘Ac’)). (A) Hemocyte types: Granulocyte
(‘GR’), plasmatocyte (‘PL’), and oenocytoid (‘OE’). Cytoplasm was stained with FITC against F-actin while nucleus was
stained with DAPI. Scale bar indicates 10 μm. (B) Comparison of the total hemocyte count (THC) between two
species. ‘NS’ stands for no significance. (C) Differential hemocyte counts. The statistical analysis was performed by Χ2
test to compare the hemocyte composition between two species. (D) Hemocyte-spreading behavior. Each larva was
injected with 1 μL of P. larvae (‘Pl’, 5 × 104 cells). Each measurement for the spreading behavior used 100 randomly
chosen hemocytes. Each treatment was replicated three times by individual sample preparation. Different letters above
the standard deviation bars indicate significant difference among means at Type I error = 0.05. Fig 2. Hemocytes and their behavior (A. mellifera (‘Am’) and A. cerana (‘Ac’)). (A) Hemocyte types: Granulocyte
(‘GR’), plasmatocyte (‘PL’), and oenocytoid (‘OE’). Cytoplasm was stained with FITC against F-actin while nucleus was
stained with DAPI. Scale bar indicates 10 μm. (B) Comparison of the total hemocyte count (THC) between two
species. ‘NS’ stands for no significance. (C) Differential hemocyte counts. 3.2. Comparative analysis of immune responses of two honeybees The statistical analysis was performed by Χ2
test to compare the hemocyte composition between two species. (D) Hemocyte-spreading behavior. Each larva was
injected with 1 μL of P. larvae (‘Pl’, 5 × 104 cells). Each measurement for the spreading behavior used 100 randomly
chosen hemocytes. Each treatment was replicated three times by individual sample preparation. Different letters above
the standard deviation bars indicate significant difference among means at Type I error = 0.05. https://doi.org/10.1371/journal.pone.0290929.g002 https://doi.org/10.1371/journal.pone.0290929.g002 there was no difference in the numbers of nodules between the injections of live and dead P. larvae (Fig 3B). Two AMPs were assessed in their expression levels after the bacterial infection of P. larvae
(Fig 3C). The bacterial infection significantly up-regulated the gene expression of apolipo-
phorin III (ApoLpIII), but not that of defensin in both honeybee species. 3.3. Eicosanoids mediate both cellular and humoral immune responses in
honeybees Immune responses are mediated by eicosanoids in insects [29]. To test this hypothesis in hon-
eybees, eicosanoid biosynthesis was inhibited by dexamethasone (DEX, a specific inhibitor of
PLA2). DEX treatment significantly suppressed the formation of nodules in response to P. lar-
vae infection in both species (Fig 4A). However, arachidonic acid (AA, a catalytic product of 7 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 PLOS ONE Honeybee immunity against the American founbrood disease Fig 3. Immune responses of honeybees A. mellifera (‘Am’) and A. cerana (‘Ac’). (A) Cellular immune response
observed by nodule formation upon infection of P. larvae by bacterial injection to L5 instar larvae at different time
points. Each larva was injected with 1 μL of P. larvae (5 × 104 cells). Each treatment was replicated three times. At 8 h
after injection, the total number of nodules was counted. PBS was injected for controls. ‘FB’ and ‘TR’ stands for fat
body and trachea, respectively. Arrows indicate nodules. (B) Comparison of live and heat-killed P. larvae in forming
nodules in the honeybee larvae. Heat-killing (‘Hk’) treatment used 98˚C for 20 min. Control (‘CON’) used sterilized
PBS for injection. Different letters above the standard deviation bars indicate significant difference among means at
Type I error = 0.05. (C) Humoral immune response assessed by expression of two AMP genes: apolipophorin III
(‘ApoLpIII’) and defensin (‘Def’) at 8 h after bacterial injection. Each treatment was replicated three times. Asterisk
stands for significant difference while ‘NS’ is no significant difference. Fig 3. Immune responses of honeybees A. mellifera (‘Am’) and A. cerana (‘Ac’). (A) Cellular immune response
observed by nodule formation upon infection of P. larvae by bacterial injection to L5 instar larvae at different time
points. Each larva was injected with 1 μL of P. larvae (5 × 104 cells). Each treatment was replicated three times. At 8 h
after injection, the total number of nodules was counted. PBS was injected for controls. ‘FB’ and ‘TR’ stands for fat
body and trachea, respectively. Arrows indicate nodules. (B) Comparison of live and heat-killed P. larvae in forming
nodules in the honeybee larvae. Heat-killing (‘Hk’) treatment used 98˚C for 20 min. Control (‘CON’) used sterilized
PBS for injection. Different letters above the standard deviation bars indicate significant difference among means at
Type I error = 0.05. 3.3. Eicosanoids mediate both cellular and humoral immune responses in
honeybees (C) Humoral immune response assessed by expression of two AMP genes: apolipophorin III
(‘ApoLpIII’) and defensin (‘Def’) at 8 h after bacterial injection. Each treatment was replicated three times. Asterisk
stands for significant difference while ‘NS’ is no significant difference. https://doi.org/10.1371/journal.pone.0290929.g003 https://doi.org/10.1371/journal.pone.0290929.g003 PLA2) significantly rescued the inhibitory activity of DEX in both species. Similarly, ApoLpIII
expression was also modulated by DEX and AA in both species (Fig 4B). PLA2) significantly rescued the inhibitory activity of DEX in both species. Similarly, ApoLpIII
expression was also modulated by DEX and AA in both species (Fig 4B). 3.4. PLA2 orthologs encoded in in two honeybee genomes From each honeybee genome, four PLA2 genes (PLA2A ~ PLA2D) were obtained and showed
an orthologous relationship (Fig 5A). The four PLA2s were classified into secretory (sPLA2),
calcium-independent (iPLA2), and lysosomal (LPLA2). PLA2A and PLA2B are secretory, while
PLA2C is calcium-independent and PLA2D is lysosomal. PLA2A and PLA2B have signal pep-
tides in their N termini and calcium-binding domains in addition to a catalytic domain (Fig
5B), which represent the typical domain composition of most sPLA2s [40]. In contrast, PLA2C
has five ankyrin repeats in addition to a catalytic domain [41]. PLA2D was classified into
Group XV PLA2s specific to lysosomal PLA2s [42]. 8 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 PLOS ONE Honeybee immunity against the American founbrood disease Fig 4. Eicosanoid mediation of the immune responses in honeybee species: A. mellifera (‘Am’) and A. cerana
(‘Ac’). L5 instar larvae were immune-challenged by injection with 1 μL of P. larvae (5 × 104 cells). (A) Inhibitory effect
of dexamethasone (‘DEX’, 1 μg/larva) on nodule formation in response to bacterial infection. Rescue by addition of
arachidonic acid (1 μg/larva). (B) Influence of DEX on ApoLpIII gene expression upon bacterial infection. Different
letters above the standard deviation bars indicate significant differences among means at Type I error = 0.05. htt
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0290929 004 Fig 4. Eicosanoid mediation of the immune responses in honeybee species: A. mellifera (‘Am’) and A. cerana
(‘Ac’). L5 instar larvae were immune-challenged by injection with 1 μL of P. larvae (5 × 104 cells). (A) Inhibitory effect
of dexamethasone (‘DEX’, 1 μg/larva) on nodule formation in response to bacterial infection. Rescue by addition of
arachidonic acid (1 μg/larva). (B) Influence of DEX on ApoLpIII gene expression upon bacterial infection. Different
letters above the standard deviation bars indicate significant differences among means at Type I error = 0.05. https://doi.org/10.1371/journal.pone.0290929.g004 3.6. Induction of PLA2 expression upon P. larvae infection Upon infection with P. larvae, PLA2 activities were significantly up-regulated in larval and adult
stages of the two honeybees (Fig 7A). The induction of elevated enzyme activity was further sup-
ported by up-regulation of PLA2 gene expression in A. mellifera (Fig 7B) and A. cerana. 3.5. Expression profile of four PLA2 genes Fig 6 shows that the four PLA2 genes were expressed in different developmental stages of both
honeybee species. Of note, PLA2D was highly expressed in all developmental stages. In addi-
tion, PLA2C was also relatively highly expressed like PLA2D in both species. In contrast, the
Group III PLA2A and Group XII PLA2B were expressed at low levels in all developmental
stages in both species. However, PLA2B was highly induced in adults. PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 3.7. RNAi against PLA2 genes increased the virulence of P. larvae To clarify the role of immune-associated PLA2(s) in honeybees, gene-specific dsRNAs were
injected into larvae to suppress target PLA2 genes. dsRNA effectively suppressed the PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 9 / 19 PLOS ONE Honeybee immunity against the American founbrood disease Fig 5. Orthologs of four PLA2 genes encoded in honeybee species: A. mellifera (‘Am’ in yellow arrows) and A. cerana (‘Ac’ in orange arrows). (A) Phylogenetic tree of the honeybee PLA2s with different PLA2, groups delineated. Honeybee PLA2s were clustered in four types: secretory (sPLA2), lysosomal (LPLA2), Ca2+-independent cellular
(iPLA2), and Ca2+-dependent cellular (cPLA2). (B) Variation of functional domains among the honeybee PLA2s. Cysteine (‘C’) residues are denoted. htt
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0290929 005 Fig 5. Orthologs of four PLA2 genes encoded in honeybee species: A. mellifera (‘Am’ in yellow arrows) and A. cerana (‘Ac’ in orange arrows). (A) Phylogenetic tree of the honeybee PLA2s with different PLA2, groups delineated. Honeybee PLA2s were clustered in four types: secretory (sPLA2), lysosomal (LPLA2), Ca2+-independent cellular
(iPLA2), and Ca2+-dependent cellular (cPLA2). (B) Variation of functional domains among the honeybee PLA2s. Cysteine (‘C’) residues are denoted. https://doi.org/10.1371/journal.pone.0290929.g005 https://doi.org/10.1371/journal.pone.0290929.g005 expression levels of target genes in both species (S1 Fig). Under these conditions, the honeybee
larvae were significantly suppressed in their ability to form nodules in response to immune
challenge with P. larvae (Fig 8A). Although all four RNAi treatments were effective at sup-
pressing the cellular immune response, RNAi treatments against PLA2A or PLA2B appeared to
be more potent than those of PLA2C or PLA2D. Similarly, all four RNAi treatments were effec-
tive at suppressing induction of ApoLpIII in the face of bacterial challenge (Fig 8B). In this
humoral immune response, RNAi treatments against PLA2A or PLA2B were much more
potent at suppressing AMP gene expression than that of PLA2C or PLA2D. PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 4. Discussion Immunosuppression makes honeybees highly susceptible to pathogens and can lead to colony
collapse [43]. For example, the honeybees that are malnourished as a result of protein defi-
ciency in their diet may be altered in specific components of the immune system, which can
lead to fatal immunosuppression [44]. The American foulbrood disease caused by P. larvae PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 10 / 19 PLOS ONE Honeybee immunity against the American founbrood disease Fig 6. Expression profile of four PLA2s (PLA2A, PLA2B, PLA2C, PLA2D) in different developmental stages of
honeybee species: A. mellifera (‘Am’) and A. cerana (‘Ac’). Different letters above standard deviation bars indicate
significant difference among means at Type I error = 0.05. Ribosomal protein RPL32 was used as a reference gene. Each treatment was replicated three times with independent sample preparations. https://doi.org/10.1371/journal.pone.0290929.g006 Fig 6. Expression profile of four PLA2s (PLA2A, PLA2B, PLA2C, PLA2D) in different developmental stages of
honeybee species: A. mellifera (‘Am’) and A. cerana (‘Ac’). Different letters above standard deviation bars indicate
significant difference among means at Type I error = 0.05. Ribosomal protein RPL32 was used as a reference gene. Each treatment was replicated three times with independent sample preparations. https://doi.org/10.1371/journal.pone.0290929.g006 https://doi.org/10.1371/journal.pone.0290929.g006 becomes serious when honeybees are also suffering from immunosuppression after exposure
to sublethal doses of insecticides [24]. This investigation has shown that eicosanoids play a cru-
cial role in defending honeybees against American foulbrood disease. Asian honeybees were shown to be more susceptible to the American foulbrood pathogen,
P. larvae than the European honeybees. This supports the earlier comparative pathogenicity
testing performed by Krongdang et al. [45]. However, bacterial virulence may be altered by the
gut microbiota. In a similar Asian honeybee, A. cerana japonica, five species of gut bacteria
exhibited a strong antagonistic activity against P. larvae growth and their relative abundance
may modulate its bacterial virulence [46]. Both honeybee species exhibited similar hemocyte compositions in THC and DHC, in
which granulocytes were predominant among the three different hemocytes that were identi-
fied. They also showed cellular immune responses upon challenge such as hemocyte-spreading
behavior and nodule formation against P. larvae infection. In addition, they showed up-regula-
tion of specific AMPs such as apolipophorin III (ApoLpIII). Collectively, these observations
suggest that the two honeybee species share cellular and humoral immune responses. 4. Discussion In PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 11 / 19 PLOS ONE Honeybee immunity against the American founbrood disease Fig 7. Induction of PLA2 enzyme activities and gene expression in response to im
with 1 μL of P. larvae (5 × 104 cells) in honeybee species: A. mellifera (‘Am’) and A
PLA2 enzyme activities in larvae and adults. The enzyme was extracted from the who
venom gland-removed adults. Different letters above standard deviation bars indicat
means at Type I error = 0.05. Induction of gene expression of four different PLA2s aft
and adult (‘A’) of Am (B) and Ac (C). The mRNA expression levels of each PLA2 gen
Honeybee immunity against t Fig 7. Induction of PLA2 enzyme activities and gene expression in response to immune-challenge by injection
with 1 μL of P. larvae (5 × 104 cells) in honeybee species: A. mellifera (‘Am’) and A. cerana (‘Ac’). (A) Induction in
PLA2 enzyme activities in larvae and adults. The enzyme was extracted from the whole body of L5 instar larvae and th
venom gland-removed adults. Different letters above standard deviation bars indicate significant difference among
means at Type I error = 0.05. Induction of gene expression of four different PLA2s after immune challenge in the larva
and adult (‘A’) of Am (B) and Ac (C). The mRNA expression levels of each PLA2 gene were measured at 8 h after Fig 7. Induction of PLA2 enzyme activities and gene expression in response to immune-challenge by injection
with 1 μL of P. larvae (5 × 104 cells) in honeybee species: A. mellifera (‘Am’) and A. cerana (‘Ac’). (A) Induction in
PLA2 enzyme activities in larvae and adults. The enzyme was extracted from the whole body of L5 instar larvae and the
venom gland-removed adults. Different letters above standard deviation bars indicate significant difference among
means at Type I error = 0.05. Induction of gene expression of four different PLA2s after immune challenge in the larva
and adult (‘A’) of Am (B) and Ac (C). The mRNA expression levels of each PLA2 gene were measured at 8 h after Fig 7. Induction of PLA2 enzyme activities and gene expression in response to immune-challenge by injection
with 1 μL of P. larvae (5 × 104 cells) in honeybee species: A. mellifera (‘Am’) and A. cerana (‘Ac’). (A) Induction in
PLA2 enzyme activities in larvae and adults. 4. Discussion The enzyme was extracted from the whole body of L5 instar larvae and the
venom gland-removed adults. Different letters above standard deviation bars indicate significant difference among
means at Type I error = 0.05. Induction of gene expression of four different PLA2s after immune challenge in the larva
and adult (‘A’) of Am (B) and Ac (C). The mRNA expression levels of each PLA2 gene were measured at 8 h after Fig 7. Induction of PLA2 enzyme activities and gene expression in response to immune-challenge by injection
with 1 μL of P. larvae (5 × 104 cells) in honeybee species: A. mellifera (‘Am’) and A. cerana (‘Ac’). (A) Induction in
PLA2 enzyme activities in larvae and adults. The enzyme was extracted from the whole body of L5 instar larvae and the
venom gland-removed adults. Different letters above standard deviation bars indicate significant difference among
means at Type I error = 0.05. Induction of gene expression of four different PLA2s after immune challenge in the larva
and adult (‘A’) of Am (B) and Ac (C). The mRNA expression levels of each PLA2 gene were measured at 8 h after PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 12 / 19 PLOS ONE Honeybee immunity against the American founbrood disease bacterial infection. Each treatment was replicated three times with independent sample preparations. Asterisks above
standard deviation bars indicate significant difference among means at Type I error = 0.05. Ribosomal protein RPL32
was used as a reference gene. Each treatment was replicated three times with independent sample preparations. https://doi.org/10.1371/journal.pone.0290929.g007 bacterial infection. Each treatment was replicated three times with independent sample preparations. Asterisks above
standard deviation bars indicate significant difference among means at Type I error = 0.05. Ribosomal protein RPL32
was used as a reference gene. Each treatment was replicated three times with independent sample preparations. https://doi.org/10.1371/journal.pone.0290929.g007 https://doi.org/10.1371/journal.pone.0290929.g007 general, bees have an innate immune system supplemented by physical barriers (integument
and peritrophic matrix), that include cellular, and humoral responses to defend against patho-
gens and parasites [47]. In particular, hemocytes represent the primary immune effectors for
cellular immunity by exhibiting phagocytosis, nodule formation, and encapsulation, as well as
the initiation of phenoloxidase (PO) that regulates coagulation or melanization [35]. Hemo-
cytes of the honeybee are classified by their morphology with behavioral differences in lectin-
binding and phagocytosis [48]. PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 4. Discussion larvae, suggesting a crucial role in defending against the bacterial pathogen. ApoLpIII was also
demonstrated in A. cerana against other insect pathogens such as Bacillus thuringiensis and
Beauveria bassiana [52]. This supports the immune-associated defense role of ApoLpIII
against P. larvae infection in both honeybee species. In addition to the AMPs, vitellogenin (Vg,
a yolk protein) plays a crucial role in defence against pathogens and neutralizing oxidative
stress in honey bees [53]. Vg is expressed in fat body and venom glands of worker bees and Vg
gene expression was highly up-regulated in response to pathogen infection and oxidative stress
in A. cerana [54]. PLA2 is associated with the cellular and humoral immune responses of honeybees. Dexa-
methasone treatment inhibited nodule formation and ApoLpIII expression while the addition
of arachidonic acid (= a catalytic product of PLA2) to the inhibitor treatment significantly res-
cued the immune responses. Eicosanoids are a subgroup of the oxygenated C20 polyunsatu-
rated fatty acids and mediate many physiological processes in insects and other invertebrates
[55]. They are usually synthesized from phospholipids by the catalytic activity of PLA2 [56]. This suggests that the eicosanoids produced by PLA2 catalysis promote the immune responses
in honeybees. Four PLA2 genes are encoded by the two honeybee genomes. Of them, PLA2A is known to
be a honeybee venom component. PLA2 and hyaluronidase enzymes account for 11~15% of
the bee venom dry weight in A. mellifera [57,58]. Another sPLA2 is PLA2B, which is classified
in Group XII. Similar Group XII PLA2s were reported in other insects such as the bug, Rhod-
nius prolixus [59] and a moth, Acrolepiopsis sapporensis [60] and known to be associated with
eicosanoid biosynthesis for reproduction. Most insects encode iPLA2s, which are further
divided into ankyrin-possessing or non-ankyrin iPLA2s [29]. PLA2C is classified into ankyrin-
possessing iPLA2. PLA2D is classified as a lysosomal PLA2 (LPLA2) and is the first of this type
known in insects. LPLA2 is localized in the lysosome or late endosome in mammals [42] and
its catalytic activity is optimal at acidic pH and is calcium-independent. Studies on the LPLA2
null mouse suggest a role for the enzyme in the catabolism of pulmonary surfactant and it also
has a role in host defense [61]. Our current study indicates that PLA2D is the same in both
honeybee species. 4. Discussion At least three types of hemocytes were observed here through
all developmental stages and they included granulocytes, plasmatocytes, and oenocytoids [49]. However, the hemocyte composition varies with developmental stages and castes, and it is the
phagocytic granulocytes that are most abundant at the larval stage but significantly diminish Fig 8. Functional association of four honeybee PLA2s with immune responses in A. mellifera (‘Am’) and A. cerana
(‘Ac’). Individual RNAi treatments were applied to specifically suppress PLA2-A using dsPLA2A, PLA2-B using
dsPLA2B, PLA2-C using dsPLA2C, or PLA2-D using dsPLA2D. After 24 h post-injection of dsRNA, the immune
challenge was performed by injecting 1 μL of P. larvae (5 × 104 cells) into larvae or adults. Effects of the RNAi
treatments on nodule formation (A) and ApoLpIII expression (B). GFP was used as a control dsRNA (‘dsCON’). Each
treatment was replicated three times. Ribosomal protein gene RL32 was used as an internal control for RT-qPCR. https://doi.org/10.1371/journal.pone.0290929.g008 Fig 8. Functional association of four honeybee PLA2s with immune responses in A. mellifera (‘Am’) and A. cerana
(‘Ac’). Individual RNAi treatments were applied to specifically suppress PLA2-A using dsPLA2A, PLA2-B using
dsPLA2B, PLA2-C using dsPLA2C, or PLA2-D using dsPLA2D. After 24 h post-injection of dsRNA, the immune
challenge was performed by injecting 1 μL of P. larvae (5 × 104 cells) into larvae or adults. Effects of the RNAi
treatments on nodule formation (A) and ApoLpIII expression (B). GFP was used as a control dsRNA (‘dsCON’). Each
treatment was replicated three times. Ribosomal protein gene RL32 was used as an internal control for RT-qPCR. https://doi.org/10.1371/journal.pone.0290929.g008 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 13 / 19 PLOS ONE Honeybee immunity against the American founbrood disease after adult emergence. Our current hemocyte analysis in both honeybee species supports the
observation of Gabor et al. [49] at the larval stage of A. mellifera. In addition to the hemocytic
activity, PO activity is required for nodulation. A serine protease called AccSp10 was identified
in A. cerana and acted as a humoral factor to defend the microbial pathogens by suppressing
bacterial growth and mediating melanization through activating PO activity [50]. In A. melli-
fera, 57 genes encode serine peptidases, however, only 6 show chymotrypsin-like specificity
[51]. Thus, the two honeybee species studies here are genetically well programmed for express-
ing PO activity. Our AMP analysis indicated that ApoLpIII was highly up-regulated against P. PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 4. Discussion PLA2C and PLA2D were highly expressed in larvae and pupae compared to
the two other sPLA2 genes in both species. This suggests that iPLA2 and LPLA2 of these honey-
bees may have important roles during immature development. All PLA2 genes were inducible in response to infection by P. larvae in both honeybees. Under RNAi inhibition of all four, the immune responses measured by nodulation and ApoL-
pIII expression were significantly suppressed. The immunosuppressive effects were more
noticeable in the RNAi treatments specific to sPLA2 genes compared to iPLA2 or LPLA2 genes. This strongly suggests that the two sPLA2s are highly associated with immune responses in
both honeybee species. This study demonstrates the physiological role of PLA2 in synthesizing eicosanoids, which
in turn, mediate immune responses of the two honeybee species. This allows us to construct an
eicosanoid signaling pathway in immune mediation from the honeybee genomes (Fig 9). Upon gut infection by virus, bacteria or fungi, a damage signal is triggered by the release of 14 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 PLOS ONE Honeybee immunity against the American founbrood disease Fig 9. Immune responses mediated by eicosanoids in honeybee species, A. mellifera (A) and A. cerana (B). Upon
immune challenge, a damage signal is triggered by dorsal switch protein 1 (DSP1). Release of DSP1 activates
phospholipase A2 (PLA2) to initiate eicosanoid biosynthesis. The catalytic activity of PLA2 produces arachidonic acid
(AA), which is oxygenated by cyclooxygenase (COX) to produce prostaglandin H2 (PGH2). PGH2 is then isomerized
to PGE2 by PGE2 synthase (PGES). AA is alternatively oxygenated by lipoxygenase (LOX) to produce leukotriene (LT)
or oxygenated by epoxygenase (EPX) to epoxyeicosatrienoic acid (EET). These eicosanoids including PGE2 mediate
cellular immune responses via phenoloxidase-activating protease (PAP) and phenoloxidase (PO), and humoral
immune responses by antimicrobial peptides (AMPs) through defensin 1 (Def 1), defensin 2 (Def 2), apolipophorin III
(ApoLpIII), lysozyme (Lyz), transferrin (Tf), and melittin (Mel). GenBank accessions are described in each signal
components. Fig 9. Immune responses mediated by eicosanoids in honeybee species, A. mellifera (A) and A. cerana (B). Upon
immune challenge, a damage signal is triggered by dorsal switch protein 1 (DSP1). Release of DSP1 activates
phospholipase A2 (PLA2) to initiate eicosanoid biosynthesis. The catalytic activity of PLA2 produces arachidonic acid
(AA), which is oxygenated by cyclooxygenase (COX) to produce prostaglandin H2 (PGH2). PGH2 is then isomerized
to PGE2 by PGE2 synthase (PGES). 4. Discussion AA is alternatively oxygenated by lipoxygenase (LOX) to produce leukotriene (LT)
or oxygenated by epoxygenase (EPX) to epoxyeicosatrienoic acid (EET). These eicosanoids including PGE2 mediate
cellular immune responses via phenoloxidase-activating protease (PAP) and phenoloxidase (PO), and humoral
immune responses by antimicrobial peptides (AMPs) through defensin 1 (Def 1), defensin 2 (Def 2), apolipophorin III
(ApoLpIII), lysozyme (Lyz), transferrin (Tf), and melittin (Mel). GenBank accessions are described in each signal
components. https://doi.org/10.1371/journal.pone.0290929.g009 DSP1 from the midgut epithelium in insects [27,62,63]. DSP1 activates PLA2 to catalyze AA
release from phospholipids. AA is then used for synthesis of various eicosanoids through dif-
ferent oxygenases, with the resulting eicosanoids mediating cellular and humoral immune
responses. Critically, all of the enzymes and effectors in the eicosanoid immune signaling path-
way have now been shown to be encoded in the honeybee genomes. DSP1 from the midgut epithelium in insects [27,62,63]. DSP1 activates PLA2 to catalyze AA
release from phospholipids. AA is then used for synthesis of various eicosanoids through dif-
ferent oxygenases, with the resulting eicosanoids mediating cellular and humoral immune
responses. Critically, all of the enzymes and effectors in the eicosanoid immune signaling path-
way have now been shown to be encoded in the honeybee genomes. Supporting information S1 Fig. Suppression of PLA2 gene expression by individual RNAi treatment. (A) Change of
PLA2 expression levels in A. mellifera after injection (1 μg/larva) of dsRNA (‘dsPLA2-A’,
‘dsPLA2-B’, ‘dsPLA2-C’, ‘dsPLA2-D’). (B) Change of PLA2 expression levels in A. cerana after
injection (1 μg/larva) of dsRNA (‘dsPLA2-A’, ‘dsPLA2-B’, ‘dsPLA2-C’, ‘dsPLA2-D’). GFP was
used as a control dsRNA (‘dsCON’). S1 Table. List of primers used in this study. (DOCX) S1 Table. List of primers used in this study. (DOCX) Writing – review & editing: Chuleui Jung, Yonggyun Kim. Writing – review & editing: Chuleui Jung, Yonggyun Kim. Project administration: Yonggyun Kim. Project administration: Yonggyun Kim. Resources: Chuleui Jung, Yonggyun Kim. Resources: Chuleui Jung, Yonggyun Kim. Software: Gahyeon Jin, Md Tafim Hossain Hrithik, Eeshita Mandal, Eui-Joon Kil. Supervision: Eui-Joon Kil, Chuleui Jung, Yonggyun Kim. Validation: Gahyeon Jin, Md Tafim Hossain Hrithik, Eeshita Mandal, Yonggyun Kim. Visualization: Gahyeon Jin, Md Tafim Hossain Hrithik. Writing – original draft: Gahyeon Jin, Md Tafim Hossain Hrithik, Eeshita Mandal, Eui-Joon
Kil, Yonggyun Kim. Writing – review & editing: Chuleui Jung, Yonggyun Kim. Author Contributions Conceptualization: Yonggyun Kim. 15 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929
February 6, 2024 PLOS ONE Honeybee immunity against the American founbrood disease Data curation: Gahyeon Jin, Md Tafim Hossain Hrithik, Eeshita Mandal, Eui-Joon Kil, Chu-
leui Jung. Formal analysis: Gahyeon Jin, Md Tafim Hossain Hrithik, Eeshita Mandal, Eui-Joon Kil,
Chuleui Jung. Funding acquisition: Chuleui Jung, Yonggyun Kim. Investigation: Gahyeon Jin, Md Tafim Hossain Hrithik, Eui-Joon Kil, Chuleui Jung, Yong-
gyun Kim. Methodology: Gahyeon Jin, Md Tafim Hossain Hrithik, Eeshita Mandal, Eui-Joon Kil, Chu-
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Is there a role for <scp>IGF</scp>‐1 in the development of second primary cancers?
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Cancer medicine
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Shanmugalingam, T., Bosco, C., Ridley, A. J., & Van Hemelrijck, M. (2016). Is there a role for IGF-1 in the development of second primary
cancers? Cancer Medicine, 5(11), 3353-3367. https://doi.org/10.1002/cam4.871 Publisher's PDF, also known as Version of record
License (if available):
CC BY
Link to published version (if available):
10.1002/cam4.871 Link to publication record on the Bristol Research Portal
PDF-document This is the final published version of the article (version of record). It first appeared online via Wiley at
https://doi.org/10.1002/cam4.871 . Please refer to any applicable terms of use of the publisher. Shanmugalingam, T., Bosco, C., Ridley, A. J., & Van Hemelrijck, M.
(2016). Is there a role for IGF-1 in the development of second primary
cancers? Cancer Medicine, 5(11), 3353-3367.
https://doi.org/10.1002/cam4.871 Shanmugalingam, T., Bosco, C., Ridley, A. J., & Van Hemelrijck, M. (2016). Is there a role for IGF-1 in the development of second primary
cancers? Cancer Medicine, 5(11), 3353-3367. https://doi.org/10.1002/cam4.871 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited. University of Bristol – Bristol Research Portal
General rights This document is made available in accordance with publisher policies. Please cite only the
published version using the reference above. Full terms of use are available:
http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Cancer Medicine 2016; 5(11):3353–3367 doi: 10.1002/cam4.871 doi: 10.1002/cam4.871 doi: 10.1002/cam4.871 Correspondence Thurkaa Shanmugalingam, Division of Cancer
Studies, Cancer Epidemiology Group,
Research Oncology, King’s College London,
3rd Floor, Bermondsey Wing, Guy’s Hospital,
London SE1 9RT, United Kingdom. Tel: +44 0 20 7188 7904;
Fax: +44 0 20 7188 9986;
E mail: thurkaa t shanmugalingam@kcl ac uk Thurkaa Shanmugalingam, Division of Cancer
Studies, Cancer Epidemiology Group,
Research Oncology, King’s College London,
3rd Floor, Bermondsey Wing, Guy’s Hospital,
London SE1 9RT, United Kingdom. Tel: +44 0 20 7188 7904; Fax: +44 0 20 7188 9986; Fax: +44 0 20 7188 9986; E-mail: thurkaa.t.shanmugalingam@kcl.ac.uk E-mail: thurkaa.t.shanmugalingam@kcl.ac.uk Abstract Breast cancer, colorectal cancer, IGF-1, lung
cancer, prostate cancer, second primary cancer Cancer survival rates are increasing, and as a result, more cancer survivors are
exposed to the risk of developing a second primary cancer (SPC). It has been
hypothesized that one of the underlying mechanisms for this risk could be
mediated by variations in insulin-like growth factor-1 (IGF-1). This review sum-
marizes the current epidemiological evidence to identify whether IGF-1 plays a
role in the development of SPCs. IGF-1 is known to promote cancer develop-
ment by inhibiting apoptosis and stimulating cell proliferation. Epidemiological
studies have reported a positive association between circulating IGF-1 levels and
various primary cancers, such as breast, colorectal, and prostate cancer. The
role of IGF-1 in increasing SPC risk has been explored less. Nonetheless, several
experimental studies have observed a deregulation of the IGF-1 pathway, which
may explain the association between IGF-1 and SPCs. Thus, measuring serum
IGF-1 may serve as a useful marker in assessing the risk of SPCs, and therefore,
more translational experimental and epidemiological studies are needed to further
disentangle the role of IGF-1 in the development of specific SPCs. Funding Information This research was supported by the
Experimental Cancer Medicine Centre at
King’s College London, Cancer Research UK
(AJR), and also by the National Institute for
Health Research (NIHR) Biomedical Research
Centre based at Guy’s and St Thomas’ NHS
Foundation Trust and King’s College London. Received: 9 October 2015; Revised: 8 June
2016; Accepted: 24 June 2016 Cancer Medicine 2016; 5(11):3353–3367 Cancer Medicine Open Access IGF-1 in First Primary Cancers This section provides an overview of evidence for the
emerging role of IGF-1 in the development of first pri-
mary cancers, with a focus on epidemiological studies
(Table 1) as well as experimental studies investigating the
underlying biological mechanisms. y
g
g
IGF-1 is a single-chain polypeptide growth factor [28–30]
that is related to insulin and IGF-2 [31]. IGF-1 stimulates
cell growth, proliferation, and differentiation, and is essen-
tial for normal organismal growth and development [32,
33]. IGF-1 binds to the insulin-like growth factor 1 recep-
tor (IGF-1R), which is a tyrosine kinase receptor [34]. IGF-1 has a higher binding affinity than IGF-2 for IGF-1R. IGF-1R initiates a cascade of downstream signal transduc-
tion pathways known to be involved in cell growth, pro-
liferation, and cancer, including Ras/Raf/ERK and PI3K/
Akt/mTOR [35]. The majority of IGF-1 found in the
circulation is produced by the liver, functioning as an
endocrine hormone. IGF-1 is also produced in other organs
where autocrine or paracrine mechanisms have a role [36]. Ample evidence indicates that IGF-1 and IGF-1R are
important for growth and survival of cancer cells [37,
38] (Fig. 1). The expression of the IGF-1 gene is primarily
regulated by growth hormone (GH), and to a smaller
extent by various other hormones [35]. By contrast, IGF-1
that is synthesized locally in an autocrine or paracrine
manner may stimulate growth of some cancers [36]. The
circulating levels of IGF-1 change markedly with age,
peaking at puberty, and slowly declining with increasing
age; this fluctuation is regulated by GH, which itself has
mitogenic and proliferative properties [35]. However, in
other cell types, for example, cartilage cells, the growth-
stimulating effects of IGF-1 are GH-independent [39]. Furthermore, GH deficiency is the most common disorder
seen in survivors of childhood cancer, and there are con-
cerns regarding its use in treating cancer survivors as it
might increase the risk of SPCs [40]. Although IGF-1
possesses antiapoptotic, cell survival, and transforming
activities, it is not classed as an oncogene. Recently, several studies have identified IGF-1 to be
associated with an increased risk of developing a number
of common cancers, including lung [21], breast [22],
colorectal [23], and prostate [24]. Circulating levels of
IGF-1 have been linked to the development of SPCs in
men with head and neck squamous cell carcinoma [20]. Introduction cancers (SPCs). Therefore, identifying which cancer sur-
vivors have a high risk of developing SPCs is crucial. It is well known that smoking [4, 5], obesity [6, 7],
and insulin resistance [8, 9] are risk factors for the
development of first primary cancers. However, the role
of these risk factors in the development of SPCs in
cancer survivors is less clear. There is some evidence
that SPCs may be the result of genetic and hormonal
risk factors [10–12], of late effects of chemo- and radio-
therapy [2, 13], smoking and alcohol effects [14, 15],
as well as nonmodifiable variables such as age and gen-
der. For instance, a study based on the Swedish Family-
Cancer Database concluded that, compared with the cancers (SPCs). Therefore, identifying which cancer sur-
vivors have a high risk of developing SPCs is crucial. Within the last 40 years, cancer survival rates have doubled
in the UK [1], mainly due to advances in treatment, and
the increased detection of cancer at an early stage [2]. In England and Wales, approximately 50% of adult cancer
patients diagnosed in 2010 to 2011 are predicted to survive
10 years or more [1]. In the US, the 5-year relative sur-
vival rate for all cancers diagnosed in 2004–2010 was
68%, an increase from 49% in 1975–1977 [3]. Side-effects
of cancer treatment and possible underlying etiological
mechanisms, such as IGF-1 metabolism, are thought to
be implicated in the development of second primary It is well known that smoking [4, 5], obesity [6, 7],
and insulin resistance [8, 9] are risk factors for the
development of first primary cancers. However, the role
of these risk factors in the development of SPCs in
cancer survivors is less clear. There is some evidence
that SPCs may be the result of genetic and hormonal
risk factors [10–12], of late effects of chemo- and radio-
therapy [2, 13], smoking and alcohol effects [14, 15],
as well as nonmodifiable variables such as age and gen-
der. For instance, a study based on the Swedish Family-
Cancer Database concluded that, compared with the 3353 IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. “prostate cancer”, “colorectal cancer”, and “meta analysis”,
combined with the keywords “second primary cancer”
and “IGF 1”. All references of the selected articles were
checked using hand searches. Introduction general population, males and females diagnosed with
an initial primary cancer were 1.3–1.6 times more likely
to develop a second cancer, respectively [16]. Epidemiological evidence suggests that survivors of cer-
tain types of cancers have a higher risk of developing
SPCs. For example, contralateral breast cancer is the most
common SPC that develops in patients diagnosed with a
first breast cancer, accounting for approximately 50% of
all SPCs [17]. Furthermore, breast cancer has emerged
as the most common solid cancer among female survivors
of Hodgkin’s lymphoma (diagnosed in childhood), which
is largely due to the high-dose chest irradiation for
Hodgkin’s lymphoma [2, 18]. In addition, urological can-
cers (bladder, kidney, testes, and penile cancers) are con-
sistently more prevalent among men with prostate cancer
[19]. Indeed, it has been hypothesized that variations in
the insulin-like growth factor (IGF) pathway, specifically
IGF-1 and its binding protein 3 (IGFBP-3), could account
for the increased risk of SPCs [20]. IGF-1 in First Primary Cancers However, so far a role of IGF-1 in development of SPCs
following diagnosis of prostate cancer, breast cancer, colo-
rectal cancer, or lung cancer has not been analyzed. In
addition, a disorder known as Laron syndrome which is
associated with low circulating levels of IGF-1 and IGFBP-3
[25] are protected from developing cancer, but instead
can develop diabetes and cardiovascular disease [26]. With the increase in number of cancer survivors, the
long-term health outcomes of this population need to be
carefully examined. Approximately, one in five cancers is
diagnosed in those with a previous diagnosis of cancer,
and hence, these “second primary cancers” are a leading
cause of morbidity and mortality among cancer survivors
[27]. It is therefore of interest to investigate the role of
IGF-1 in the development of various SPCs as it can help
us understand the potential underlying mechanism for
carcinogenesis. This review therefore aims to identify
whether IGF-1 plays a role in the development of SPCs,
by assessing epidemiological evidence available to date. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Breast cancer We used a computerized literature search database
(PubMed and EMBASE) to identify full text and abstract
studies of English language, using human subjects and
published between the years 1999 and 2015. Searches were
performed with and without the Medical Subject Heading
(MeSH) terms for “cancer”, “breast cancer”, “lung cancer”, Findings to date on the role of IGF-1 in breast cancer
development vary depending on the study. An early case–
control study conducted in 1993 demonstrated that cir-
culating levels of IGF-1 were higher in women with breast
cancer compared to women without breast cancer [41]. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 3354 IGF-1 in the development of first and second primary cancers IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. Table 1. Studies of cancer risk related to IGF-1 level. Author (Year)
Control (n)
Cases (n)
Cancer risk related to IGF-1 level
Reference
Breast cancer
Peyrat (1993)
92
44
Median concentrations: 26 ng/mL (BCa)
versus 20 ng/mL (controls)
[38]
Endogenous Hormones and Breast
Cancer Collaborative Group
(2010)
1839
1032
OR for BCa in the highest versus lowest
fifth of IGF1 concentration was 1.28
(95% CI: 1.14–1.44; P < 0.0001)
[39]
Rinaldi (2006)
312
202
Highest versus lowest quintile OR 1.38
(95% CI: 1.02–1.86; P = 0.01) for
women who develop breast cancer after
50 years of age
[40]
Kaaks (2014)
259
193
OR=1.41 (95% CI: 1.01–1.98; P = 0.01 for
the highest versus lowest quartile, for
ER+ breast tumors overall (pre- and
postmenopausal women combined)
[41]
Baglietto (2007)
42961
119
HR for BCa comparing the fourth with the
first quartiles was 1.20 (95% CI:
0.87–1.65). [42]
19542 versus 736
68 versus 9
HR for BCa in older women comparing the
fourth with the first quartiles (+60 years)
was 1.61 (95% CI: 1.04–2.51) versus
0.60 (95% CI: 0.25–1.45) for younger
women (<50 years)
Renehan (2004)
Meta-analysis of 4
studies
Meta-analysis of 4
studies
High concentrations of IGF-1 were
associated with an increased risk of
premenopausal BCa (OR comparing 75th
with 25th percentile 1.65, 95% CI:
1.26–2.08; P < 0.001)
[44]
Shi (2004)
1306
779
Premenopausal women: Nearly 40%
increase in BCa risk among those who
had higher IGF-1 in the circulation
(overall OR 1.39, 95% CI: 1.16–1.66). [45]
1552
911
No association in postmenopausal women
(overall OR 0.93, 95% CI: 0.80–1.10). (Continued) Breast cancer Sugumar (2004)
1471
764
Subjects with higher circulating levels of
IGF-1 had increased risk of premenopau-
sal BCa with an OR of 1.74 (95% CI:
0.97–3.13; P = 0.06)
[46]
Schernhammer (2006)
158
79
RR for top versus bottom quartile of IGF-1
was 0.98 (95% CI: 0.69–1.39; P = 0.77)
[47]
Hankinson (1998)
92
46
Postmenopausal women: No association
between IGF-1 concentrations and BCa
risk (top vs. bottom quintile of IGF-1,
RR = 0.85 [95% CI: 0.53–1.39]). [22]
35
35
RR of BCa among premenopausal women
by IGF-1 concentration (top vs. bottom
tertile) was 2.33 (95% CI: 1.06–5.16;
P = 0.08)
Lung cancer
Ahn (2006)
101
38
OR for LCa risk by IGF-1 concentrations
(highest vs. lowest quartile) was 0.69
(95% CI: 0.41–1.15); P = 0.26
[51]
London (2002)
159
51
OR for LCa risk by IGF-1 concentrations
(highest vs. lowest quartile) was 0.73
(95% CI: 0.43–1.24); P = 0.80
[53]
Lukanova (2001)
47
23
OR for LCa risk by IGF-1 concentrations
[54] Table 1. Studies of cancer risk related to IGF-1 level. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 3355 IGF-1 in the development of first and second primary cancers IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. Author (Year)
Control (n)
Cases (n)
Cancer risk related to IGF-1 level
Reference
Morris (2006)
11,072
843
Meta-analysis: OR for LCa risk by IGF-1
concentrations (highest vs. lowest
quartile) was 1.02 (95% CI: 0.80–1.31);
P = 0.64
[55]
Yu (1999)
54
74
High plasma levels of IGF-1 were
associated with an increased risk of LCa
(OR = 2.06; 95% CI: 1.19–3.56;
P = 0.01)
[21]
Chen (2009)
Meta-analysis of 6
studies
Meta-analysis of 6
studies
Pooled OR for LCa risk by IGF-1 concentra-
tions (highest vs. lowest quartile) was
0.87 (95% CI: 0.60–1.13)
[56]
Cao (2012)
Meta-analysis of 6
studies
Meta-analysis of 6
studies
OR for LCa risk by IGF-1 concentrations
(highest vs. lowest quartile) was 1.05
(95% CI: 0.80–1.37); P = 0.74
[57]
Prostate cancer
Mantzoros (1997)
52
51
Increment of 60 ng mL corresponded to
an OR of 1.91 (95% CI: 1.00–3.73;
P = 0.05)
[62]
Colorectal cancer
Nomura (2003)
282
177 (colon cancer)
105 (rectal cancer)
Weakly positive association of IGF-I with
colon cancer. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Breast cancer Colon cancer cases in third
(IGF-1 of 137–174 ng/mL) and fourth
quartile (IGF-1 > 174 ng/mL) had
increased risk compared with controls
(OR of 2.2 and 1.8, respectively)
No association of IGFI with rectal cancer
[68]
Palmqvist (2002)
336
110 (colon cancer)
58 (rectal cancer
Increase in colon cancer risk with
increasing levels of IGF-1 (OR of 2.30 and
2.66 for third and fourth quartile,
respectively)
Rectal cancer risk was inversely related to
levels of IGF-1 (OR of 0.33 and 0.33 for
third and fourth quartile, respectively)
[69]
Tripkovic (2007)
52
52
Increase in IGF-1 level was followed by a
3.15-fold increased risk for developing
colon cancer with levels of
IGF-1 > 310 ng/mL, whereas twice as
many controls exhibited levels of
IGF-1 < 107 ng/mL
[70]
Ma (1999)
318
193
Men in the highest quintile for IGF-I had
an increased risk of colorectal cancer
compared with men in the lowest
quintile (RR = 2.51; 95% CI: 1.15–5.46;
P = 0.02)
[71]
Kaaks (2000)
200
102
Colorectal cancer risk showed a modest
but statistically nonsignificant positive
association with levels of IGF-I
[72]
BCa, breast cancer; OR, odds ratio; CI, confidence intervals; HR, hazard ratios; RR, relative risk; LCa, lung cancer. 1B
t
d
l
l t d f
th 2284
ith IGF I
d
Table 1. Studies of cancer risk related to IGF-1 level. (Continued) Table 1. Studies of cancer risk related to IGF-1 level. (Continued) Control (n)
Cases (n)
Cancer risk related to IGF-1 level
Reference
11,072
843
Meta-analysis: OR for LCa risk by IGF-1
concentrations (highest vs. lowest
quartile) was 1.02 (95% CI: 0.80–1.31);
P = 0.64
[55]
54
74
High plasma levels of IGF-1 were
associated with an increased risk of LCa
(OR = 2.06; 95% CI: 1.19–3.56;
P = 0.01)
[21]
Meta-analysis of 6
studies
Meta-analysis of 6
studies
Pooled OR for LCa risk by IGF-1 concentra-
tions (highest vs. lowest quartile) was
0.87 (95% CI: 0.60–1.13)
[56]
Meta-analysis of 6
studies
Meta-analysis of 6
studies
OR for LCa risk by IGF-1 concentrations
(highest vs. lowest quartile) was 1.05
(95% CI: 0.80–1.37); P = 0.74
[57]
52
51
Increment of 60 ng mL corresponded to
an OR of 1.91 (95% CI: 1.00–3.73;
P = 0.05)
[62]
282
177 (colon cancer)
105 (rectal cancer)
Weakly positive association of IGF-I with
colon cancer. Breast cancer Colon cancer cases in third
(IGF-1 of 137–174 ng/mL) and fourth
quartile (IGF-1 > 174 ng/mL) had
increased risk compared with controls
(OR of 2.2 and 1.8, respectively)
No association of IGFI with rectal cancer
[68]
336
110 (colon cancer)
58 (rectal cancer
Increase in colon cancer risk with
increasing levels of IGF-1 (OR of 2.30 and
2.66 for third and fourth quartile,
respectively)
Rectal cancer risk was inversely related to
levels of IGF-1 (OR of 0.33 and 0.33 for
third and fourth quartile, respectively)
[69]
52
52
Increase in IGF-1 level was followed by a
3.15-fold increased risk for developing
colon cancer with levels of
IGF-1 > 310 ng/mL, whereas twice as
many controls exhibited levels of
IGF-1 < 107 ng/mL
[70]
318
193
Men in the highest quintile for IGF-I had
an increased risk of colorectal cancer
compared with men in the lowest
quintile (RR = 2.51; 95% CI: 1.15–5.46;
P = 0.02)
[71]
200
102
Colorectal cancer risk showed a modest
but statistically nonsignificant positive
association with levels of IGF-I
[72]
nfidence intervals; HR, hazard ratios; RR, relative risk; LCa, lung cancer. culated from the 2284 women with IGF-I measured. GF-1 level. (Continued) BCa, breast cancer; OR, odds ratio; CI, confidence intervals; HR, hazard ratios; RR, relative risk; LCa, lung cancer. 1Breast cancer cases and person-years calculated from the 2284 women with IGF-I measured. 2Breast cancer cases and person-years. Since then, several epidemiological studies have reported
that higher circulating levels of IGF-1 are associated with
an increased risk of breast cancer [22, 42–45]. This may
arise as higher levels of IGF-1 are associated with accel-
eration of early carcinogenesis [36]. More recently, three meta-analyses demonstrated positive associations between
IGF-1 and risk of breast cancer among premenopausal,
but not postmenopausal women [46–48]. A study by
Schernhammer et al. aims to explain these observations
by showing that premenopausal women with high IGF-1 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 3356 IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. Figure 1. The effects of IGF-1 and IGF-1R on normal and cancerous cells. Cancer cell
Normal cell
IGF-1
IGF-1
IGF-1R
IGF-1R
Binds to
Binds to
Proliferation
Proliferation
Apoptosis
Apoptosis
Stimulate,
Inhibit,
Increased levels
Transformation Binds to Binds to IGF-1R Proliferation Apoptosis Proliferation Apoptosis Cancer cell Transformation Increased levels Stimulate, Figure 1. Breast cancer The effects of IGF-1 and IGF-1R on normal and cancerous cells. particularly in premenopausal women. Moreover, experi-
mental evidence suggests that a link between IGF-1 and
estrogen may explain this positive association, but perhaps
only in breast cancers that express the estrogen
receptor. levels were at risk of higher IGF-1R activation in mam-
mary epithelial cells, which is suggested to increase survival
of these cells with accumulating DNA damage, thereby
facilitating stepwise carcinogenesis [49]. These results may
indicate the importance of IGF-1 levels in younger women
in early life or its possible interaction with other hormones
such as estrodial [36] and growth hormone [39]. In con-
trast to these studies, a large prospective study pooling
two Swedish cohorts found no association between cir-
culating IGF-1 and risk of breast cancer, regardless of
menopausal status [50]. It is unclear why there is such
a discrepancy in study findings. However, differences in
findings may be due to timing of blood sampling, the
patient cohort or the subset of breast cancer. Prospective
studies are advantageous over retrospective studies since
blood samples to measure IGF-1 levels are collected before
the clinical diagnosis of cancer and hence reverse causa-
tion (i.e., effects of an undiagnosed cancer on levels of
IGF-1) is less likely to play a role [50]. levels were at risk of higher IGF-1R activation in mam-
mary epithelial cells, which is suggested to increase survival
of these cells with accumulating DNA damage, thereby
facilitating stepwise carcinogenesis [49]. These results may
indicate the importance of IGF-1 levels in younger women
in early life or its possible interaction with other hormones
such as estrodial [36] and growth hormone [39]. In con-
trast to these studies, a large prospective study pooling
two Swedish cohorts found no association between cir-
culating IGF-1 and risk of breast cancer, regardless of
menopausal status [50]. It is unclear why there is such
a discrepancy in study findings. However, differences in
findings may be due to timing of blood sampling, the
patient cohort or the subset of breast cancer. Prospective
studies are advantageous over retrospective studies since
blood samples to measure IGF-1 levels are collected before
the clinical diagnosis of cancer and hence reverse causa-
tion (i.e., effects of an undiagnosed cancer on levels of
IGF-1) is less likely to play a role [50]. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Lung cancer Studies to date have investigated the association between
IGF-1 and lung cancer. Several studies have shown that
circulating IGF-1 levels were not associated with an
increased risk of lung cancer [53–57]. One case–control
study found a positive association between IGF-1 and
risk of lung cancer (OR: 2.06; 95% CI: 1.19–3.56) [21]. Furthermore, this study identified that the levels of IGF-1
and IGF-2 in plasma were not influenced by cigarette
smoking [21]. IGFBP-3 is the main IGF-1-binding protein in blood. IGFBP-3 is generally considered to act as a tumor sup-
pressor gene by reducing the ability of IGF-1 to promote
cell survival and proliferation [58]. Although epidemio-
logical studies overall found no association for IGF-1, a
reduced risk of lung cancer is reported with higher cir-
culating levels of IGFBP-3, when comparing the highest
quartile versus lowest quartile of IGFBP-3 in a Chinese
prospective study (OR: 0.50, 95% CI: 0.25–1.02) [55]. Moreover, several meta-analyses have also reported an
inverse association between IGFBP-3 and risk of lung
cancer [58, 59]. Estrogen plays an important role in the etiology of
breast cancer, and there are experimental studies reporting
cross-talk between IGF-1 and the estrogen receptor (ER)
in mammary cells, possibly through synergistic effects that
contribute to breast carcinogenesis [44, 51]. Stewart et al. showed that estrogen increases IGF-1 binding and IGF-1R
mRNA levels in the estrogen-sensitive MCF-7 cell line by
7- and 6.5-fold, respectively [52]. This suggests that one
potential mechanism by which estrogen stimulates breast
cancer cell proliferation may involve sensitization of IGF-1
[52]. It is possible that both IGF-1 and IGFBP-3 contribute
to the development of lung cancer. Cell culture studies
have found that lung cancer cell lines, regardless of their Thus far, epidemiological evidence overall suggests a
positive association between IGF-1 and breast cancer risk, 3357 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. histological subtypes, have the capacity to express IGF-1
and its binding protein, IGFBP-3, both in tumors and
blood [60, 61]. vivo studies have shown significantly reduced proliferation
rates in PC-3 prostate cancer cell lines in IGF-1-deficient
hosts, compared to control hosts [66]. Exogenous IGF-1
increased the invasive potential of the DU145 prostate
cancer cell line, which was dependent on IGF-1R, the
ERK MAPK pathway, and the PI3K pathway [67]. Prostate cancer Associations between prostate cancer and IGF-1 have been
studied extensively, and consistently show a positive asso-
ciation. Since 1993, it has been investigated whether higher
circulating IGF-1 levels are associated with an increased
risk of prostate cancer [62, 63]. Early studies failed to
demonstrate an association between IGF-1 and prostate
cancer risk. The first significant positive association between
IGF-1 and prostate cancer was examined in a case–control
study by Mantzoros et al. By comparing men with prostate
cancer to healthy controls, the odds ratio per 60 ng/mL
increment in circulating levels of IGF-1 was 1.91 (95%
CI: 1.00–3.73) [64]. Furthermore, the authors also men-
tioned that this association is further reinforced by the
lack of association between IGF-1 and benign prostatic
hyperplasia. Lung cancer Furthermore, prostate cancer epithelial cells can stimulate
their own growth by synthesizing and responding to IGF-1
in an autocrine manner (Fig. 2), as opposed to paracrine
signaling [68]. Thus, until now, there is little evidence for a link
between IGF-1 and lung cancer risk, but an inverse asso-
ciation between IGFBP-3 and lung cancer risk has been
observed. These epidemiological observations are consistent
with experimental data, which demonstrates that IGFBP-3
block the mitogenic and antiapoptotic effects of IGF-1
on lung cancer cells [21, 55]. Overall, epidemiological and experimental evidence to
date suggest a positive association between circulating
IGF-1 and prostate cancer risk. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Colorectal cancer Overall, studies have provided data showing that colorectal
cancer is positively associated with IGF-1 levels [23, 69–72]. Early studies in the late 1990s, suggested that high cir-
culating IGF-1 concentrations are associated with an
increasing risk of colorectal cancer [23, 73]. Nomura et al. evaluated the association between IGF-1 and colon and
rectal cancer separately [69]. They showed a higher risk
of colon cancer for those with IGF-1 levels in the third
(137–174 ng/mL) and fourth quartiles (IGF-1 > 174 ng/
mL) as compared to the controls (OR of 2.2 and 1.8,
respectively). There was no association between IGF-1 and
rectal cancer. However, another study found a decreased
risk of rectal cancer with high levels of circulating IGF-1
[70]. The authors commented that this may be due to
rectal cancer presenting at an earlier stage than colon
cancer, which may have masked the association with IGF-1 IGF-1 is known to stimulate the growth of prostate
cancer cells by inducing cell proliferation and inhibiting
apoptosis [65]. The effect of IGF-1 on prostate cancer
cell lines has been extensively explored. For example, in Figure 2. The autocrine and paracrine actions of IGF-1. Stromal cell
IGF-1R
IGF-1
Paracrine
action
Cancer cell Figure 2. The autocrine and paracrine actions of IGF-1. Stromal cell
IGF-1R
IGF-1
Paracrine
action
Cancer cell 3358
© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Figure 2. The autocrine and paracrine actions of IGF-1. IGF-1R
Cancer cell
IGF-1
Autocrine
action
Stromal cell
IGF-1R
IGF-1
Paracrine
action
Cancer cell IGF-1R
Cancer cell
IGF-1
Autocrine
action Stromal cell Cancer cell IGF-1 Cancer cell Figure 2. The autocrine and paracrine actions of IGF-1. Figure 2. The autocrine and paracrine actions of IGF-1. 3358 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. levels [70]. On the other hand, data from two nested
case–control study showed no statistically significant asso-
ciation between IGF-1 and colorectal cancer risk [74, 75]. lymphoma during radiation treatment, or the duration
and dose of treatment [88, 89]. Even in the absence of
treatment effects, in general, younger women are at a
greater risk of developing a second primary breast cancer
than older women [90, 91]. For instance, a cohort study
by Hancock et al. Colorectal cancer Therefore,
further research is required to investigate the role of obe-
sity, ethnicity, and dietary habits possibly as confounders
to IGF-1 and colorectal cancer risk. Breast cancer The risk of developing a second primary breast cancer
in a patient diagnosed with a first breast cancer has been
studied extensively. It has been shown that in women
with breast cancer, the risk of developing a new primary
breast cancer in the contralateral breast is much higher
than for healthy women developing a first breast cancer
[84, 85]. Only a small portion of this large risk is attrib-
utable to effects of treatment: lifestyle and genetic factors
also need to be taken into account [84], and possibly
the role of IGF-1 in increasing breast cancer risk. IGF-1 in Second Primary Cancers This section summarizes studies on the emerging role of
IGF-1 in the development of SPCs with a focus on the
definition of SPC as well as their potential link with IGF-1
in the case of patients with primary breast, lung, prostate,
and colorectal cancer. SPCs are defined as malignant tumors diagnosed at the
same time as the primary tumor or later, which are in
a different organ [82] and are not a metastasis or recur-
rence of the original primary cancer [83]. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Colorectal cancer which reviewed women treated for
Hodgkin’s disease between 1961 and 1990 (mean follow-
up, 10 years) concluded that the greatest risk was seen
in young women treated before the age of 15 years (RR:
136, 95% CI: 34–371), with a significant decline in rela-
tive risk with advancing age (above 30 years of age, RR:
0.7, 95% CI: 0.2–1.8) [92]. The epidemiological data on IGF-1 and colorectal cancer
is supported by laboratory evidence. The IGF-1R is
expressed in both normal colonic mucosa and human
colorectal cancers [70]. IGF-1 has been speculated to cause
proliferation of colorectal cancer cells and promote over-
expression of the IGF-1R in several studies, with no uni-
form consensus [76–79]. Furthermore, in acromegaly, a
condition that is associated with high IGF-1 levels, studies
have shown that there is increased proliferation of normal
colonic epithelium with an increased risk of developing
colorectal adenomas and cancers in acromegaly patients
[80, 81]. From a biological point of view, the pubertal growth
of the mammary gland is mediated predominantly by the
actions of IGF-1 and GH via estrogen [93]. It is postu-
lated that this mechanism may make younger women
more prone to developing a secondary breast cancer due
to the increased levels of both IGF-1 and estrogen during
puberty, and the promoting effects of IGF-1 [41, 94]. According to the evidence from in situ hybridization, in
breast cancer, IGF-1 is predominantly expressed in the
stromal cells (mainly fibroblasts) and very rarely in the
breast epithelium [28, 95]. This supports the concept of
a paracrine role of IGF-1 in breast cancer (Fig. 2). It is
possible that there is also an endocrine role, given that
circulating IGF-1 in the bloodstream is implicated in the
malignant transformation of breast tissue [28, 43, 96]. IGF-1 functions to protect breast cancer cells from apop-
tosis and induces survival [28], suggesting that locally
synthesized IGF-1 influences the growth of human breast
cancer cells. It can therefore be hypothesized that IGF-1
produced by the stromal cells is increased in breast cancer
(Fig. 2). This may then promote growth of a second
primary breast cancer by entering the blood stream and
acting in an endocrine fashion [94, 97]. In colorectal cancer, the circulating levels of IGF-1 is
particularly influenced by nutritional status. Lung cancer The lungs are often regarded as one of the most common
organs to develop a SPC [11, 98, 99]. Common causes
of a secondary lung cancer include a resected primary
lung cancer, treatment-related complications in breast
cancer and head and neck cancers [99–101], as well as
continued smoking [102, 103]. In a US study, Johnson
reported a 2–14% risk of developing a second lung cancer
per person per year, with the risk increasing from twofold
to sevenfold after 10 years of initial lung cancer diagnosis
[104]. Breast cancer is also a common SPC in itself. For
instance, it is the most common SPC that develops in
young women treated for Hodgkin’s lymphoma with
supradiaphragmatic irradiation [86]. The estimates of
cumulative risks of developing breast cancer ranged from
35% by 40 years of age [87] to 48% by 40 years of age
[88] after treatment. Much of this variation may be due
to the differences in the age of diagnosis of Hodgkin’s Head and neck cancer patients are at an increased risk
of developing lung cancer with a standardized incidence
ratio (SIR) of 3.75 (95% CI: 3.01–4.62) [98]. In addition,
according to data from the Surveillance, Epidemiology,
and End Results (SEER) registries, approximately 5% of 3359 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. cancers [111, 112]; this seems to be influenced by pelvic
radiation therapy for prostate cancer [108]. Moreover,
diagnostic bias is thought to play a role due to anatomy. However, Chrouser et al. did not observe an increased
risk of bladder cancer after radiotherapy for prostate cancer
[113], and there are some uncertainties in relation to the
possible mechanism for the lack of association observed
in this study. It is possible that there may have been an
increased risk in this study that was not detected due to
a short mean follow-up period of 7.1 years or likelihood
of underreporting SPCs. Based on the current evidence,
it seems that the risk of developing a SPC after prostate
cancer is higher, particularly for other urological
cancers. breast cancer survivors are diagnosed with a second pri-
mary lung cancer [11]. Additionally, treatment of breast
cancer patients with radiation postmastectomy has been
shown to approximately double the risk of second primary
lung cancer, especially in the ipsilateral lung [99]. Prostate cancer The risk of all SPCs following a diagnosis of a first pri-
mary prostate cancer has been studied with variable results. In 1999, a Swiss study based on data collected from the
Cancer Registries of the Swiss Cantons of Vaud and
Neuchâtel investigated the risk of SPCs in prostate cancer
survivors between 1974 and 1994. They found a signifi-
cantly reduced incidence rate of all cancers in men diag-
nosed with prostate cancer, compared with the general
population (SIR: 0.7, 95% CI: 0.6–0.8) [107]. More recently,
in 2014, a cohort study from the Swiss Canton of Zurich
investigated the risk of SPCs in prostate cancer survivors
between 1980 and 2010 [19]. They found an increased
risk of SPCs among men with prostate cancer, compared
to the general population (SIR: 1.11, 95% CI: 1.06–1.17). The inconsistency between these two studies may be due
to the diagnosis of prostate cancer at an advanced stage
with shorter survival in the earlier years of study [19]. Therefore, the chance of developing a SPC was lower
than what it is currently, when prostate cancer is gener-
ally diagnosed at a less advanced stage. In contrast to the biological mechanisms of breast and
lung cancer, prostate cancer epithelial cells can stimulate
their own growth by synthesizing and responding to IGF-1
[65, 68]. Furthermore, there is evidence that IGF-1 enhances
the adhesion of prostate cancer cells and this promotes
prostate cancer metastasis, possibly through the actions
of IL-17 [118]. The potential data does not suggest a
direct causative role for IGF-1 signaling in the progression
and invasiveness of prostate cancer. The IGF-1 pathway
activates a number of downstream signaling pathways,
including the phosphatidylinositol-3 kinase (PI3-K) path-
way, the protein kinase C pathway, the CREB pathway,
and the mitogen-activated protein kinase (MAPK) pathway. These pathways contribute to prostate cancer through
deregulation and constitutive activation of the pathway
[67]. While the etiology of IGF-1 in second primary pros-
tate cancer is unknown, it is plausible that those who
develop prostate cancer may possess a common genetic,
hormonal, or environmental factor that protects them
from developing a SPC [119]. Prostate cancer survivors
have a lower risk of developing cancers of the stomach,
lung, and pancreas [108, 120], raising the question of
whether these patients are “protected” against these malig-
nancies, or whether it is simply that they are above the When looking at specific cancer types, Davies et al. Lung cancer The
rates of second primary lung cancer among women diag-
nosed with breast cancer before the age of 50 years is
rising significantly, with the increase being as early as
1 year after breast cancer diagnosis [105]. Radiotherapy
treatment for breast cancer seems unlikely as the sole
cause of this rise (see methodological section) because a
long-term latency period (5–10 years) is usually associated
with radiotherapy treatment [105]; proteins or hormones
such as IGF-1 should also be considered as predisposing
factors. Prostate cancer is also commonly observed as a SPC
in itself. Kok et al. concluded that in the first year fol-
lowing a first cancer diagnosis, male cancer survivors have
a 30% increased risk of developing prostate cancer as a
SPC, partly due to increased diagnostic activity of the
urological organs or incidental finding following health
check-ups [114]. Other studies have also shown an excess
risk of developing prostate cancer as a SPC after a diag-
nosis of a bladder cancer as a first cancer [115, 116]. In
addition, cancer survivors diagnosed with a first primary
urological cancer may request for prostate-specific antigen
(PSA) testing as a consequence of anxiety or persisting
urological symptoms [114]. Furthermore, survivors of
melanoma are also at increased risk of developing prostate
cancer [117]. When considering the biological effects of IGF-1 in
second primary lung cancer, it has been shown that lung
mesenchymal cells locally synthesize IGF-1 which acts on
the bronchial epithelium in a paracrine manner [56]. A
possible mechanism explaining the role of IGF-1 in second
primary lung cancer is that higher IGF-1 levels detected
in lung cancer are probably regulated by the levels of
tissue-derived IGF-1, but not circulating IGF-1 [106]. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Colorectal cancer With
the exposure to radiotherapy, one hypothesis suggests that
in individuals with higher IGF bioactivity, there is enhanced
survival of partially transformed cells which leads to a
larger pool of targets for subsequent “hits” initiating colo-
rectal carcinogenesis via the process of stepwise carcino-
genesis and malignant transformation. A second hypothesis
suggests that the time needed for the progression of a
fully transformed cell to fully developed cancer is inversely
associated with IGF bioactivity [69]. Secondly, when evaluating the effect of IGF-1 on SPCs,
one has to consider sources of errors that cause misclas-
sification of this biomarker. Nondifferential misclassifica-
tion of IGF-1 may occur due to laboratory errors (e.g.,
specimen collection, processing, and storage) or changes
in IGF-1 assays [126]. In addition, a single measurement
of IGF-1 may not reflect the actual underlying levels. Repeated measurements would reflect long-term exposure
and may be useful in the context of carcinogenesis [127]. Aside from misclassification of data for IGF-1, it is also
possible to have misclassification related to the SPCs
because it is not always possible from a pathological point
of view to make a distinction between local recurrences,
metastases, or a true SPC. In normal colonic tissue, IGF-1 binds with high affinity
to the IGF-1R and activates specific insulin receptor sub-
strates, which can modulate several downstream pathways
involved in gene transcription, cell proliferation, and
apoptosis [124]. Although the etiology of IGF-1 in second
primary colorectal cancer is unknown, based on findings
from normal colonic tissues, we can speculate about the
potential complexity of this carcinogenic mechanism. With
the exposure to radiotherapy, one hypothesis suggests that
in individuals with higher IGF bioactivity, there is enhanced
survival of partially transformed cells which leads to a
larger pool of targets for subsequent “hits” initiating colo-
rectal carcinogenesis via the process of stepwise carcino-
genesis and malignant transformation. A second hypothesis
suggests that the time needed for the progression of a
fully transformed cell to fully developed cancer is inversely
associated with IGF bioactivity [69]. Thirdly, when studying the association between IGF-1
and SPCs, one has to be aware of potential confounders
such as smoking or treatment. In the case of lung cancer,
the effect of current and past smoking needs to be removed
to maximize the efficiency of the study [56] as some
studies have shown that smoking decreases the levels of
IGF-1 [128], while others have found no relationship
[129]. Colorectal cancer It is therefore possible that cigarette-related car-
cinogen exposure may overshadow the more subtle effects
of IGF-1 on cancer development, which could explain
the general lack of an association between IGF-1 and risk
of lung cancer [54]. As a result, smoking may have an
effect on IGF-1 levels as well as the risk of developing
a SPC, but it is unlikely to be an intermediate in the
pathway between IGF-1 and SPCs [130]. Furthermore,
treatment received for the primary cancer may confound
the association between IGF-1 and SPCs [114, 130]. Similarly to smoking, treatment may also alter the effect
of IGF-1 as well as the risk of developing a SPC, but it
is again unlikely to be an intermediate in the pathway
between IGF-1 and SPCs [130]. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. IGF-1 in the development of first and second primary cancers age at which the risk of these tumors typical peak, which
is at an earlier age [119]. Even though several studies suggest a link between IGF-1
and development of SPCs, several methodological issues
need to be considered when assessing these epidemiologi-
cal findings. Firstly, diagnostic bias may occur when the
SPC is the main outcome of interest, as it may be detected
following a diagnostic intervention related to the first
primary tumor [115]. Aside from diagnostic activity, treat-
ment related to this first primary tumor may also increase
the risk of developing a second primary tumor (e.g.,
chemotherapy and radiation therapy) [115, 125]. Methodological considerations for
epidemiological studies investigating the
link between IGF-1 and risk of second
primary cancers In the clinical setting, it may be problematic to absolutely
define whether the second tumor is in fact a SPC or a
recurrence or a metastasis, and a definitive diagnosis may
only be possible histologically. Whether the results showed
in this review were strictly according to the standard are
unclear, so therefore we need to consider the results with
caution. Prostate cancer reported that survivors of prostate cancer had a 40% lower
risk of developing a SPC compared to the general male
US population; the risk was lower for leukemias and
cancers of the oral cavity, stomach, colon, liver, lung,
and pancreas [108]. However, they observed a higher risk
of developing bladder [109, 110], renal, and endocrine © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 3360 Colorectal cancer Several studies have demonstrated an increased risk of
developing secondary colorectal cancer following radio-
therapy exposure, in particular, rectal cancer following
radiation for prostate cancer and colorectal cancer fol-
lowing abdominopelvic radiation for cervical cancer. Brenner et al. investigated the risk in prostate cancer
patients who underwent radiotherapy or surgery and
reported a significantly increased risk of rectal cancer in
the radiotherapy group, particularly for long-term survi-
vors, when comparing with the surgery group [121]. Furthermore, Baxter et al. observed a significant increase
in the development of rectal cancer postradiation for
prostate cancer [122]. However, radiation did not promote
development of cancer in the remainder of the colon,
suggesting that the effect of radiation is limited directly
to irradiated tissue. In addition to prostate cancer patients,
cervical cancer patients also seem to be at risk of devel-
oping colon cancer, as observed by Chatruvedi et al. [123]. Several studies have demonstrated an increased risk of
developing secondary colorectal cancer following radio-
therapy exposure, in particular, rectal cancer following
radiation for prostate cancer and colorectal cancer fol-
lowing abdominopelvic radiation for cervical cancer. Brenner et al. investigated the risk in prostate cancer
patients who underwent radiotherapy or surgery and
reported a significantly increased risk of rectal cancer in
the radiotherapy group, particularly for long-term survi-
vors, when comparing with the surgery group [121]. Furthermore, Baxter et al. observed a significant increase
in the development of rectal cancer postradiation for
prostate cancer [122]. However, radiation did not promote
development of cancer in the remainder of the colon,
suggesting that the effect of radiation is limited directly
to irradiated tissue. In addition to prostate cancer patients,
cervical cancer patients also seem to be at risk of devel-
oping colon cancer, as observed by Chatruvedi et al. [123]. In normal colonic tissue, IGF-1 binds with high affinity
to the IGF-1R and activates specific insulin receptor sub-
strates, which can modulate several downstream pathways
involved in gene transcription, cell proliferation, and
apoptosis [124]. Although the etiology of IGF-1 in second
primary colorectal cancer is unknown, based on findings
from normal colonic tissues, we can speculate about the
potential complexity of this carcinogenic mechanism. Conflict of Interest None declared. 14. McLaughlin, V. H., A. Trentham-Dietz, J. M. Hampton,
P. A. Newcomb, and B. L. Sprague. 2014. Lifestyle
factors and the risk of a second breast cancer after
ductal carcinoma in situ. Cancer Epidemiol. Biomark. Prev. 23:450–460. Acknowledgments 11. Mariotto, A. B., J. H. Rowland, L. A. Ries, S. Scoppa,
and E. J. Feuer. 2007. Multiple cancer prevalence: a
growing challenge in long-term survivorship. Cancer
Epidemiol. Biomark. Prev. 16:566–571. This research was supported by the Experimental Cancer
Medicine Centre at King’s College London, Cancer Research
UK (AJR) and also by the National Institute for Health
Research (NIHR) Biomedical Research Centre based at
Guy’s and St Thomas’ NHS Foundation Trust and King’s
College London. The views expressed are those of the
author(s) and not necessarily those of the NHS, the NIHR,
or the Department of Health. 12. Travis, L. B., W. Demark Wahnefried, J. M. Allan, M. E. Wood, and A. K. Ng. 2013. Aetiology, genetics and
prevention of secondary neoplasms in adult cancer
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10:289–301. 13. Rheingold, S. R., A. I. Neugut, and A. T. Meadows. 2003. Therapy-related secondary cancers. 6th ed. BC
Decker. Medicine H-FC, editor. Conclusion In spite of a consistent positive observation between IGF-1
and risk of first primary cancers (especially breast, prostate,
and colorectal), the evidence for the role of IGF-1 in the
development of SPCs is less clear. Some of the evidence
we gathered came from targeting the IGF system in cell 3361 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. IGF-1 in the development of first and second primary cancers T. Shanmugalingam et al. study of obesity and cancer risk (Sweden). Cancer
Causes Control 12:13–21. culture studies, and therefore, we need to see the results
with caution on whether it can be compared to clinical
situations. However, the relevant influences of these path-
ways in SPCs are unknown. This lack of an association
may be partly explained by methodological issues. With
respect to the biological pathway, there is consistent evi-
dence for the mitogenic role of IGF-1 in carcinogenesis
by increasing cell proliferation and inhibiting apoptosis. However, experimental studies highlight uncertainties
regarding the role of IGF-1 in the development of SPCs. More observational studies are needed to further under-
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Sustainability in the Aerospace, Naval, and Automotive Supply Chain 4.0: Descriptive Review
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Received: 9 November 2020; Accepted: 7 December 2020; Published: 10 December 2020 Received: 9 November 2020; Accepted: 7 December 2020; Published: 10 December 2020 Received: 9 November 2020; Accepted: 7 December 2020; Published: 10 December 2020 Received: 9 November 2020; Accepted: 7 December 2020; Published: 10 December 2020 Abstract: The search for sustainability in the Supply Chain (SC) is one of the tasks that most concerns
business leaders in all manufacturing sectors because of the importance that the Supply Chain has as
a transversal tool and due to the leading role that it has been playing lately. Of all the manufacturing
sectors, this study focuses on the aerospace, shipbuilding, and automotive sectors identified as
transport. The present study carries out a descriptive review of existing publications in these three
sectors in relation to the sustainability of the Supply Chain in its 4.0 adaptation as an update in
matters that are in constant evolution. Among the results obtained, Lean practices are common to
the three sectors, as well as different technologies focused on sustainability. Furthermore, the results
show that the automotive sector is the one that makes the greatest contribution in this sense through
collaborative programs that can be very useful to the other two sectors, thus benefiting from the
consequent applicable advantages. Meanwhile, the Aerospace and Shipbuilding sectors do not seem
to be working on promoting a sustainable culture in the management of the Supply Chain or on
including training programs for their personnel in matters related to Industry 4.0. Keywords: sustainability; supply chain management; manufacturing system; automotive; aerospace;
shipbuilding; transports Materials 2020, 13, 5625; doi:10.3390/ma13245625 www.mdpi.com/journal/materials Review
Sustainability in the Aerospace, Naval, and
Automotive Supply Chain 4.0: Descriptive Review Magdalena Ramirez-Peña *
, Pedro F. Mayuet
, Juan Manuel Vazquez-Martinez
and
Moises Batista *
School of Engineering, University of Cádiz, Avenue Universidad de Cádiz, 10, 11519 Puerto Real-Cadiz, Spain;
pedro.mayuet@uca.es (P.F.M.); juanmanuel.vazquez@uca.es (J.M.V.-M.)
* Correspondence: magdalena.ramirez@uca.es (M.R.-P.); moises.batista@uca.es (M.B.);
Tel.: +34-956-483-200 (M.B.) Magdalena Ramirez-Peña *
, Pedro F. Mayuet
, Juan Manuel Vazquez-Martinez
and
Moises Batista *
School of Engineering, University of Cádiz, Avenue Universidad de Cádiz, 10, 11519 Puerto Real-Cadiz, Spain;
pedro.mayuet@uca.es (P.F.M.); juanmanuel.vazquez@uca.es (J.M.V.-M.)
* Correspondence: magdalena.ramirez@uca.es (M.R.-P.); moises.batista@uca.es (M.B.);
Tel.: +34-956-483-200 (M.B.) materials materials 1. Introduction pp
pp y
p
g
p
roles and responsibilities of each member and the expected outcome of each process. Therefore, Supply Chain activities are framed within three macro processes: CRM, Customer
Relationship Management; ISCM, Internal SCM; and SRM, Supplier Relationship Management. Therefore, Supply Chain activities are framed within three macro processes: CRM, Customer
Relationship Management; ISCM, Internal SCM; and SRM, Supplier Relationship Management. Figure 1
details these three framework processes [1]. Figure 1 details these three framework processes [1]. Figure 1. Macro Supply Chain processes adapted from [1]. Figure 1. Macro Supply Chain processes adapted from [1]. Figure 1. Macro Supply Chain processes adapted from [1]. Information is disrupted as it moves up the chain because the information shared in the stages
is incomplete. The lack of coordination can be called “the whip effect”. This lack of coordination
damages relations between the different stages where there is a tendency to blame other stages
Information is disrupted as it moves up the chain because the information shared in the stages is
incomplete. The lack of coordination can be called “the whip effect”. This lack of coordination damages
relations between the different stages where there is a tendency to blame other stages thinking that
theirs is doing well, which causes a loss of trust between the different stages and makes coordination
efforts difficult [5]. thinking that theirs is doing well, which causes a loss of trust between the different stages and makes
coordination efforts difficult [5]. In the present case, transport companies tend to report on greenhouse gas emissions, fuel
In the present case, transport companies tend to report on greenhouse gas emissions,
fuel consumption, and transport efficiency. From an environmental perspective, they report on four
categories: energy consumption, water consumption, greenhouse gas emissions, and waste generation. consumption, and transport efficiency. From an environmental perspective, they report on four
categories: energy consumption, water consumption, greenhouse gas emissions, and waste
generation. The role of sustainability in the Supply Chain today has become crucially important in both its
The role of sustainability in the Supply Chain today has become crucially important in both its design
and the operations that concern it while improving its performance [6]. The framework presented by the
United Nations World Summit in 2005 identifies three pillars on which sustainable economic, environmental,
and social development rests. design and the operations that concern it while improving its performance [6]. 1. Introduction The framework
presented by the United Nations World Summit in 2005 identifies three pillars on which sustainable
economic, environmental, and social development rests. In order to build a more sustainable Supply Chain, companies must clearly define the reasons for
developing more sustainable approaches to fuel interest from customers who are reluctant to pay more
for sustainable products [7]. In order to build a more sustainable Supply Chain, companies must clearly define the reasons
for developing more sustainable approaches to fuel interest from customers who are reluctant to pay
more for sustainable products [7]. Therefore the aim of this article is to explore the advances that exist in the three manufacturing
Therefore, the aim of this article is to explore the advances that exist in the three manufacturing
sectors: Aerospace, Shipbuilding, and Automotive in terms of sustainable Supply Chain management. At the same time, common areas and possible synergies between the three sectors will be identified. 1. Introduction It can be said that a Supply Chain is composed of all the interested parties: customers, suppliers,
manufacturers, transporters, warehousemen, etc. Each organization includes all the functions involved
in it starting from the development of the new product, marketing, manufacturing, finance, to customer
service and whose purpose is to satisfy the needs of the customer while generating profits in the
process for itself [1]. Each Supply Chain will be divided into different stages, and within each stage, several actors can
coexist, so it should really be called a Supply Network. All stages are connected through the flow of
products, information, and funds—in both directions—aimed at maximizing the total value generated
by the Supply Chain. The success of a Supply Chain should not be measured at each stage but in its
total profitability. Therefore, the success of a Supply Chain lies in the efficiency of its management [2]. p
y
,
pp y
y
g
[ ]
In addition, Supply Chain must adapt both to changes in technology and to customer requirements
in order to remain competitive. The manufacturing Supply Chain is of the pull type as the processes
are carried out in response to the request of the customer, which is also known as a reactive process [3]. Each connection between the stages of the Supply Chain (supplier–manufacturer–distributor–
retailer–customer) has the processes required for each process cycle (sales order cycle, replenishment
l
f
t
i
l
t
l )
d th
ti
di id d i t In addition, Supply Chain must adapt both to changes in technology and to customer requirements
in order to remain competitive. The manufacturing Supply Chain is of the pull type as the processes
are carried out in response to the request of the customer, which is also known as a reactive process [3]. Each connection between the stages of the Supply Chain (supplier–manufacturer–distributor–
retailer–customer) has the processes required for each process cycle (sales order cycle, replenishment
cycle, manufacturing cycle, procurement cycle), and these connection processes are divided into Materials 2020, 13, 5625; doi:10.3390/ma13245625 www.mdpi.com/journal/materials Materials 2020, 13, 5625
cycle, manufacturin
processes at the sam 2 of 23
o sub-
ems to sub-processes at the same time [4]. The cycle view is useful when establishing information systems to
support Supply Chain operations when considering operational decisions because it establishes the
roles and responsibilities of each member and the expected outcome of each process. Therefore, the aim of t
sectors: Aerospace, Shipb
2. Materials and Methods management. At the same time, common areas and possible synergies between the three sectors will
be identified. The methodology carried out in this work is shown in Figure 2. It is a descriptive review in order
to provide the existing advances on Supply Chain in the three big sectors that compose the transport
manufacturing such as aerospace, naval, and automotive. Many advances are being made in each
of these areas individually with respect to sustainability-focused supply chain management, but a
descriptive review of the three areas together will provide an update for people working in the same
fields in different areas. It is in this sense where it is intended to highlight that synergies are possible. Both aerospace and shipbuilding coincide in the type of production, while the automotive sector is 3 of 23
em to
ork is Materials 2020, 13, 5625
with the studies pub
update the various mass production. However, there are related fields that would take advantage of advances in each
production system or even sector. Hence, the managers dedicated to these fields can be nourished
with the studies published by the scientific community serving as a strategic tool that allows them to
update the various aspects addressed in the study. Therefore, the aim is to find out what work is being
done in these three areas in terms of the progress of Industry 4.0 (I4.0) together with sustainability [8]. sustainability [8]. The study will be developed in five stages, starting with the definition of the research objective,
mentioned above, until the evidence is reported once it has been analyzed [9]. To carry out the bibliographic search, Scopus was used as the main database in which the main
journals and conference papers will be studied, as well as some book chapters and review articles. Figure 2. Research methodology for this study. Figure 2. Research methodology for this study. Figure 2. Research methodology for this study. Figure 2. Research methodology for this study. To establish the search strategy, the descriptors “Supply Chain Management”, “4.0”, and each
of the three sectors—“aerospace”, “shipbuilding”, “automotive”—were used as arguments. No
The study will be developed in five stages, starting with the definition of the research objective,
mentioned above, until the evidence is reported once it has been analyzed [9]. 3. Results and
3.1. Aerospace 3.1. Aerospace
Starting with the aerospace sector and considering the above-mentioned search arguments, a
Starting with the aerospace sector and considering the above-mentioned search arguments, a total
of 18 articles are established that deal with this industry, of which 13 include sustainability in their
content. Only two articles have been excluded because the criteria established were not met. total of 18 articles are established that deal with this industry, of which 13 include sustainability in
their content. Only two articles have been excluded because the criteria established were not met. After evaluating the adaptation that this type of industry whose production engineer-to-order
is characterized by the activities that must be added in order to comply with the established lead
time, whether in terms of commercial management, procurement, production, or logistics and
distribution in the case of the Supply Chain, the difficulty that smaller companies face in adapting to
Industry 4.0 becomes clear. While large companies are more aware of the changes they must make in
this adaptation, the Supply Chain is made up of these and other smaller and less developed
i
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th i t
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After evaluating the adaptation that this type of industry whose production engineer-to-order is
characterized by the activities that must be added in order to comply with the established lead time,
whether in terms of commercial management, procurement, production, or logistics and distribution
in the case of the Supply Chain, the difficulty that smaller companies face in adapting to Industry
4.0 becomes clear. While large companies are more aware of the changes they must make in this
adaptation, the Supply Chain is made up of these and other smaller and less developed companies in
terms of both resources and organizational capacity for the integration of Industry 4.0, one of their
concerns being the susceptibility to external breakdown [10]. companies in terms of both resources and organizational capacity for the integration of Industry 4.0,
one of their concerns being the susceptibility to external breakdown [10]. Engineers, to order environments, develop Lean methodologies to accelerate delivery time
among other techniques. Lean practices such as Just In Time (JIT) and Visual Management show how
certain areas improve the potential impacts of business performance as well as the overall Supply
Chain [11]. Therefore, the aim of t
sectors: Aerospace, Shipb
2. Materials and Methods exclusion criteria were established with respect to time due to the inclusion of the term 4.0 as a
descriptor that acts as a limiter. To carry out the bibliographic search, Scopus was used as the main database in which the main
journals and conference papers will be studied, as well as some book chapters and review articles. A total of 297 articles were found, to which the criteria of scientific quality were applied. Subsequently, duplicates were eliminated, and the abstracts and conclusions were not read until the
To establish the search strategy, the descriptors “Supply Chain Management”, “4.0”, and each of
the three sectors—“aerospace”, “shipbuilding”, “automotive”—were used as arguments. No exclusion
criteria were established with respect to time due to the inclusion of the term 4.0 as a descriptor that
acts as a limiter. A total of 297 articles were found, to which the criteria of scientific quality were applied. Subsequently, duplicates were eliminated, and the abstracts and conclusions were not read until the
articles were selected to be read in their entirety. The distribution of publications used in the study is
shown in Figure 3. 4 of 23 Materials 2020, 13, 5625
shown in Figure 3. Figure 3. Articles considered from each sector. Figure 3. Articles considered from each sector. 3. Results and Discussion 3. Results and
3.1. Aerospace 3. Results and
3.1. Aerospace In addition to Lean, Green is another paradigm that focuses on the requirements that I4.0
makes, from product and process design, production planning and control, and communication with
suppliers. Furthermore, the flexibility in the development of shared communication with suppliers
is a fundamental requirement for the competitiveness of the Supply Chain [12]. To achieve this
required competitiveness, in addition to enhancing management and sustainability in the Supply
Engineers, to order environments, develop Lean methodologies to accelerate delivery time among
other techniques. Lean practices such as Just In Time (JIT) and Visual Management show how
certain areas improve the potential impacts of business performance as well as the overall Supply
Chain [11]. In addition to Lean, Green is another paradigm that focuses on the requirements that
I4.0 makes, from product and process design, production planning and control, and communication
with suppliers. Furthermore, the flexibility in the development of shared communication with
suppliers is a fundamental requirement for the competitiveness of the Supply Chain [12]. To achieve
this required competitiveness, in addition to enhancing management and sustainability in the Supply
Chain, aerospace companies demonstrate the impact of product life management (PLM) systems by
managing the entire product life cycle, from the first marketing idea to the after-sales service [13]. required competitiveness, in addition to enhancing management and sustainability in the Supply
managing the entire product life cycle, from the first marketing idea to the after-sales service [13]. With regard to the digitalization of the framework processes of the Supply Chain, technologies such
as the Internet of Things become important for companies in the sector interested in the transformation With regard to the digitalization of the framework processes of the Supply Chain, technologies such
as the Internet of Things become important for companies in the sector interested in the transformation 5 of 23 Materials 2020, 13, 5625 of Industry 4.0. Management principles that improve performance throughout the company focused
on the involvement of employees in decision making, and two applications are the most suitable for
the implementation of this technology: TQM (total quality management), which is a CRM (Customer
Relationship Management) application that allows centralizing in a single database all interactions
between a company and customers and the management of relationships with suppliers, and SRM
(Supplier Relationship Management), with the intention of establishing positive relationships with the
company. 3. Results and
3.1. Aerospace In addition, these are also used in the reduction of carbon emissions and the adoption of
Green concepts [14]. It could be said that the Internet of Things collaborates closely on energy management in smart
factories, smart logistics and transport, and creating smart business models. This is done in four
main areas: (1) designing incentive mechanisms to promote green consumer behavior; (2) improving
visibility throughout the product life cycle; (3) increasing system efficiency while reducing development
and operational costs; and (4) encouraging sustainability monitoring and reporting performance in
Supply Chain networks [15]. Moreover, the Internet of Things becomes more important in terms of the need to be able to visualize
information in real time [16], as well as the existing improvement in after-sales services achieved
through the sensors placed in its products, together with the Big Data technology, which reports on
their performance, defects, and usage patterns in the hands of the customer. This fact has changed the
business model, and the manufacturer has become the solution to the problem [17]. In this way, the importance of Big Data is confirmed due to the critical challenge that these
factories have to process so much information. These intelligent systems are capable of monitoring
and controlling the processes of the Supply Chain as well as providing information on breakdowns
for the entire system of planning and control of production and finally providing useful solutions to
employees [16]. With respect to Additive Manufacturing, it plays an important role in the viability of a complex
product. Together with the freedom of product design, the ability to customize and the variety of products
are determining factors in the competitiveness of the Supply Chain [18]. The environmental impact,
health, and safety seems to be contemplated in this technology that marks a trend in terms of resource
consumption [16,18]. The impact of the technology on production strategy, technical requirements and
distribution is still to be resolved [18]. Within the study that allows the development of a conceptual model of the Supply Chain
using Blockchain technology, it becomes evident that, as in companies in the aerospace sector, the top
management is responsible for making strategic decisions and therefore for designing and implementing
sustainability in the organization [15,19]. 3.2. Shipbuilding In the case of shipbuilding, there are only five articles that meet the search criteria from which
only one had to be removed, which will be analyzed below. In the same way as in the previous section
covering the aerospace sector, this section also considers the Engineer-to-order type of production. Thus, this sector faces the same problem of susceptibility to external breakdowns due to the difficulty
of small enterprises to adapt to Industry 4.0 [10]. In the same way, the Lean methodology provides
benefits in the Engineer-to-order environment, together with the relationship between digital and
information technology of the I4.0, which is considered as an established term in the Lean Supply
Chain [20]. In addition to the Lean paradigm, there are other Supply Chain Paradigms studied for the
shipbuilding sector such as Green, Agile, and Resilient, which in combination with the enabling
technologies stand out from the others in Big Data Analysis focused on the reduction of emissions. Data processing enables the reliability and security of data to quantify CO2 emissions from ships and
provide information on energy efficiency parameters [21]. Other techniques include optimizing the
energy efficiency of the ship by analyzing the energy transfer between the hull, propeller, and main Materials 2020, 13, 5625 6 of 23 engine; analyzing the optimal engine speeds [22]. After analyzing the data collected, including sea
currents, waves, and winds, along with engine logging data, location, and speed, it is possible to
predict ship performance, reduce fuel consumption, and thus reduce emissions [23], even by analyzing
historical data as a basis for estimating future accidents [24]. Other technologies focused on Supply Chain sustainability are Cloud Computing, Cybersecurity,
and Blockchain [25]. Cloud Computing studies the optimization of virtual machine placement. This is a
great challenge in terms of the number of physical machines with the aim of reducing energy costs and
waste of resources, in addition to minimizing operating expenses dedicated to the target platform [26],
in collaboration with other technologies allowing a rapid diagnosis of system efficiency, in particular
engine breakdown [27], in addition to collaborating in the sustainable development of the marine
economy [28]. Cybersecurity has an important role to ensure the safe operation of ships, in addition to improving
the environmental safety of the oceans. 3.2. Shipbuilding With the intention of complying with international regulations,
the available resources are studied by analyzing the methods and policies of maritime cybersecurity
that guarantee these aspects [29]. At the same time, there are publications that aim to inform staffto
help protect cyberspace from adversaries through an introductory view of systems that help manage
cyberspace security that simplifies the complexity of cyberspace and the variety of possible attacks. As for the energy efficiency of cryptocurrencies, Blockchain technology tries to implement and
change to more efficient algorithms such as the Proof of Stake (PoS), leaving behind the use of the
Proof by Work (PoW) algorithm used to achieve energy sustainability [30]. In the aeronautical sector, these technologies have also taken on a leading role with regard to the
sustainability of the Supply Chain, but there is no evidence of this from Cybersecurity. This does not
mean that the sector has not focused on the study of this technology; there is evidence related to the
characterization of digital manufacturing systems, identification of threats and vulnerability, control,
and determination of risks [31]. There are studies that show the benefits of Blockchain, Internet of Things, and Fog Computing
technologies in the application to a system that allows the identification and tracking of the pipes of a
ship during its construction [32]. Likewise, no publications on Fog Computing technology have been
published in the aeronautical sector as a technology that drives the Supply Chain and its sustainability,
although in the same way as Cybersecurity, it does in other areas [33]. The Internet of Things has also been applied in other sector companies revealing a great impact on
the performance of the Supply Chain and highlighting the potential for improvement not only in the
economic but also in the environmental and social sustainability aspects. Its use allows a sustainable
development in collaboration with the strategic and organizational management of the companies. In addition, it offers solutions attending to criteria such as the management of services or operations
from the perspective of business based on intelligent operations [34]. 3.3. Automotive In the case of the automotive industry, there are a total of 54 publications, of which two have
been eliminated and three have been evaluated in the sectors studied. In this case, and to consider the
difference of the previous sectors, the type of production corresponds to the mass production; however,
the Lean methodology is also present in this type of production. One of the improvements in the operation and control of the plant is done through the relationships
of the key performance indicators (KPIs). This performance measurement system of a Lean production
system provides answers at the strategic, tactical, and operational levels in the implementation of
I4.0 projects [35]. In addition to the contribution of Lean guaranteeing an efficient use of resources,
in combination with Agile, they act as drivers for the general improvement of performance. As a
decision support tool for decision making by identifying potential I4.0 technologies, the Lean–Agile
combination adopts strategies that help achieve the overall objectives of the organization [36]. Materials 2020, 13, 5625 7 of 23 Another possible combination with Lean that is used as a lever to strengthen relationships is
with Green practices. The result in this case would be Green Supply Chain Management, where Lean
facilitates the collaboration with suppliers and environmental programs. At the same time, following
a process innovation strategy based on I4.0 technologies, in addition to improving the Lean effect,
leads to better economic results. However, companies will have to choose to obtain better performance
by charging suppliers in environmental programs or by investing in I4.0 technologies, but not in
both [37]. This is because innovation in technologies does not have the same impact on the Green
Supply Chain; if the intention is to improve performance by targeting technologies, then Green is not
being improved and vice-versa. g
p
In the same way, it has been demonstrated that I4.0 technologies do not improve the performance
of the Lean Supply Chain, and it can be negative to think that better results will be achieved by
acquiring a technology than through management practices [38]. However, there are other studies that
indicate that the Green and Lean approach can improve the content of I4.0 by adapting product and
process design, manufacturing planning and control, cooperation with suppliers, shared information
and customer energy and value through flexibility and process re-engineering, with communication
between Supply Chain players being essential. 3.3. Automotive All this makes the Supply Chain more flexible and
visible and can be made possible through I.40 enabling technologies [12]. Supported by these information and communication technologies and Lean Manufacturing
management methods, a new generation of manufacturing systems is born, which is called a Small
Scale Intelligent Manufacturing System that is capable of generating value and meeting customer
demands. In addition, in order to carry out Green Manufacturing, a Closed-loop Supply Chain model
was developed [39]. This concept of Closed-Loop is not new; it was introduced by Solvang in 2007,
defining it as a Supply Chain without waste [40], and it is related to a more current concept such as the
circular economy. This circular economy is favored by the interconnectivity promoted by Industry 4.0 allowing
for real-time data collection, communication, and data analysis [41], although the transition between
Industry 3.0 and 4.0 presents barriers between the Circular Supply Chain and Industry 4.0 [42]. Among the barriers to implementation of I4.0 are the workforce capable of understanding Industry 4.0,
ineffective legislation and control, and short-term corporate objectives. These barriers, combined with
the lack of funding for I4.0 initiatives, are causing organizations to develop an integrated strategic
approach that is capable of utilizing the improved knowledge of I4.0 and the circular economy in order
to take advantage of the increased profits from products and process designs that promote energy
efficiency [43]. To achieve the effectiveness of Industry 4.0 in the sustainability of the Supply Chain, initiatives are
identified from the organizational, legal, and ethical perspective and technological strategies. Within these
technological strategies are the need for integration of technological platforms, data-sharing protocols,
and a lack of internet-based network infrastructure [44]. Data-based technology and operations provide
opportunities for new methods and operations to become an adopter of Industry 4.0 [45]. In order to know the facilitators of the sustainable Supply Chain, Figure 4 shows the most
significant ones looking for the highest demand for digital, horizontal, and vertical integration
and End-to-End. The framework of Supply Chain processes in which the Internet of Things becomes highly
important had already been appreciated earlier in the aerospace sector [14]. And the impact it has on
the performance of the Supply Chain by improving economic, environmental and social sustainability
aspects in shipbuilding [14]. 3.3. Automotive It could therefore be said that the Internet of Things and environmentally
friendly practices are the most influential factors in becoming a sustainable and industry-compliant
organization 4.0 [46]. This is not the case with Additive Manufacturing, despite the fact that its adoption has many effects
from the viability of a complex product, the freedom of design or the ability to mass customize, there are Materials 2020, 13, 5625
addition to not bei
sectors 8 of 23
er two still contradictions with regard to the complexity and flexibility of the Supply Chain in addition to not
being profitable in the automotive industry [18]. Just the opposite of the other two sectors. Furthermore, there are studies that show how simulation boosts the flexibility and efficiency of
the automotive Supply Chain by using simulation based on multi-objective optimization and
developing a decision support model [47]. This flexible simulation-based approach allows risks to be Furthermore, there are studies that show how simulation boosts the flexibility and efficiency of the
automotive Supply Chain by using simulation based on multi-objective optimization and developing
a decision support model [47]. This flexible simulation-based approach allows risks to be assessed
prior to implementation with a positive impact on Supply Chain risk management, saving many real
resources, which makes the Supply Chain more sustainable [48,49]. assessed prior to implementation with a positive impact on Supply Chain risk management, saving
many real resources, which makes the Supply Chain more sustainable [48,49]. Another way to achieve sustainability in production is through the use of Just in Time material
in the assembly lines; this is achieved by implementing decentralized logistics areas known as
supermarkets. At the same time, it was observed how the cost of shipping material across the Another way to achieve sustainability in production is through the use of Just in Time material
in the assembly lines; this is achieved by implementing decentralized logistics areas known as
supermarkets. At the same time, it was observed how the cost of shipping material across the assembly
line is the most influential factor in reducing the total cost of the supermarket. It was through simulation
that the optimum location of these supermarkets on the assembly lines was optimized [50]. Hence,
the simulation allows us to optimize from a particular point of view any necessary movement by
making iterations until the optimal solution is reached. 3.3. Automotive This same concept is used in previous sectors,
but there are no simulation-related applications for it. p
pp
g
assembly line is the most influential factor in reducing the total cost of the supermarket. It was
through simulation that the optimum location of these supermarkets on the assembly lines was
optimized [50]. Hence, the simulation allows us to optimize from a particular point of view any
necessary movement by making iterations until the optimal solution is reached. This same concept is
used in previous sectors, but there are no simulation-related applications for it. The simulation also served as a semantic validator of Big Data, due to the fact that the Big Data
technology showed indetermination when analyzing the data that could be solved through
simulation. This shows that Big Data technology requires improvement [51]. However, it is the The simulation also served as a semantic validator of Big Data, due to the fact that the Big Data
technology showed indetermination when analyzing the data that could be solved through simulation. This shows that Big Data technology requires improvement [51]. However, it is the analysis of Big
Data that drives artificial intelligence to achieve sustainable manufacturing and circular economy
capabilities [52]. g
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[
]
analysis of Big Data that drives artificial intelligence to achieve sustainable manufacturing and
circular economy capabilities [52]. Figure 4. Sustainable Supply Chain facilitators. Figure 4. Sustainable Supply Chain facilitators. Figure 4. Sustainable Supply Chain facilitators. The expected connection in the automotive factories make the amount of shared data very large
through the activities of the Supply Chain and in the interaction of product and service in the cloud. Thi
h
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The expected connection in the automotive factories make the amount of shared data very
large through the activities of the Supply Chain and in the interaction of product and service in the
cloud. This shows the need to implement Cybersecurity through the integration of Supply Chain
management—marketing integration [53]. In addition to marketing integration, the other areas
addressed within the Supply Chain also benefit from Cybersecurity. 3.3. Automotive Another barrier is found at the level of
employees and middle management in the face of increased control and performance measurement in
real time, fearing changes in management [60,61] in addition to the lack of knowledge on the part of
the managers of knowing if they will return the investment and will obtain benefit nor in time [62]. It
could be said that one of the biggest problems the automotive industry faces is in management and
organization [63]. There are also barriers due to lack of knowledge of I4.0 by suppliers [61]. The lack
of technological infrastructure also makes implementation more difficult considering that there is no
management support for the implementation of I4.0 [61,62]. On the other hand, there are findings that show that neither customer loyalty nor satisfaction is
relevant to the success of Supply Chain management. The customer experience will be a differentiator
in the future, and it will work to maintain the support of the rest of the factors [64]. However, there are still areas to be exploited that can be beneficial in the automotive industry [65]. In order to help the leaders of the companies make their plants intelligent, it is clear that there is a
need for integration, collaboration, and transparency of all the members of the chain [66]. Leaders are
encouraged to establish sustainable policies, training programs focused on I4.0 and to consider I4.0 as
a strategic decision to improve costs, reduce resources and energy consumption, and contribute to the
development of healthy societies [44]. However, this integration, behavior, and trust will be reflected
when it is manifested by including the concept in the vision and mission of their organizations [13]. One of the proposals still to be developed is the servicing of Supply Chain management with
respect to I4.0 applications [67]. Another is the implementation of I4.0 concepts at multiple levels of
the Supply Chain. Within this multi-stage implementation proposal, they discourage talkers that go
from a cultural, multifunctional approach and continuous improvement. It proposes to start from the
focus organization for later integration of the partner organizations until arriving at the intelligent
factory where the Supply Chains are connected among themselves and with their systems and the
machines are linked to a common network system [68]. 3.3. Automotive This shows the need to implement Cybersecurity through the integration of Supply Chain
An adaptation of cloud computing with the use of robots, cloud robotics, are key to the virtual creation
and integration of computational and physical processes resulting in the Cyber–Physical–Systems key to the
transition to the sustainable digital world [54]. These systems make it necessary to analyze Cybersecurity
risks in a globalized Supply Chain. Some occur due to cyber-attacks that cause an operational disruption in
the SC; others cause an operational disruption affecting the entire Supply Chain, and others are produced
by an inappropriate interaction between man and machine [55]. In addition to Cybersecurity, security in Materials 2020, 13, 5625 9 of 23 the traceability of operations is also necessary, for which a reference architecture of the applicability of
Blockchain technology is necessary as well [56]. However, it seems that most companies prefer the implementation of only one technology to the
adoption and integration of several. Most of them invest in the Internet of Things, Cloud Computing,
or Radio Frequency Identification due to the optimization of resources, ease of access from anywhere,
or for decision-making based on visibility. Others choose Big Data Analytics because of the speed in
detecting failures with a better customer service and reduction of preventive maintenance. Furthermore,
some companies rely on Blockchain to improve the traceability and transparency, which increases trust
with stakeholders [57]. Several of these technologies such as Robotics, Automated Guided Vehicles,
or Additive Manufacturing help reduce wasted resources and emissions by setting up a collaborative
program. Thereby, when innovation costs are shared, the motivation to invest more is greater, and this
translates into better Supply Chain performance [58]. It can be said that either the actors in the Supply
Chain work collaboratively and support each other, or there will be no success in the performance
of the Supply Chain [38]. It seems fundamental for the growth of Industry 4.0 and the coordination
between the entities of the Supply Chain to establish models in daily environments, competition, and
cost-sharing contracts [59]. In spite of seeking solutions such as collaboration, there is a lot of resistance that companies
encounter when it comes to putting into practice the management changes that a sustainable Supply
Chain carries out. As mentioned above, the size of companies has an influence, making it easier for
larger companies to implement changes than for smaller ones. 3.3. Automotive Finally, the proposal relating to installations
and the application to the recovery of the value of the product at the end of its life cycle could be
mentioned [69]. Figure 5 shows, as a summary, the technologies that each of the sectors studied considers applicable
to boost sustainability in the Supply Chain. It shows how only Big Data and the Internet of Things are
common to all three sectors. Similarly, Figure 6 shows the methodologies and practices that each of the
sectors studied apply to the sustainable Supply Chain. Materials 2020, 13, 5625
he sectors studied apply to the sustainable Supply Chain. Figure 5. Focus technologies in Supply Chain Management (SCM) digitali
Figure 6. Sustainable Supply Chain-driving methodologies. Overview
st section a summary of the most significant aspects of each sector
Figure 5. Focus technologies in Supply Chain Management (SCM) digitalization. Figure 5. Focus technologies in Supply Chain Management (SCM) digita
Figure 6. Sustainable Supply Chain-driving methodologies. s Overview
ast section, a summary of the most significant aspects of each secto
Table 1, in order to establish a comparison between them. The faci
he automotive industry supply chain are taken as a reference. Figure 6. Sustainable Supply Chain-driving methodologies. 3.4. Key Points Overview
In this last section, a summary of the most significant aspects of each sector studied is
as shown in Table 1, in order to establish a comparison between them. The facilitators refer
Figure 4 for the automotive industry supply chain are taken as a reference. 10 of 23 Materials 2020, 13, 5625 Figure 5. Focus technologies in Supply Chain Management (SCM) digitalization. Focus technologies in Supply Chain Management (SCM) digi Figure 5. Focus technologies in Supply Chain Management (SCM) digitalization. g
pp y
g
g Figure 5. Focus technologies in Supply Chain Management (SCM) digitalization. gure 5. Focus technologies in Supply Chain Management (SCM) digit
Figure 6. Sustainable Supply Chain-driving methodologies. Figure 6. Sustainable Supply Chain-driving methodologies. igure 5. Focus technologies in Supply Chain Management (SCM) digit
Figure 6. Sustainable Supply Chain-driving methodologies. Figure 6. Sustainable Supply Chain-driving methodologies. 6. Sustainable Supply Chain-driving method
Figure 6. Sustainable Supply Chain-driving methodologies. 3.4. Key Points Overview 3.4. Key Points Overview Figure 6. Sustainable Supply Chain-driving methodologies. 3.3. Automotive O
i
s Overview
ast section, a summary of the most significant aspects of each sector studie
T bl 1 i
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th
Th f
ilit t
In this last section, a summary of the most significant aspects of each sector studied is included,
as shown in Table 1, in order to establish a comparison between them. The facilitators referred to in
Figure 4 for the automotive industry supply chain are taken as a reference. 11 of 23 Materials 2020, 13, 5625 Table 1. Comparison of SC facilitators. Table 1. Comparison of SC facilitators. 3.3. Automotive Aerospace
Shipbuilding
Automotive
Methodologies
Lean practices improve SC
performance [11,12,70–77]
Lean strengthens the probability of success of
Supply Chain Management [78,79]
Lean provides competitive advantages, quality, and
flexibility performance [80,81] and improves dealer service
through an inventory management model [82]
Agile practices evaluate new event with
restructuring suggestions [75,83–85]
The Agile methodology identifies improvements
in the relationship between the shipyard and its
suppliers [85,86]
Agile provides competitive advantages, quality, and
flexibility performance [80,87] and is used as a strategy for
supplier selection [88]
Green practices make an important
contribution to SC sustainability and
suppliers [73,75,86,89–91]
Green practices contribute to a sense of social
responsibility and competitive advantage [86,92]
Green practices improve the relationship between
companies and green suppliers, improves the capacity to
develop green products, and increases the competitiveness
of companies in the market [93] and minimizes the total
cost [94]
Resilient initiatives improve SC
sustainability and social improvements
in safety and environmental
health [70,72,73,83,95]
The resilient paradigm is compromised by the
social and functional aspects of the I4.0
performance model [96]
Resilient methodologies to SC are preferable to focus on
minimizing costs [97], improve the selection of sustainable
and appropriate suppliers, and maximize value by
developing close and long-term relationships [98]
Models
Closed-Loop SC models help increase
profits by transforming and
remanufacturing waste [86]
Closed-Loop SC models help increase profits by
transforming and remanufacturing waste [86]
Adding value to remanufacturing practices [99]
collaborating with environmental management [100]
No evidence of Circular Economy
Circular economy helps to reduce CO2
emissions [101]
Circular economy provides priority solution measures to
formulate effective strategies to overcome failures in the
adoption of SC management [63]
Environmental sustainability by
applying the product life cycle
management system [90,102,103]
Product lifecycle management (PLM)
contributes to efficient control and distribution,
minimizes costs, and reduces lead times [104]
Product life-cycle management (PLM) approach supports
decision making [105], reduces the time to market, and
satisfies the end customer needs [106]
Infrastructure
Use of technological platforms to
improve logistics capacity [107] and to
develop the reference architecture and
define the standards to exchange
electronic information securely [108]
The use of technological platforms achieves an
important integration and collaboration with its
suppliers and customers [109]
Through a platform with several simulation components,
the control of the manufacturing systems is
established [110]. Shipbuilding Aerospace Aerospace
Shipbuilding
Automotive
Data Sharing Package allows the
reduction of SC inefficiencies [112]
Lightweight data format for the visualization of
3D product information and the collaboration of
all SC agents in all phases of the ship
lifecycle [113]
Data-sharing protocols based on Blockchain technology
provide reliability [114]. Data sharing on production
planning and scheduling using IoT can reduce product
preparation and delivery time [115]
Analysis, design, and performance
improvement of the SC by applying the
SC Operations Reference Model (SCOR)
using the internet [116,117]
Web-based software framework that enables
electronic collaboration between companies
working together for ship repair [118,119],
An open communication infrastructure
guarantees the success of SC [120]
Providing benefits to remanufacturing practices through
the use of Big Data using the Internet [99]
Technologies
IoT: Registering and verifying the
identity of the machines simplifying the
management of the assets within the
connected SC [121]
IoT: Identification and tracking of the pipes of a
ship during its construction [32], offering
solutions for the management of services and
operations [34]
IoT: Allowing connectivity for later analysis through
simulation [110]. This exchange of data on production
planning and scheduling using IoT can reduce product
preparation and delivery time [115,122]
Simulation: To accurately model or
predict the effects of joining and fixing
parts [123], analyzing SC
performance [124], for decision
support [125]
Simulation: Management tool [107], to solve
complicated problems of SC management [126],
identify the critical control point to mitigate the
effects caused by the disproportion in the
logistic flow [127]
Simulation: As a training tool for ship design
processes [128] and as a tool for decision making [105]
Big Data Analytics: Support for dynamic
production capacity and decision
making of the SC [129]
Big Data Analytics: Used to optimize the design
of a vessel and to maximize efficiency and safety
in an existing one [130], focused on the
reduction of emissions [21,22]
Big Data Analytics: Providing advantages to
remanufacturing practices [99]
Artificial Intelligence: Adaptive resource
management based on multi-agent
technology [131], to produce more
affordable parts, faster, and with less
weight [132]
Artificial Intelligence: Using control architecture
and programming of the production plant [133],
focused on reducing CO2 [23]
Artificial Intelligence: new dimension of the relationship
between financing and production [134]. Solves problems
in the management of the SC that can track, communicate,
analyze, and ensure the overall sustainability of the
system [135]. 3.3. Automotive In addition, an integrated platform based
on a cyber–physical system provides optimal use of
manufacturing resources in dynamic, real-time
environments to increase efficiency and responsiveness to
uncertain market changes [111] Automotive 12 of 23 Materials 2020, 13, 5625 Table 1. Cont. Shipbuilding Automotive Shipbuilding Automotive Aerospace Aerospace
Shipbuilding
Automotive
Cloud Computing: Providing unlimited
processing to SC management [141]
Cloud Computing: Improving economic, energy,
and environmental aspects [96]
Cloud Computing: Allows the collection, supply, and
analysis of relevant data in all companies that make up the
SC [122,142]
Additive Manufacturing: Supporting
sustainability in CS through material
recycling [143], remanufacturing of
high-value parts on the reverse logistics
supply chain [144]
Additive Manufacturing: Enabling design
flexibility, reducing waste, and integrating
subassemblies [145], Negative aspect: increased
delivery time, shipping cost, inventory
requirements, and transportation
vulnerability [146]
Additive Manufacturing: Used during the supply stage; it
changes complex subsets into a single integrated
structure [147]
Blockchain: Ensuring traceability by
certified agents in the SC [148,149]
Blockchain: strengthening production security
in the collaborative development process,
improving the integrity and traceability of
Supply Chain data [150]
Blockchain: provides reliability in the creation of protocols
to share processes, business logic, and financial
ledgers [114]. Guarantees the security, transparency, and
visibility of the network from the origin of the SC, the
reengineering of the business processes to the improvement
of the security [151]
llaborative
Programs
Use of system of systems to address
multi-system integration problems
associated with SC [152], Collaborative
Aerospace Life Cycle Systems Program
that integrates from the beginning of the
aerospace design process [153]
Information systems for project management
with integrated approach [154], high integration
and collaboration between design,
manufacturing, and management
functions [109]
Logistics integration through collaborative supply chain
innovation [155]
Gaining transparency between the
central company and its suppliers,
exchanging high-quality information
leads to significant improvements in
overall SC performance [156]
Through transparency, collaborative risk
management in SC management shows
collaborative control mechanisms [157]
Through the Blockchain technology, the security,
transparency, and visibility of the network is
guaranteed [151]. Focal companies increase multi-tier SC management
transparency for sustainability [158]
Through the implementation of
sustainable policies with long-term
strategies among the agents involved in
SC [159]
Through carbon policies based on the
sustainability characteristics of the region, the
level of design of Supply Chain networks is
improved, cost is reduced, and the
environmental impact is improved [160]
The application of Green strategies to the management of
CS helps companies establish innovative and effective
policies [161]. Shipbuilding Facilitate the execution of mechanism
design-based negotiations [136]
Cybersecurity: To derive the behavior of
programs with hidden malicious
operations and supporting workforce
productivity [137], providing operational
certainty of SC systems [138]
Cybersecurity: Improving economic, energy,
and environmental aspects [96]
Cybersecurity: Threat deterrence and mitigation
function [139]. Provides mechanisms for identifying
generic and manufacturing-specific vulnerabilities [140] Big Data Analytics: Used to optimize the design
of a vessel and to maximize efficiency and safety
in an existing one [130], focused on the
reduction of emissions [21,22] Artificial Intelligence: new dimension of the relationship
between financing and production [134]. Solves problems
in the management of the SC that can track, communicate,
analyze, and ensure the overall sustainability of the
system [135]. Facilitate the execution of mechanism
design-based negotiations [136] Artificial Intelligence: Using control architecture
and programming of the production plant [133],
focused on reducing CO2 [23] Cybersecurity: To derive the behavior of
programs with hidden malicious
operations and supporting workforce
productivity [137], providing operational
certainty of SC systems [138] Cybersecurity: Threat deterrence and mitigation
function [139]. Provides mechanisms for identifying
generic and manufacturing-specific vulnerabilities [140] Cybersecurity: Improving economic, energy,
and environmental aspects [96] 13 of 23 Materials 2020, 13, 5625 Table 1. Cont. Shipbuilding Closed-Loop SC provides recommendations
for sustainable policies [100]
No evidence of the I4.0 training
programs despite potential benefits to SC
management [162]
No evidence of I4.0 training programs despite
potential benefits to SC management [162]
Design of training tools for ship design processes through
the use of simulation [128] Cloud Computing: Allows the collection, supply, and
analysis of relevant data in all companies that make up the
SC [122,142] Cloud Computing: Providing unlimited
processing to SC management [141] Cloud Computing: Improving economic, energy,
and environmental aspects [96] Additive Manufacturing: Enabling design
flexibility, reducing waste, and integrating
subassemblies [145], Negative aspect: increased
delivery time, shipping cost, inventory
requirements, and transportation
vulnerability [146] Additive Manufacturing: Supporting
sustainability in CS through material
recycling [143], remanufacturing of
high-value parts on the reverse logistics
supply chain [144] Additive Manufacturing: Used during the supply stage; it
changes complex subsets into a single integrated
structure [147] Additive Manufacturing: Used during the supply stage; it
changes complex subsets into a single integrated
structure [147] Blockchain: provides reliability in the creation of protocols
to share processes, business logic, and financial
ledgers [114]. Guarantees the security, transparency, and
visibility of the network from the origin of the SC, the
reengineering of the business processes to the improvement
of the security [151] Blockchain: strengthening production security
in the collaborative development process,
improving the integrity and traceability of
Supply Chain data [150] Blockchain: Ensuring traceability by
certified agents in the SC [148,149] Use of system of systems to address
multi-system integration problems
associated with SC [152], Collaborative
Aerospace Life Cycle Systems Program
that integrates from the beginning of the
aerospace design process [153] Information systems for project management
with integrated approach [154], high integration
and collaboration between design,
manufacturing, and management
functions [109] Gaining transparency between the
central company and its suppliers,
exchanging high-quality information
leads to significant improvements in
overall SC performance [156] Through transparency, collaborative risk
management in SC management shows
collaborative control mechanisms [157] Through carbon policies based on the
sustainability characteristics of the region, the
level of design of Supply Chain networks is
improved, cost is reduced, and the
environmental impact is improved [160] The application of Green strategies to the management of
CS helps companies establish innovative and effective
policies [161]. Shipbuilding Closed-Loop SC provides recommendations
for sustainable policies [100] Through the implementation of
sustainable policies with long-term
strategies among the agents involved in
SC [159] No evidence of the I4.0 training
programs despite potential benefits to SC
management [162] Design of training tools for ship design processes through
the use of simulation [128] 14 of 23 Materials 2020, 13, 5625 Table 1. Cont. Aerospace
Shipbuilding
Automotive
Multi-Stage
Implementation
No evidence of culture in the sector in
relation to SC
No evidence of culture in the sector in relation to
SC
Implementing Green practices in the management of SC
collaborates in the implementation of socio-cultural
responsibility [163]
No evidence of multifunctional approach
in the sector in relation to SC
No evidence of multifunctional approach in the
sector in relation to SC
Multifunctional approach using Closed-Loop SC [164]
Continuous improvement of the quality
of products and processes [165] system
to define a Lean workflow [166]
Through collaborative tools that allow
completely managing the SC in continuous
improvement [154]
Continuous improvement to reduce stocks [167], evaluating
the performance of the downstream supply chain [168,169] Table 1. Cont. Table 1. Cont. Shipbuilding 4. Conclusions In the aerospace sector, there is a tendency among companies that are committed to a sustainable
4.0 Supply Chain to be concerned that the breakdown will come from small external companies in
the Supply Chain. Even so, they adopt Lean and Green methodologies considering their impact
on performance. With regard to the macro processes described in the introduction, the aerospace sector is committed
to managing them through the Internet of Things applications, improving both the relations between
the participants and with regard to the adoption of sustainable actions. In addition, there is evidence
of the use of other technologies such as Big Data, Additive Manufacturing, and Blockchain, which are
also focused on the implementation of sustainability in the Supply Chain. Similarly, in the shipbuilding sector, there is also evidence of the concern about the ruptures
caused by the smaller companies that make up the sector. In this case, the paradigms studied for this
sector coincide with the aerospace sector, and the Agile and Resilient paradigms are added as well. Similarly, in the shipbuilding sector, there is also evidence of the concern about the ruptures
caused by the smaller companies that make up the sector. In this case, the paradigms studied for this
sector coincide with the aerospace sector, and the Agile and Resilient paradigms are added as well. There is little evidence of the implementation of different technologies in this sector, although the
Internet of Things seems to be the most remarkable p
g
p
g
There is little evidence of the implementation of different technologies in this sector, although the
Internet of Things seems to be the most remarkable. In the case of the automotive industry and changing from production to mass production,
they coincide with the Lean, Green, and Agile paradigms, although there is controversy in particular
regarding Lean Supply Chain and 4.0 technologies where management practices are preferable. With regard to technologies, it could be said that this sector is one of the ones that has most
implemented its applications in most of them, highlighting on the one hand the additive manufacturing
as, despite the advantages it has, it does not seem to give benefits in this sector. On the other hand,
Simulation stands out as providing flexibility and efficiency to the automotive Supply Chain and as a
facilitator together with other technologies. Shipbuilding Implementing Green practices in the management of SC
collaborates in the implementation of socio-cultural
responsibility [163] Materials 2020, 13, 5625 15 of 23 Acknowledgments: Universidad de Cádiz (UCA) and Dpt. of Mechanical Engineering and Industrial Design
supported this work. Author Contributions: M.R.-P. and M.B. conceptualized the paper. M.B. and P.F.M. approved the experimental
procedure; M.R.-P. and J.M.V.-M. analyzed the data; M.R.-P. wrote the paper; M.B. and J.M.V.-M. revised the paper;
P.F.M. supervised the paper. All authors have read and agreed to the published version of the manuscript. References 1. Chopra, S.; Meindl, P. Administración de la Cadena de Suministro; Cruz Castillo, L.M., Gutierrez Hernández, B.,
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manner among the participants in the chain and also in the implementation by stages. Furthermore,
the sector has identified the barriers that prevent it from successfully implementing technologies that
make the Supply Chain sustainable, and it mainly identifies the human factor in this. Despite comparing sectors with different production systems, it can be seen how all three rely
on Lean practices as necessary to make the Supply Chain sustainable. Even the automotive sector,
being the one that presents more publications, prefers Lean management practices to the benefits that
Industry 4.0 technologies could bring. It could be said that Lean practices should be intrinsic to the
company and that any technology to be implemented should not displace these practices. With regard to technologies, all three sectors reveal a strong interest in the Internet of Things as
being paramount for the sustainability of the Supply Chain. At the same time, Big Data and Blockchain
are two technologies that also demonstrate contributions to sustainability and therefore focus on all
three sectors. However, additive manufacturing is appropriate for the aerospace and shipbuilding
sector, while the automotive sector does not find the full benefit. The technology that this sector is
interesting in is Simulation, contributing considerably directly to the Supply Chain and indirectly as
support to other technologies. Finally, the contribution of the automotive sector to collaborative approaches to change management
to smart factories should be highlighted, which at the same time would help alleviate the concern of
the aerospace and shipbuilding sectors about the source of external breakdowns of components in the
Supply Chain. Author Contributions: M.R.-P. and M.B. conceptualized the paper. M.B. and P.F.M. approved the experimental
procedure; M.R.-P. and J.M.V.-M. analyzed the data; M.R.-P. wrote the paper; M.B. and J.M.V.-M. revised the paper;
P.F.M. supervised the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: Universidad de Cádiz (UCA) and Dpt. of Mechanical Engineering and Industrial Design
supported this work Acknowledgments: Universidad de Cádiz (UCA) and Dpt. of Mechanical Engineering and Industrial Design
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Submicroscopic deletion of 5q involving tumor suppressor genes (CTNNA1, HSPA9) and copy neutral loss of heterozygosity associated with TET2 and EZH2 mutations in a case of MDS with normal chromosome and FISH results
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* Correspondence: morteza.x.hemmat@questdiagnostics.com
1Cytogenetics Department, Quest Diagnostics Nichols Institute, 33608 Ortega
Hwy, 92675 San Juan Capistrano, CA, USA
Full list of author information is available at the end of the article © 2014 Hemmat 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/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. Abstract Advances in genome-wide molecular cytogenetics allow identification of novel submicroscopic DNA copy number
alterations (aCNAs) and copy-neutral loss of heterozygosity (cnLOH) resulting in homozygosity for known gene
mutations in myeloid neoplasms. We describe the use of an oligo-SNP array for genomic profiling of aCNA and
cnLOH, together with sequence analysis of recurrently mutated genes, in a patient with myelodysplastic syndrome
(MDS) presenting with normal karyotype and FISH results. Oligo-SNP array analysis revealed a hemizygous deletion
of 896 kb at chromosome 5q31.2, representing the smallest 5q deletion reported to date. The deletion involved
multiple genes, including two tumor suppressor candidate genes (CTNNA1 and HSPA9) that are associated with
MDS/AML. The SNP-array study also detected 3 segments of somatic cnLOH: one involved the entire long arm of
chromosome 4; the second involved the distal half of the long arm of chromosome 7, and the third encompassed
the entire chromosome 22 (UPD 22). Sequence analysis revealed mutations in TET2 (4q), EZH2 (7q), ASXL1
(20q11.21), and RUNX1 (21q22.3). Coincidently, TET2 and EZH2 were located at segments of cnLOH resulting in their
homozygosity. Loss of heterozygosity affecting these two chromosomes and mutations in TET2 and EZH2 are indicative
of a myelodysplastic syndrome with a poor prognosis. Deletion of the tumor suppressor genes CTNNA1 and HSPA9 is
also likely to contribute to a poor prognosis. Furthermore, the original cnLOHs in multiple chromosomes and additional
cnLOH 14q in the follow-up study suggest genetic evolution of the disease and poor prognosis. This study attests to
the fact that some patients with a myelodysplastic syndrome who exhibit a normal karyotype may have underlying
genetic abnormalities detectable by chromosomal microarray and/or targeted mutation analyses. (20q11.21), and RUNX1 (21q22.3). Coincidently, TET2 and EZH2 were located at segments of cnLOH resulting in their
homozygosity. Loss of heterozygosity affecting these two chromosomes and mutations in TET2 and EZH2 are indicative
of a myelodysplastic syndrome with a poor prognosis. Deletion of the tumor suppressor genes CTNNA1 and HSPA9 is
also likely to contribute to a poor prognosis. Furthermore, the original cnLOHs in multiple chromosomes and additional
cnLOH 14q in the follow-up study suggest genetic evolution of the disease and poor prognosis. This study attests to
the fact that some patients with a myelodysplastic syndrome who exhibit a normal karyotype may have underlying
genetic abnormalities detectable by chromosomal microarray and/or targeted mutation analyses. Submicroscopic deletion of 5q involving tumor
suppressor genes (CTNNA1, HSPA9) and copy
neutral loss of heterozygosity associated with
TET2 and EZH2 mutations in a case of MDS with
normal chromosome and FISH results Morteza Hemmat1*, Weina Chen2, Arturo Anguiano1, Mohammed El Naggar1, Frederick K Racke1, Dan Jones3,
Yongbao Wang3, Charles M Strom1, Karl Chang1 and Fatih Z Boyar1 Morteza Hemmat1*, Weina Chen2, Arturo Anguiano1, Mohammed El Naggar1, Frederick K Racke1, Dan Jones3,
Yongbao Wang3, Charles M Strom1, Karl Chang1 and Fatih Z Boyar1 Abstract Keywords: Copy neutral loss of heterozygosity (cnLOH), Uniparental disomy (UPD), MDS, TET2, EZH2, RUNX1, EZH2,
ASXL1, CTNNA1, HSPA9 Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Morphologic and immunophenotypic findings An 88-year-old woman presented with leukocytosis
(16.2×109/L) including minimal monocytosis (1.0×109/L),
mild anemia (HGB 105 g/L), and moderate thrombocytopenia
(79×109/L). Bone marrow morphologic and flow cytometric
examinations revealed hypercellular bone marrow with
granulocytic predominance with left-shifted and unusual Background heterozygosity (LOH) is an indicator of neoplastic evo-
lution and disease progression [12,13]. Copy-neutral
LOH (cnLOH) arises either via a hemizygous deletion in
one homolog and duplication of the other, or uniparental
disomy (UPD). Both types of somatic LOH have been ob-
served in studies of various cancer types and may explain
some of the mechanisms by which tumor suppressor
genes (TSGs) are inactivated or activating mutations in
oncogenes are duplicated. Acquired UPD (aUPD) is now
understood to be common in oncogenesis and appears to Recent advances in genome-wide molecular cytogenetics
allow the identification of novel molecular abnormalities
[1-8]. Emerging data demonstrate that myelodysplastic
syndrome (MDS) exhibits abundant CNAs and cnLOH,
often in the setting of a normal karyotype [9-11]. Loss of Page 2 of 9 Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 maturation, and occasional atypical granulocytes and
megakaryocytes, but no significant increase in mono-
cytes or myeloblasts (Figure 1). A myeloid neoplasm
positive for CD13 and CD33 was diagnosed, best clas-
sified as MDS, refractory cytopenia with multilineage
dysplasia. Data on therapeutic interventions were not
available for this study. At the 5-month follow-up, the
overall morphologic and immunophenotypic findings
were similar to those at initial presentation. be a mechanism to increase the allelic burden of the
mutated genes [14-24]. Thus, it is important to know the
regions of cnLOH to determine new regions containing
potential mutational targets affecting disease pathogenesis
and treatment outcome [25,26]. Application of SNP-array technology has led to the iden-
tification of recurrent regions of cnLOH in a majority of
the chromosomes [4,13,25,27-30] and recurrent pathogenic
mutations. These findings have greatly advanced our under-
standing of the molecular mechanisms of cancer evolution
and have led to the development of therapeutics and diag-
nostic tests. In this respect, mutation analysis helps further
stratify neoplasms and their treatment outcome [31-34]. Chromosome and FISH study Bone marrow cells were cultivated for 24 and 48 hours
in RPMI 1640 or Marrow Max Medium with 10% fetal calf
serum (Life Technologies, Grand Island, NY 14072, USA). In total, 20 mitoses were analyzed according to the
ISCN (International System for Human Cytogenetic
Nomenclature), with a resolution of 300 bands per
haploid karyotype. FISH analyses using a panel of MDS
probes (−5/5q-, −7/7q-, +8 and 20q-; Vysis, Downers
Grove, IL, USA) and BlueGnome probes RP11-114B12
(Illumina, San Diego, CA, USA) for the deleted region
5q31.2 were performed on interphase cells according
to the manufacturer’s protocol. Subsequently, 200 cells
were examined carefully. In this study, we used SNP array-based genomic
profiling to detect acquired copy number alterations
(aCNA) and cnLOHs, together with sequence analysis
of genes commonly mutated in MDS, in a patient with
normal chromosome and MDS FISH (fluorescence in
situ hybridization) panel results. Clinical presentation Morphologic and immunophenotypic findings Oligo SNP array Microdeletion/microduplication screening was performed
using an SNP-array platform (CytoScan HD SNP array; Figure 1 Morphologic and immunophenotypic findings. A) At presentation, there was granulocytic predominance with progressive
maturation and no significant increase in blasts or monocytes. The core biopsy shows hypercellular bone marrow with left-shifted granulocytic
predominance. Flow cytometry reveals granulocytic predominance (in green) but no significant increase in monocytes (in blue) or myeloblasts
(in red). B) At follow-up, the morphologic and immunophenotypic findings are similar to those at presentation. Figure 1 Morphologic and immunophenotypic findings. A) At presentation, there was granulocytic predominance with progressive
maturation and no significant increase in blasts or monocytes. The core biopsy shows hypercellular bone marrow with left-shifted granulocytic
predominance. Flow cytometry reveals granulocytic predominance (in green) but no significant increase in monocytes (in blue) or myeloblasts
(in red). B) At follow-up, the morphologic and immunophenotypic findings are similar to those at presentation. Figure 1 Morphologic and immunophenotypic findings. A) At presentation, there was granulocytic predominance with progressive
maturation and no significant increase in blasts or monocytes. The core biopsy shows hypercellular bone marrow with left-shifted granulocytic
predominance. Flow cytometry reveals granulocytic predominance (in green) but no significant increase in monocytes (in blue) or myeloblasts
(in red). B) At follow-up, the morphologic and immunophenotypic findings are similar to those at presentation. Page 3 of 9 Page 3 of 9 Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Page 3 of 9 Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Results and discussion Affymetrix, Santa Clara, CA), following the manufacturer’s
instructions. The CytoScan HD array has 2.67 million
probes, including 1.9 million copy number probes and
0.75 million SNP probes. Array data were analyzed
using the Chromosome Analysis Suite (ChAS) software
v 2.0 (Affymetrix). During the initial evaluation of the patient, cytogenetic
analysis revealed a normal karyotype and FISH studies were
negative for aCNAs commonly seen in MDS (MDS panel)
(Figure 2A-D). Microarray analysis revealed a microdeletion
of approximately 896 kb at the 5q31.2 chromosomal region
and three segments of somatic cnLOH for the entire
long arm of chromosome 4 (136 Mb), the distal half of
the long arm of chromosome 7 (50 Mb), and the entire
chromosome 22 (31 Mb). The microdeletion at 5q31.2
extended from 137,821,899 to 138,718,504 bp (UCSC gen-
ome Browser; http://genome.ucsc.edu/; hg19 release) and
included the ETF1, HSPA9, SNORD63, CTNNA1, LRRTM2,
SIL1, SNHG4, MATR3, SNORA74A, PAIP2, and SLC23A1
genes (Figure 3). Mutation analysis Genomic DNA was also tested for mutations in 19 genes
that are recurrently mutated in myeloid neoplasms,
including ASXL1, EZH2, RUNX1, IDH1, IDH2, KRAS,
NRAS and TET2. Sequencing was performed using a
TruSeq custom amplicon assay on the MiSeq sequencing
platform (Illumina, Hayward, CA). Analysis was performed
using SeqPilot software (JSI Medical Systems, Costa Mesa,
CA). The assay had sufficient read depth to provide a mini-
mum sensitivity of 5% to 10% for mutation detection. Of the genes deleted due to this microdeletion, two
are tumor suppressor candidate genes associated with Figure 2 Routine cytogenetic and fluorescence insitu hybridization (FISH) results. A) G-banded chromosome analysis shows a normal
female karyotype. B-D) FISH analyses show normal hybridization with an MDS panel using probes for EGR1 (5q31, red signal) and its control
probe D5S23 (5p15.2, green signal) in B; D7S486 (7q31, red signal) and its control probe D7Z1 (7centromere, green signal) in C; CEP8
(green signal) for chromosome 8 centromere and D20S108 (20q12, red signal) in D. Figure 2 Routine cytogenetic and fluorescence insitu hybridization (FISH) results. A) G-banded chromosome analysis shows a normal
female karyotype. B-D) FISH analyses show normal hybridization with an MDS panel using probes for EGR1 (5q31, red signal) and its control
probe D5S23 (5p15.2, green signal) in B; D7S486 (7q31, red signal) and its control probe D7Z1 (7centromere, green signal) in C; CEP8
(green signal) for chromosome 8 centromere and D20S108 (20q12, red signal) in D. Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Page 4 of 9 A
B
C
Figure 3 5q31.2 deletion. A) Chromosome 5 with deletion at q31.2. B) SNP-array results, including the weighted log2 ratio, copy number state,
and allele peaks at the deleted region. C) Database of genomic variants showing an 896-kb deletion in the short arm of chromosome 5 within
band q31.2 (position 137,821,899 to 138,718,504), including the CTNNA1 and HSPA9 genes. A
B
C
Figure 3 5q31.2 deletion. A) Chromosome 5 with deletion at q31.2. B) SNP-array results, including the weighted log2 ratio, copy number state,
and allele peaks at the deleted region. C) Database of genomic variants showing an 896-kb deletion in the short arm of chromosome 5 within
band q31.2 (position 137,821,899 to 138,718,504), including the CTNNA1 and HSPA9 genes. A B C Figure 3 5q31.2 deletion. A) Chromosome 5 with deletion at q31.2. Mutation analysis B) SNP-array results, including the weighted log2 ratio, copy number state,
and allele peaks at the deleted region. C) Database of genomic variants showing an 896-kb deletion in the short arm of chromosome 5 within
band q31.2 (position 137,821,899 to 138,718,504), including the CTNNA1 and HSPA9 genes. MDS/AML: CTNNA1, which encodes alpha-1 catenin,
and HSPA9, which encodes heat-shock 70-KD protein
9 (mortalin) [35]. Deletion of CTNNA1 was confirmed
by applying BlueGnome FISH probes (RP11-114B12)
(Figure 4). Alpha-catenins such as that encoded by
CTNNA1 are essential for the regulation of cell-cell and
cell-matrix interactions in tissues [36]. Loss of expression
of the CTNNA1 tumor suppressor gene in hematopoietic
stem cells may provide a growth advantage that contributes
to human MDS/AML with 5q deletion [37]. Furthermore,
loss of the CTNNA1 expression has been associated with
leukemia progression or transformation of MDS to AML
[38]. The HSPA9 gene is also located at the 5q31.2 region frequently deleted in MDS/AML, making it a candidate
tumor suppressor gene; this is consistent with the bio-
logical function of its murine homologue. Human mortalin
(encoded by HSPA9) was originally identified by its close
homology to murine mortalins, which play important
roles in cellular senescence [39]. The HSPA9 gene is a
novel negative regulator of Raf/MEK/ERK pathway that
may be a potential therapeutic target [40]. The combined size of cnLOHs spanning at least 10 Mb
across the genome was approximately 217.6 Mb (Figure 5). These were detected at the long arm of chromosomes
4, 7, and 22. The cnLOH might result from mitotic re-
combination or nondisjunction which leads to segmental Page 5 of 9 Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Figure 4 FISH inverted DAPI image showing deletion of the
CTNNA1 gene using the BlueGnome FISH probe RP11-114B12
(5q31.2, red signal). The deleted chromosome 5 is indicated by an arrow. or whole chromosomal UPD, respectively [13]. Acquired
UPD (cnLOH) at diagnosis in our case is indicative of
neoplastic evolution [12,13]. An additional UPD was identi-
fied at 14q in the follow-up study 5 months later, confirm-
ing the genetic progression of disease (Figure 6). Recent investigations have indicated that cnLOH can
be responsible for homozygosity of mutations in critical
genes within the region. Mutation analysis Reduction to homozygosity as a
consequence of cnLOH was initially thought to be a
mechanism for only inactivation of tumor suppressor
genes [14,41,42]; however, identification of cnLOH in
leukemia has shown that oncogeneic mutations are
also targeted [13,16,17]. Mutation analysis of 19 MDS-associated genes re-
vealed an ASXL1 frameshift/stop mutation (Y591*, 41%
of reads), an EZH2 point mutation (R690H, 85%), two
RUNX1 frameshift/stop mutations (c.474dupT, 19% and
c.424_425ins11bp, 7%), and two TET2 frameshift/stop
mutations (c.1510_1513delAAAA, 77% and R1465*, 10%). These mutated genes are located on chromosomes 20, 7,
21, and 4, respectively. Recent studies have shown that
TET2 mutations are present in up to 30% of MDS/MPN Figure 4 FISH inverted DAPI image showing deletion of the
CTNNA1 gene using the BlueGnome FISH probe RP11-114B12
(5q31.2, red signal). The deleted chromosome 5 is indicated by an arrow. Figure 4 FISH inverted DAPI image showing deletion of the
CTNNA1 gene using the BlueGnome FISH probe RP11-114B12
(5q31.2, red signal). The deleted chromosome 5 is indicated by an arrow Figure 5 SNP-array results at diagnosis showing loss of heterozygosity (LOH) at the long arm of chromosomes 4, 7, and 22 as purple
bars next to the corresponding chromosomes. Figure 5 SNP-array results at diagnosis showing loss of heterozygosity (LOH) at the long arm of chromosomes 4, 7, and 22 as purple
bars next to the corresponding chromosomes. Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Page 6 of 9 Figure 6 SNP-array results at the follow-up study showing loss of heterozygosity (LOH) of chromosome 14, in addition to the LOH
chromosomes 4, 7, and 22 found at initial diagnosis. LOH is shown as purple bar next to the corresponding chromosome. http://www.molecularcytogenetics.org/content/7/1/35 Figure 6 SNP-array results at the follow-up study showing loss of heterozygosity (LOH) of chromosome 14, in a
chromosomes 4, 7, and 22 found at initial diagnosis. LOH is shown as purple bar next to the corresponding chromo cases, with mutations in ASXL1, EZH2, and RUNX1 also
commonly reported [43]. The finding of mutations in
all four of these myeloid regulatory genes suggests that
they play a critical role in the pathogenesis of this case and
demonstrate that mutation analysis is useful in cytogeneti-
cally normal myeloid disorders [43]. unfavorable prognosis [48] explained by an association
with resistance to chemotherapy [49]. Mutation analysis Mutations in ASXL1 have been identified in MDS,
AML, chronic myeloid leukemia, chronic myelomonocytic
leukemia (CMML), and juvenile myelomonocytic leukemia
[50-54], and act as a tumor suppressor in myeloid malig-
nancies [50]. Mutations in ASXL1, TET2, and EZH2 have
been found in 41% of MDS cases in a Chinese population
[44], similar to the data reported in patients of European
decent with MDS [21,23,24,55,56]. The cnLOH affecting chromosome 7q and homozygous
EZH2 mutation have been reported in 10% of AML
and MDS cases. They have been associated with a poor
prognosis [9,26,44] and clonal evolution [45,46], supporting
the possible role of EZH2 as a tumor suppressor gene
for myeloid malignancies. Conclusion Akagi T, Ogawa S, Dugas M, Kawamata N, Yamamoto G, Nannya Y, Sanada
Miller CW, Yung A, Schnittger S, Haferlach T, Haferlach C, Koeffler HP: Frequent
genomic abnormalities in acute myeloid leukemia/myelodysplastic
syndrome with normal karyotype. Haematologica 2009, 94:213–223. 6. Akagi T, Ogawa S, Dugas M, Kawamata N, Yamamoto G, Nannya Y, Sanada
Miller CW, Yung A, Schnittger S, Haferlach T, Haferlach C, Koeffler HP: Frequent
genomic abnormalities in acute myeloid leukemia/myelodysplastic
syndrome with normal karyotype. Haematologica 2009, 94:213–223. 7. Maciejewski JP, Tiu RV, O’Keefe C: Application of array based whole
genome scanning technologies as a cytogenetic tool in haematological
malignancies. Br J Hematol 2009, 146:479–488. 7. Maciejewski JP, Tiu RV, O’Keefe C: Application of array based whole
genome scanning technologies as a cytogenetic tool in haematological
malignancies. Br J Hematol 2009, 146:479–488. 8. Tiu RV, Gondek LP, O’Keefe CL, Huh J, Sekeres MA, Elson P, McDevitt MA,
Wang XF, Levis MJ, Karp JE, Advani AS, Maciejewski JP: New lesions
detected by single nucleotide polymorphism array-based chromosomal
analysis have important clinical impact in acute myeloid leukemia. J Clin
Oncol 2009, 27:5219–5226. 8. Tiu RV, Gondek LP, O’Keefe CL, Huh J, Sekeres MA, Elson P, McDevitt MA,
Wang XF, Levis MJ, Karp JE, Advani AS, Maciejewski JP: New lesions
detected by single nucleotide polymorphism array-based chromosomal
analysis have important clinical impact in acute myeloid leukemia. J Clin
Oncol 2009, 27:5219–5226. Received: 30 March 2014 Accepted: 19 May 2014
Published: 27 May 2014 17. Kralovics R, Guan Y, Prchal JT: Acquired uniparental disomy of chromosome
9p is a frequent stem cell defect in polycythemia vera. Exp Hematol 2002,
30(3):229–36. Acknowledgments The authors would like to express their thanks to Maryam Talai and Robert
Mcgough (Quest Diagnostics) for their technical support. The authors would like to express their thanks to Maryam Talai and Robert
Mcgough (Quest Diagnostics) for their technical support. Authors’ contributions 12. Andersen CL, Wiuf C, Kruhøffer M, Korsgaard M, Laurberg S, Ørntoft TF:
Frequent occurrence of uniparental disomy in colorectal cancer. Carcinogenesis 2007, 28(1):38–48. 12. Andersen CL, Wiuf C, Kruhøffer M, Korsgaard M, Laurberg S, Ørntoft TF:
Frequent occurrence of uniparental disomy in colorectal cancer. Carcinogenesis 2007, 28(1):38–48. MH, First author; performed chromosome, FISH, and microarray analysis,
interpretation of the results, drafting and finalization of the manuscript. WC
performed the morphology and immunophenotypic analysis. DJ and YW
performed the genomic sequencing. FR performed the comprehensive
review of the clinical and laboratory findings. AA reviewed and reported the
SNP-array analyses. MEN reviewed the manuscript. FZB reviewed and finalized
the manuscript. All authors read and approved the final manuscript. 13. Raghavan M, Smith LL, Lillington DM, Chaplin T, Kakkas I, Molloy G, Chelala
C, Cazier JB, Cavenagh JD, Fitzgibbon J, Lister TA, Young BD: Segmental
uniparental disomy is a commonly acquired genetic event in relapsed
acute myeloid leukemia. Blood 2008, 112(3):814–21. 14. Flotho C, Steinemann D, Mullighan CG, Neale G, Mayer K, Kratz CP,
Schlegelberger B, Downing JR, Niemeyer CM: Genome-wide single-nucleotide
polymorphism analysis in juvenile myelomonocytic leukemia identifies
uniparental disomy surrounding the NF1 locus in cases associated with
neurofibromatosis but not in cases with mutant RAS or PTPN11. Oncogene 2007, 26(39):5816–21. Abbreviations bb e at o s
aCNA: Acquired copy number alteration; cnLOH: Loss of heterozygosity;
aUPD: Acquired uniparental disomy. aCNA: Acquired copy number alteration; cnLOH: Loss of heterozygosity;
aUPD: Acquired uniparental disomy. 11. Tiu RV1, Gondek LP, O’Keefe CL, Elson P, Huh J, Mohamedali A, Kulasekararaj
A, Advani AS, Paquette R, List AF, Sekeres MA, McDevitt MA, Mufti GJ,
Maciejewski JP: Prognostic impact of SNP array karyotyping in
myelodysplastic syndromes and related myeloid malignancies. Blood 2011, 117(17):4552–60. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Ethical approval and consent 9. Heinrichs S, Kulkarni RV, Bueso-Ramos CE, Levine RL, Loh ML, Li C, Neuberg
D, Kornblau SM, Issa JP, Gilliland DG, Garcia-Manero G, Kantarjian HM, Estey EH,
Look AT: Accurate detection of uniparental disomy and microdeletions by
SNP array analysis in myelodysplastic syndromes with normal cytogenetics. Leukemia 2009, 23(9):1605–13. 9. Heinrichs S, Kulkarni RV, Bueso-Ramos CE, Levine RL, Loh ML, Li C, Neuberg
D, Kornblau SM, Issa JP, Gilliland DG, Garcia-Manero G, Kantarjian HM, Estey EH,
Look AT: Accurate detection of uniparental disomy and microdeletions by
SNP array analysis in myelodysplastic syndromes with normal cytogenetics. Leukemia 2009, 23(9):1605–13. These studies were performed on anonymized samples
received in the clinical laboratory and thus were exempted
from the requirement for consent by an opinion for the
Western Institutional Review Board. 10. Thiel A1, Beier M, Ingenhag D, Servan K, Hein M, Moeller V, Betz B,
Hildebrandt B, Evers C, Germing U, Royer-Pokora B: Comprehensive array
CGH of normal karyotype myelodysplastic syndromes reveals hidden
recurrent and individual genomic copy number alterations with
prognostic relevance. Leukemia 2011, 25:387–399. Author details
1 15. Fitzgibbon J, Smith LL, Raghavan M, Smith ML, Debernardi S, Skoulakis S,
Lillington D, Lister TA, Young BD: Association between acquired
uniparental disomy and homozygous gene mutation in acute myeloid
leukemias. Cancer Res 2005, 65:9152–9154. 1Cytogenetics Department, Quest Diagnostics Nichols Institute, 33608 Ortega
Hwy, 92675 San Juan Capistrano, CA, USA. 2University of Texas southwestern
Medical Center, 5323 Harry Hines Blvd, 75235 Dallas, TX, USA. 3Quest Diagnostics Nichols Institute, 14225 Newbrook Drive, 20151 Chantilly, VA, USA. 16. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli
A, Cazzola M, Skoda RC: A gain-of-function mutation of JAK2 in
myeloproliferative disorders. N Engl J Med 2005, 352(17):1779–90. Received: 30 March 2014 Accepted: 19 May 2014
Published: 27 May 2014 4.
Gupta M, Raghavan M, Gale RE, Chelala C, Allen C, Molloy G, Chaplin T,
Linch DC, Cazier JB, Young BD: Novel regions of acquired uniparental 20.
Dunbar AJ, Gondek LP, O’Keefe CL, Makishima H, Rataul MS, Szpurka H,
Sekeres MA, Wang XF, McDevitt MA, Maciejewski JP: 250 K single Conclusion In contrast to the mutated TET2 and EZH2 genes,
no LOH was found for the other two mutated genes
(RUNX1 and ASXL1). RUNX1 mutations have been
proposed as clinically useful biomarkers to follow disease
progression from MDS to AML as well as to monitor
minimal residual disease (MRD) [47]. Moreover, RUNX1
mutations were demonstrated to be frequent in de novo
AML with non-complex karyotypes and conferred an In conclusion, our study identified four large cnLOH and
a microdeletion at 5q31 harboring two tumor suppres-
sor genes (CTNNA1 and HSPA9) in an MDS case with
an apparently normal karyotype. The regions of cnLOH at
chromosomes 4, 7, 14, and 22 confirm and extend previous
studies, supporting that cnLOHs in myeloid disorders are
common and nonrandom. Moreover, the presence of an
additional aUPD 14q at follow up, along with persistence of Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Hemmat et al. Molecular Cytogenetics 2014, 7:35
http://www.molecularcytogenetics.org/content/7/1/35 Page 7 of 9 Page 7 of 9 the 5q31 microdeletion and all cnLOHs detected at pres-
entation, indicates genetic progression of the disease. The
deletion of tumor suppressor genes CTNNA1 and HSPA9,
along with mutation of candidate myeloid regulatory genes
TET2 and EZH2, supports the diagnosis of MDS and likely
portends a poor prognosis. disomy discovered in acute myeloid leukemia. Genes Chromosomes
Cancer 2008, 47:729–739. 5. Kawamata N, Ogawa S, Zimmermann M, Kato M, Sanada M, Hemminki K,
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series of patients with MDS may yield information on
how to stratify this category of disease to seek further
molecular definition. 6. Akagi T, Ogawa S, Dugas M, Kawamata N, Yamamoto G, Nannya Y, Sanada
Miller CW, Yung A, Schnittger S, Haferlach T, Haferlach C, Koeffler HP: Frequent
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http://www.molecularcytogenetics.org/content/7/1/35 doi:10.1186/1755-8166-7-35
Cite this article as: Hemmat et al.: Submicroscopic deletion of 5q
involving tumor suppressor genes (CTNNA1, HSPA9) and copy neutral
loss of heterozygosity associated with TET2 and EZH2 mutations in a
case of MDS with normal chromosome and FISH results. Molecular
Cytogenetics 2014 7:35. doi:10.1186/1755-8166-7-35
Cite this article as: Hemmat et al.: Submicroscopic deletion of 5q
involving tumor suppressor genes (CTNNA1, HSPA9) and copy neutral
loss of heterozygosity associated with TET2 and EZH2 mutations in a
case of MDS with normal chromosome and FISH results. Molecular
Cytogenetics 2014 7:35. doi:10.1186/1755-8166-7-35 Submit your next manuscript to BioMed Central
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Newer Oral Anticoagulants in the Treatment of Acute Portal Vein Thrombosis in Patients with and without Cirrhosis
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International journal of hepatology
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cc-by
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Hindawi
International Journal of Hepatology
Volume 2018, Article ID 8432781, 9 pages
https://doi.org/10.1155/2018/8432781 Hindawi
International Journal of Hepatology
Volume 2018, Article ID 8432781, 9 pages
https://doi.org/10.1155/2018/8432781 Hindawi Review Article
Newer Oral Anticoagulants in the Treatment of Acute Portal
Vein Thrombosis in Patients with and without Cirrhosis P. Priyanka
,1 J. T. Kupec
,2 M. Krafft,2 N. A. Shah,3 and G. J. Reynolds 1Department of Medicine, West Virginia University Hospitals, Morgantown, WV, USA
2Department of Medicine, Section of Digestive Diseases, West Virginia University Hospitals, Morgantown, WV, USA
3Department of Medicine, Section of Hematology, West Virginia University Hospitals, Morgantown, WV, USA 1Department of Medicine, West Virginia University Hospitals, Morgantown, WV, USA
2Department of Medicine, Section of Digestive Diseases, West Virginia University Hospitals, Morgantown, WV, USA
3Department of Medicine, Section of Hematology, West Virginia University Hospitals, Morgantown, WV, USA Correspondence should be addressed to P. Priyanka; priyanka.priyanka@hsc.wvu.edu Received 6 April 2018; Accepted 30 April 2018; Published 5 June 2018 Academic Editor: Dirk Uhlmann Copyright © 2018 P. Priyanka et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Newer oral anticoagulants (NOACs) are being utilized increasingly for the treatment of venous thromboembolism
(VTE). NOAC use is the standard of care for stroke prophylaxis in nonvalvular atrial fibrillation and treatment of acute VTE
involving extremities and pulmonary embolism. In contrast, most guidelines in the literature support the treatment of acute portal
vein thrombosis (PVT) with low molecular weight heparin (LMWH) and vitamin K antagonists (VKA). Literature evaluating
NOAC use in the treatment of acute portal vein thrombosis is sparse. This review focuses on the safety and efficacy of the use of
NOACs in the treatment of acute PVT in patients, with or without concomitant cirrhosis, based on the most recent data available in
the current literature. Methods. A systematic review was conducted through a series of advanced searches in the following medical
databases: PubMed, BioMed Central, Cochrane, and Google Scholar. Keywords utilized were as follows: NOAC, DOAC (direct oral
anticoagulants), portal vein thrombosis, rivaroxaban, apixaban, dabigatran, and edoxaban. Articles related to newer anticoagulant
use in patients with portal vein thrombosis were included. Results. The adverse events, including bleeding events (major and minor)
and the failure of anticoagulation (propagation of thrombus or recurrence of PVT), are similar between the NOACs and traditional
anticoagulants for the treatment of acute PVT, irrespective of the presence of cirrhosis. Conclusions. Newer oral anticoagulants are
safe and efficacious alternatives to traditional anticoagulation with low molecular weight heparin and vitamin K antagonists in the
treatment of acute portal vein thrombosis with or without cirrhosis. 3. Drawbacks of Traditional Anticoagulants
Use in PVT with Cirrhosis for stroke prophylaxis in nonvalvular atrial fibrillation,
venous thromboembolism (VTE) prophylaxis in orthopedic
surgery patients, and the treatment of VTE involving the
extremities and acute pulmonary embolism (PE) [16]. Despite
this progress, anticoagulation of portal vein thrombosis
with NOACs has remained controversial. Traditionally, low
molecular weight heparin (LMWH) and vitamin K antago-
nists (VKAs) have been used mainly due to lower costs, more
physician experience with these agents, and easy reversibility
in case of severe bleeding [5, 6]. The role of NOACs for the
PVT treatment is still undefined in the current literature.h Despite their acceptance as the standard of care for many
thromboembolic events, there are some disadvantages of
using LMWH and VKAs in the treatment of PVT.fi g
For example, in patients with cirrhosis, the efficacy of
unfractionated heparin (UFH) and LMWH may be signifi-
cantly decreased (up to 40%) due to lower levels of antithrom-
bin synthesis by the liver [24]. Additionally, LMWH has
limited use in concurrent renal impairment. Coagulopathy from liver disease frequently results in an
elevated INR and thus utilizing the INR to guide dosing
of VKAs is particularly challenging. Following the INR as
a monitoring parameter was only studied in noncirrhotic
population and is being used by extrapolation for VKA
monitoring in patients with cirrhosis [8]. Elevated INR
levels associated with cirrhosis can offer a false measure of
the therapeutic efficacy. Conversely, when VKAs artificially
elevate the INR, this can directly affect the model for end-
stage liver disease (MELD) score, impacting transplantation
eligibility. Protein C, a vitamin K dependent anticoagulant
factor, is reduced in cirrhosis and may decrease the efficacy
of VKAs [8]. i
The complications of PVT (gastrointestinal bleeding,
including variceal, intestinal infarction and development of
portal hypertension, portal cholangiopathy, and subsequent
hepatic decompensation) can be quite serious [1]. Recanaliza-
tion of the thrombus occurs more frequently with the aid of
anticoagulation thereby reducing complications mentioned
above. Additionally, the development of PVT is associated
with higher morbidity and mortality after liver transplanta-
tion, complicating treatment strategies [17]. As supported by
animal data, another proposed benefit of anticoagulation in
PVT is the suggestion that fibrogenesis may be reduced by
thrombin antagonism [17, 18]. Patients with cirrhosis have been excluded ipso facto in
clinical trials of NOACs mostly due to concerns of severe
bleeding in the absence of reversal agents. 2. Methods A comprehensive, systematic search of the PubMed, Biomed
Central, Cochrane Library, and Google Scholar was per-
formed. Relevant English language articles were identified
up to February 28, 2018. Manual searching for relevant
publications from the reference section of extracted articles
was also performed.it A total of 53 manuscripts were identified after searching
the above databases. Duplicate studies were removed. Of
these articles, 43 were discarded as they were not relevant to
the topic and 10 were included (Table 1). i
Systemic clearance upon discontinuation of NOACs is
faster (5-9 hours in young adults and 11-13 hours in older
adults) and more reliable when compared to warfarin (20-
60 hours) [27]. Clinically this is significant as rivaroxaban
can be stopped much closer to an elective surgery or invasive
procedure than warfarin. All of these properties combine to
increase patient compliance, reliability, and effectiveness. Of the selected 10 articles, four studies were original
manuscripts and six were case reports. Only one study
was prospective and three were retrospective. Six studies
involved patients with cirrhosis, three articles included only
noncirrhotic patients, and one study included patients both
with and without cirrhosis. In all, a total of 119 patients, across
all studies, were included. 1. Introduction but also efficacious when compared to the traditional antico-
agulation [8–12]. Portal vein thrombosis (PVT) is defined as thrombosis within
the portal vein trunk and intrahepatic portal branches. The presence of PVT in cirrhosis varies with a reported
incidence of 8.4% to 11.2% and prevalence nearing 26% [1]. The benefits of anticoagulation are seen in both cirrhotic and
noncirrhotic patients. These benefits include a higher rate of
recanalization (42-100%), a lower thrombus extension rate,
a lower incidence of hepatic decompensation, and improved
survival [2–7]. Although the exact duration of anticoagulation for acute
portal vein thrombosis remains controversial, the American
Association for the Study of Liver Disease (AASLD) rec-
ommends at least three months of anticoagulant use in the
treatment of PVT, irrespective of the presence of cirrhosis
[13]. Most trials of treatment of PVT have effectively used
low molecular weight heparin and warfarin [5, 14, 15]. Far
fewer studies have examined the safety and efficacy of newer
oral anticoagulants (NOACs) in the treatment of this disease
process. Historically, patients with liver dysfunction were advised
to avoid newer oral anticoagulants due to the risk of excess
bleeding and reduced effectiveness. However, increasing
evidence suggests that oral anticoagulants are not only safe NOACs are one of the landmark advances in the recent
practice of medicine as they overcome numerous drawbacks
of traditional anticoagulants. NOACs are currently approved 2 International Journal of Hepatology 4. Advantages of NOACs for VTE There are benefits to considering NOACs for the treatment
of thromboembolic events. These medications can be cost-
effective and convenient. Unlike LMWH, they do not require
daily subcutaneous injections, and since their pharmacoki-
netics and pharmacodynamics are predictable, they do not
require routine dose monitoring like warfarin. In contrast
to the strict dietary precautions VKA users have to follow,
NOACs pharmacodynamics are not affected by dietary intake
[25]. In addition, NOACs act faster than VKAs. Rivaroxaban’s
onset of action is 30 minutes, faster than the 36-72 hours
for warfarin (which requires overlap with heparin therapy). Interactions of NOACs with other drugs are rare, with the
exception of a few antibiotics and antifungals, which may be
significant for patients with malignancy [26]. The purpose of this review is to evaluate the existing
literature on the use of NOACs for PVT in patients with and
without cirrhosis. 3. Drawbacks of Traditional Anticoagulants
Use in PVT with Cirrhosis Off-label use
of NOACs in VTE involving atypical sites like splanchnic
vein thrombosis, cerebral venous thrombosis, and renal and
ovarian vein thrombosis has been evaluated with excellent
efficacy and safety profile [19]. Despite the ever-increasing
evidence, NOACs have not yet found mainstream success as
an alternative for the treatment of PVT [13].h VKAs have a narrow therapeutic window as well as
numerous drug and dietary interactions and need frequent
coagulation testing and dose adjustments. Despite their
efficacy, VKAs are far from an easy means of anticoagulation. 5. Rivaroxaban Is the Most Studied NOAC for
the Treatment of PVT Recanalization rates and thrombus recurrence rates
served as efficacy endpoints. Bleeding events were utilized as
the complication endpoint. Rivaroxaban has been consistently used in the most studies
examining the role of NOACs in the treatment of PVT either International Journal of Hepatology 3 Table 1
Author
Study design
Number of patients (n)
Duration
Agent
Response
Bleeding events
Janczak [19]
(2018)
Prospective, non-cirrhotic,
Atypical sites
Total
N=36
PVT
N= 16
Rivaroxaban
Apixaban
Recurrence rate
7.3 % (n=2)
(both had malignancy)
Minor
N=1 (3.6%)
Major
N=2 (7.2%)
Qi [20]
(2017)
Case report, cirrhotic, CPT
not specified
SMV, splenic vein, N=1
11 weeks
Rivaroxaban
Recanalization
Melena and hematemesis
Nery [21]
(2017)
Case report
Non-cirrhotic
N=1
>6m
Rivaroxaban 20 mg daily
Complete recanalization L
branch,
partial recanalization right
branch
None
De Gottardi
[10]
(2016)
Retrospective, Both
cirrhotic and non-cirrhotic,
Splanchnic
Total,
N= 94
PVT
N= 80
(Non cirrh-
N=38
Cirrh-
N=22)
Non-cirrhotic
13.1 m
cirrhotic
9.6 m
Rivaroxaban
Apixaban
Dabigatran
Not studied
Cirrhosis:
Minor, n=7, Major, n=2
Non-cirrh:
Minor,
n= 4
Major, n=1
Hum [9]
(2016)
Retrospective,
Cirrhotic,
CPT A, B & C
All indications
Total,
N= 27
PVT
N=4
Rivaroxaban
15 mg bid
+/- 20 mg daily load,
Apixaban
5mg bid +/- 10mg bid load,
No bridging
Recurrence rate 4%
(n=1)
Major
N=1 (4%)
Minor,
N=7
Yang [12]
(2016)
Case report, cirrhotic
CPT A
N=1
6 m
Rivaroxaban
15 mg bid x 3 wks, then 20
mg/d
Complete recanalization
None
Intagliata [8]
(2015)
Retrospective, cirrhotic,
CPT A and B
Total
N= 20
PVT, N=12
10.6 m
Apixaban
Rivaroxaban
Not studied
Major, n=1
Martinez [22]
(2014)
Case report,
Cirrhotics,
CPT A
N=1
6 m
Rivaroxaban
20 mg /d
Complete recanalization
None
Lenz [11]
(2014)
Case report,
Cirrhotic
CPT A
N=1
5 m
Rivaroxaban 10 mg daily
Complete recanalization
None
Pannach [23]
(2013)
Case report,
Non-cirrhotic
N=1
>4 weeks
Rivaroxaban 20 mg daily
Complete recanalization
None
CPT: Child Pugh Turcotte; Cirrh: cirrhosis. International Journal of Hepatology 4 the therapeutic efficacy. This highlights the need for agent-
specific dose adjustments in the presence of liver cirrhosis. the therapeutic efficacy. This highlights the need for agent-
specific dose adjustments in the presence of liver cirrhosis. as a single agent or in combination with a second agent such
as apixaban or dabigatran [8–12, 19, 21–23]. Similarly, the
studies evaluating the pharmacodynamics and hepatotoxicity
of NOACs in patients with cirrhosis were mainly performed
with rivaroxaban [28, 29]. 6. Summary of the Data Supporting Treatment
of PVT with NOACs (Table 1) It has been postulated that reduced flow rates in the portal
vein in PVT or cirrhosis may be associated with severe
protein C deficiency contributing to a locally prothrombotic
milieu [32]. Due to the very high bioavailability of rivaroxa-
ban and other factor Xa inhibitors, local anticoagulant action
of these agents compared to systemic anticoagulants has
been proposed, as the metabolites of rivaroxaban have no
anticoagulant activity [11]. This could support the use of even
lower doses (2.5 mg once or twice daily) of rivaroxaban in
PVT. There is little scientific evidence regarding the use of NOACs
in PVT, and well-designed prospective studies are even fewer. Recently, a prospective study involving patients from the
Mayo Clinic thrombophilia registry for initial treatment of
acute venous thromboembolism of atypical location (VTE-
AL) was published [19]. Outcomes, recurrence of PVT, and
bleeding rates were similar for rivaroxaban and apixaban for
patients with VTE in a typical location (VTE-TL) treated
with NOACs compared to VTE-AL treated with enoxaparin
in 623 patients without cirrhosis. In this study group, 29
had PVT and 63 patients had VTE-AL. Another multicenter,
retrospective European study with 94 patients from 17 centers
looked at NOAC use for splanchnic vein thrombosis, cardiac
arrhythmias, peripheral deep vein thrombosis (DVT), and
PE [19]. They concluded that NOACs were an effective and
safe alternative to LMWH and VKAs, including patients with
PVT with and without underlying cirrhosis. 9. Agent-Specific Considerations in NOACs 9.1. Rivaroxaban, Apixaban, and Edoxaban. Rivaroxaban,
apixaban, and edoxaban are direct factor Xa inhibitors with
half-lives of 5-9 hours, 12 hours, and 10-15 hours, respectively. Rivaroxaban and apixaban are metabolised in the liver (67%)
and kidneys (33%) and are extensively bound to plasma
proteins. Edoxaban is metabolised hepatically and renally
equally. Each of these agents is well absorbed in gastroin-
testinal (GI) tract and has high bioavailability. Rivaroxaban
absorption occurs only in the stomach making it the better
choice for patients with gastric tubes. Apixaban is absorbed
through the entire length of GI tract, predominantly in the
distal small bowel and ascending colon. As a result, apixaban
administration may not be suitable for patients with a prox-
imal colectomy or a distal small bowel resection. Edoxaban
is absorbed mainly in the small intestine; therefore it can be
given effectively to patients with previous colectomy [33–37]. Smaller studies aimed at evaluating the risk of bleeding
in patients with PVT treated with NOACs show near equal
complications to those treated with LMWH and VKAs [8, 9]. It must be noted, however, that patients with decompensated
cirrhosis were mostly excluded from these studies with only
a few Child Pugh Turcotte (CPT) class C patients included in
just one study [9]. 10. Dabigatran PVT can be associated with thrombosis of the mesenteric
vein. Mesenteric vein thrombosis may cause impaired drug
absorption due to reduced mesenteric perfusion resulting
from severe venous congestion [19]. This could interfere with
the efficacy of NOACs. It is important to be aware of this
possibility while initiating treatment with NOACs in patients
with PVT. Unlike the factor Xa inhibitors, dabigatran has unique
pharmacodynamics. Dabigatran has poor absorption in the
gastrointestinal tract, limited hepatic metabolism, minimal
binding to plasma proteins, and a longer half-life (12-14
hours) and is eliminated almost entirely through the kidneys
(80%) [38]. Additionally, dabigatran has a different mecha-
nism of action; it is a direct thrombin inhibitor [39, 40]. It
is known to cause significant dyspepsia due to high concen-
trations in the colon secondary to poor GI absorption and
tartaric acid content of the capsule [41]. It becomes clinically
significant as simultaneous administration of pantoprazole
reduces dabigatran’s effective therapeutic area under curve
(AUC) by 22%. At this time, dose modification of dabigatran
in CPT A and B cirrhosis patients is not indicated due to
limited data available on its use in these patients. Given the progressive nature of cirrhosis, hepatic func-
tion may change over time. During long-term treatment
with NOACs, this progression could potentially be missed
as routine coagulation testing is not recommended with
NOACs. This emphasizes the need for detailed testing of
the liver function prior to initiation of treatment and also
supports periodic monitoring of coagulation parameters
while on NOACs [30]. A recent study suggests reduced in vitro anticoagulant
potency of rivaroxaban in patients with CPT B and C
cirrhosis and increased anticoagulant effect of dabigatran
proportional to the severity of liver disease [31]. This may
result in overanticoagulation or even undertreatment. Mea-
surement of factor X levels for rivaroxaban and factor II levels
in dabigatran may be necessary in cirrhosis patients to ensure 14. Adverse Events Noted in the Studies
Involving NOACs in PVT reduction in recurrent thrombi formation. NOACs appear
to perform as well as traditional anticoagulants in achieving
recanalization and have similar thrombus recurrence rates [9,
19]. In the aforementioned Mayo oncology study, recurrence
of VTE-AL was 7.3% in patients with underlying malignancy
treated with rivaroxaban [19]. This was not different from
recurrence rates noted in VTE-TL. NOAC use in splanchnic
vein thrombosis, a study by the Vascular Liver Disease
Group (VALDIG) consortium that included patients with
and without cirrhosis, showed thrombotic events in two of
58 treated patients (3.4 %), including a case of progression
of PVT [10]. No anticoagulation failure was reported in the
cirrhosis group in this study. In another study, only one failure
of anticoagulation in both the NOAC and the traditional
anticoagulant groups was observed in 27 patients with cir-
rhosis treated for various indications [9]. Additionally, no
progression of PVT was noted. Complete recanalization of
PVT has been noted in case reports; one report describes
partial recanalization [11, 12, 20–22]. 14.1. Bleeding. As with any form of anticoagulation, the risk
of bleeding should be balanced with the potential benefit
of treatment. A few studies have evaluated the overall risk
of bleeding when utilizing newer oral anticoagulants. Major
bleeding was defined as overt bleeding with a drop in the
hemoglobin greater than or equal to 2 g/dl, transfusion of
two or more units of packed red blood cells, or intracranial,
intraspinal, intraocular, retroperitoneal, pericardial, or fatal
bleeding. Any bleeding events not meeting the above criteria, but
requiring medical attention, were categorized as nonmajor
bleeding. 14.2. Major Bleeding. In the available studies, the rate of major
bleeding with NOACs was not higher than with traditional
anticoagulants. In patients treated with apixaban and rivarox-
aban from the Mayo Thrombophilia Registry, two patients
out of 36 treated with NOACs (5.55%) with underlying
malignancy had a major bleeding event [19]. In the VALDIG
study, major bleeding requiring discontinuation of NOAC
was seen in two of 258 (0.71%) patients without cirrhosis and
one of 36 (2.7%) patients with cirrhosis [10]. These included
bleeding after variceal band ligation and after hysterectomy in
the noncirrhosis cohort and lower GI bleeding in the cirrhosis
group. These rates are not higher than major bleeding rates
with traditional anticoagulants. 12. Monitoring of NOACs In 2014 guidelines from the Australasian Society of Throm-
bosis and Hemostasis, prothrombin time prolongation was
thought to be most sensitive test for rivaroxaban and edox-
aban, while activated partial thromboplastin time (aPTT)
and thrombin time (TT) were markers of dabigatran [42]. HEMOCLOT direct thrombin inhibitors assay (HYPHEN
BioMed, France, CK002K) is the recommended confirmatory
test for drug levels of dabigatran. Drug-specific anti-factor
Xa assays are recommended for apixaban, edoxaban, and
rivaroxaban for confirmation. The assays for these agents are
not currently commercially available and they are of little
use in emergent situations. Furthermore, it is important to
note that a normal aPTT with dabigatran or normal PT with
apixaban, edoxaban, or rivaroxaban does not exclude the
presence of active drug [43]. 11. Efficacy of NOACs in Acute PVT Anticoagulant efficacy can be defined as both the rate
of recanalization of the thrombus in the portal vein and 5 International Journal of Hepatology 14. Adverse Events Noted in the Studies
Involving NOACs in PVT No excessive major bleeding
rates were noted in two other studies specifically designed
to investigate the risk of bleeding in cirrhotic patients with
NOACs when compared to traditional anticoagulants [8, 9]. Due to differences in the dosing regimens and indications
for anticoagulation, investigators could not ascertain if the
risk of bleeding was uniform in their cohort. Some data
actually suggests that the risk of major bleeding was higher
with traditional anticoagulant use (28%) than NOACs (4%) in
patients with cirrhosis [9]. This reduced risk was specifically
noted for central nervous system (CNS) bleeds, as no CNS
bleeding was noted with NOAC therapy compared to three
(17%) cases for traditional anticoagulants. Only one case
report recorded hematemesis and melena but no other major
bleeding events were noted in any case reports [11, 12, 20–23]. 16. Other Limitations of NOACs 16.1. Higher Cost. New medications often face challenges with
respect to insurance coverage and overall costs to the patients
and NOACs are no exception. Higher costs are a concern with
NOACs, but it has been shown that these costs can be offset
by reduced costs of monitoring, less healthcare provider time,
and an increased convenience to the patient. As such, some
data supports the idea that rivaroxaban can be more cost-
effective than warfarin in the prevention of recurrent VTE
[47]. A recent review supported the superiority of four-factor
prothrombin complex concentrate (4F-PCC) over fresh
frozen plasma (FFPs) as a reversal agent [50]. Determining
the appropriate dose of 4F-PCC remains a challenge as
excessive use may result in a higher risk of thrombosis. Idarucizumab, a monoclonal antibody for dabigatran rever-
sal, used as a single intravenous dose has been shown to be
effective. Though available in many countries, use of both
PCC and idarucizumab remains limited as they may be cost
prohibitive [51]. Andexanet alfa, a recombinant modified
human factor Xa decoy protein, though not yet approved,
has shown potential as a possible reversal of factor Xa
inhibitors. Ciraparantag binds directly to factor Xa inhibitors,
dabigatran, LMWH, and unfractionated heparin and is under
evaluation for reversal of both direct thrombin inhibitors and
factor Xa inhibitors [51]. 16.2. Hepatotoxicity. Interestingly, no cases of hepatotoxicity
were noted in the studies focusing on NOAC use in PVT,
possibly due to smaller size of these studies. Liver toxicity of
NOACs, particularly rivaroxaban, more so than dabigatran or
apixaban, has been reported in the literature. The incidence of
hepatotoxicity in patients with chronic advanced liver disease
is estimated to be 0.1-1% and appears to be idiosyncratic in
nature [28, 29]. Liver dysfunction is usually reversible after
stopping the treatment. Though infrequent, patients should
be made aware of the possible symptoms of liver dysfunction. In addition, dabigatran is amenable to neutralization with
gastric lavage soon after ingestion and, due to its low protein
binding and lipophilic nature, hemodialysis in severe cases. 16.3. Cautious Use in Liver Dysfunction. Moderate or severe
hepatic impairment can reduce NOAC clearance and aug-
ment their pharmacodynamic effects to varying degrees. Significantly increased exposure was noted in CPT B cirrhosis
patients compared to CPT A patients for rivaroxaban [48]. 13. Indications and Duration of NOAC
Treatment in PVT AASLD guidelines recommend at least three months of anti-
coagulation with traditional anticoagulants for all patients
with acute PVT irrespective of presence of symptoms. Long-
term treatment should be considered in patients with perma-
nent risk factors for thrombosis and concomitant mesenteric
vein thrombosis, due to the risk of mesenteric infarction [40].h 14.3. Nonmajor Bleeding. Nonmajor bleeding rates were actu-
ally lower in patients with VTE-TL treated with NOAC
(8.7%) in the Mayo study than the nonmajor bleeding rate in
patients with VTE-AL treated with enoxaparin (15.6%) [19]. The VALDIG study showed similar nonmajor bleeding rates
in patients with cirrhosis (11.1%) and without cirrhosis (12%). These bleeding events included epistaxis, gingival bleeding,
GI bleeding in the noncirrhotic group and bleeding from
portal hypertensive gastropathy, lower GI bleeding, epistaxis,
and bleeding after band ligation in the cirrhosis cohort. In another study, minor bleeding events included vaginal
bleeding and GI bleeding [8]. The American College of Chest Physicians recommends
a minimum of three months of anticoagulation for symp-
tomatic patients only and no anticoagulation for asymp-
tomatic patients [16]. Indefinite anticoagulation should be
considered in patients with permanent risk factors such as
cirrhosis, malignancy, or autoimmune disorders.h The duration of anticoagulation with NOACs has not
been addressed in any guideline as NOACs are still not the
standard of care in PVT treatment. In the studies discussed
in this review, the duration of treatment varies from 5 to 13
months in duration and longer in some cases. A recent meta-analysis from 43 randomized controlled
trials has concluded that there is an increased risk of International Journal of Hepatology 6 edoxaban, indicating more consistent drug pharmacokinetics
[49]. Currently, rivaroxaban and edoxaban are recommended
to be avoided in patients with CPT B and C cirrhosis and
apixaban in patients with CPT C cirrhosis [33–35]. There are
risks of higher doses of NOACs, particularly in prolonged
exposure in patients with liver dysfunction, but the data is
limited with respect to recommending a dose adjustment in
moderate to severe liver dysfunction with any of these drugs
[11]. These factors should be considered when choosing a
NOAC in patients with cirrhosis. gastrointestinal bleeding with NOACs use with an incidence
of 1.4% [44]. Furthermore, this risk was found to be more for
rivaroxaban and dabigatran and less for edoxaban and apix-
aban. Concurrent antiplatelet therapy can further increase
the bleeding risk by five times. 16. Other Limitations of NOACs In
patients with CPT B cirrhosis, the area under the plasma con-
centration or AUC of rivaroxaban and apixaban is increased
(2.27 times and 1.09 times, respectively) and decreased
(4.8% and 5.6%) for edoxaban and dabigatran, respectively
[30]. In comparison, there was no difference in the drug
exposure between patients with mild and moderate hepatic
dysfunction after administration of a single 15 mg dose of 13. Indications and Duration of NOAC
Treatment in PVT Currently, although there
is no evidence in literature to suggest that anticoagulation
increases the risk of variceal bleeding, studies recommend
screening for varices and initiating or optimizing beta blocker
therapy or perform endoscopic variceal ligation (EVL) before
starting anticoagulation [45, 46]. 15. Other Side Effects 16.4. Cautious Use in Renal Impairment. Since all NOACs
are excreted through kidneys to some degree, caution needs
to be exercised in utilizing NOACs in the presence of renal
impairment. The Food and Drug Administration (FDA) has
approved a lower dose of dabigatran (75 mg bid), apixaban (5
mg bid or 2.5 mg bid), and rivaroxaban (15 mg od) for patients
with renal insufficiency and creatinine clearance of 15-30
ml/min [33, 34, 38]. NOACs are not indicated at this time in
end-stage renal disease (ESRD) and patients on hemodialysis. While much of the risk-benefit discussion with the patients
centers on the risk of bleeding, additional adverse effects
(AEs) of NOAC treatment should be considered. As these
drugs are relatively new, the amount of data available is
limited. Some of these AEs were elucidated by VALDIG
investigators. In patients without cirrhosis, leukopenia (n=1)
was a rare event but was not severe enough to discontinue
treatment. However, dizziness (n=1) did require discontinua-
tion of the medication [10]. 16.5. Reversal. One of the notable advantages of the tradi-
tional anticoagulants is the availability of reversal agents. This
is significant in the context of NOACs where a lack of a cost-
effective, widely available reversal agent hampers their use
with respect to major bleeding events. To reduce the risk
of excessive bleeding during surgery, adequate reversal of
NOACs is of paramount importance in patients awaiting liver
transplantation. 16.5. Reversal. One of the notable advantages of the tradi-
tional anticoagulants is the availability of reversal agents. This
is significant in the context of NOACs where a lack of a cost-
effective, widely available reversal agent hampers their use
with respect to major bleeding events. To reduce the risk
of excessive bleeding during surgery, adequate reversal of
NOACs is of paramount importance in patients awaiting liver
transplantation. 20. Conclusions and Future Directions with therapeutic dose of rivaroxaban (20 mg daily) and
apixaban (5 mg twice a day), and 25% were treated with
lower dose (rivaroxaban 10 mg daily and apixaban 5 mg
daily) successfully without excessive bleeding episodes and
recurrence rates [8]. Other studies were performed without
dose modification [9]. Case reports support a standard dose
of rivaroxaban, even in the presence of cirrhosis, without any
effect on efficacy and bleeding events [11, 12]. At this time,
there is no clear guidance in the literature about the effect
of dose modification on the efficacy and possible benefit for
cirrhosis patients. Although the current literature does not conclusively estab-
lish the role of NOACs in treatment of PVT in the presence
or absence of cirrhosis, it extends the armamentarium of safe
and efficacious options available for anticoagulation of these
patients.hf This review establishes that NOACs can be used effec-
tively and safely without any risk of increased bleeding events
in the treatment of PVT, even in patients with CPT class
A and B cirrhosis. This review also presents the evidence
that NOACs are already being increasingly used off label
for this indication despite the lack of robust data on their
safety and efficacy. This preliminary data may prompt better
quality studies in the future comparing traditional and newer
anticoagulants. Conflicts of Interest The authors declare that they have no conflicts of interest. Authors’ Contributions Traditionally, VKAs are bridged with unfractionated heparin
(UFH) or LMWH for five days. For treatment of VTE in
typical locations, NOACs do not routinely require heparin
bridging. At this time, the data regarding bridging NOACs
with UFH/LMWH is limited and is currently subject to the
physician preference.h P. Priyanka was responsible for topic selection, study concept
and design, acquisition of data, analysis and interpretation of
data, drafting of the manuscript, literature search, writing,
editing, and submitting the manuscript. J. T. Kupec con-
tributed to analysis and interpretation of data, drafting of the
manuscript, critical revision of the manuscript for important
intellectual content, and editing. M. Krafft was responsi-
ble for analysis and interpretation of data, drafting of the
manuscript, critical revision of the manuscript for important
intellectual content, and editing. N. A. Shah contributed to
critical revision of the manuscript for important intellectual
content and editing. G. J. Reynolds contributed to critical
revision of the manuscript for important intellectual content. The available studies and case reports support several
approaches to potentially utilizing another agent to bridge
patients before starting NOAC, and some of them do not
address bridging at all [8, 19]. NOAC was used as the initial
anticoagulant in one-third of patients without cirrhosis while
two-thirds were previously treated with traditional anticoag-
ulants, without any mention of bridging [8]. Other studies
did not bridge their patients with traditional anticoagulants
[9]. There are case reports in this review where patients were
bridged with heparin and some in which heparin was not
used [11, 23]. 17. Dosing of NOACs for PVT Patients The presence of cirrhosis may affect the dosing of NOACs. Yet
current data is unclear in determining the appropriate dose. In the VALDIG study, the median daily dose of all NOACs
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https://www.ams.org/proc/2011-139-05/S0002-9939-2010-10632-9/S0002-9939-2010-10632-9.pdf
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English
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Mertens’ theorem for toral automorphisms
|
Proceedings of the American Mathematical Society
| 2,011
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public-domain
| 3,086
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1. Introduction Discrete dynamical analogs of Mertens’ theorem are concerned with a map T :
X →X and are motivated by work of Sharp [6] on Axiom A flows. A set of the
form τ = {x, T(x), . . . , T k(x) = x} with cardinality k is called a closed orbit of length |τ| = k, and the results pro-
vide asymptotics for a weighted sum over closed orbits. For the discrete case of a
hyperbolic diffeomorphism T, we always have MT (N) :=
|τ|⩽N
1
eh|τ| ∼log N, where h is the topological entropy, with more explicit additional terms in many
cases. The main term log N is not really related to the dynamical system, but is
a consequence of the fact that the number of orbits of length n is 1
nehn + O(eh′n)
for some h′ < h (see [5]). Without the assumption of hyperbolicity, the asymp-
totics change significantly, and in particular depend on the dynamical system. For
quasihyperbolic (ergodic but not hyperbolic) toral automorphisms, Waddington [8]
found asymptotics for an unweighted orbit-counting sum, and Noorani [4] found an
analogue of Mertens’ theorem in the form where h is the topological entropy, with more explicit additional terms in many
cases. The main term log N is not really related to the dynamical system, but is
a consequence of the fact that the number of orbits of length n is 1
nehn + O(eh′n)
for some h′ < h (see [5]). Without the assumption of hyperbolicity, the asymp-
totics change significantly, and in particular depend on the dynamical system. For
quasihyperbolic (ergodic but not hyperbolic) toral automorphisms, Waddington [8]
found asymptotics for an unweighted orbit-counting sum, and Noorani [4] found an
analogue of Mertens’ theorem in the form (1)
MT (N) = m log N + C1 + o(1) MT (N) = m log N + C1 + o(1) (1) for some m ∈N. The constant C1 is related to analytic data coming from the
dynamical zeta function. For more general non-hyperbolic group automorphisms,
the coefficient of the main term may be non-integral (see [2] for example). In this note Noorani’s result (1) with error term O(N −1) is recovered using
elementary arguments, and the coefficient m of the main term in (1) is expressed
as an integral over a subtorus. PROCEEDINGS OF THE
AMERICAN MATHEMATICAL SOCIETY
Volume 139, Number 5, May 2011, Pages 1819–1824
S 0002-9939(2010)10632-9
Article electronically published on November 1, 2010 PROCEEDINGS OF THE
AMERICAN MATHEMATICAL SOCIETY
Volume 139, Number 5, May 2011, Pages 1819–1824
S 0002-9939(2010)10632-9
Article electronically published on November 1, 2010 PROCEEDINGS OF THE
AMERICAN MATHEMATICAL SOCIETY
Volume 139, Number 5, May 2011, Pages 1819–1824
S 0002-9939(2010)10632-9
Article electronically published on November 1, 2010 Received by the editors May 27, 2010.
2010 Mathematics Subject Classification. Primary 37C35, 11J72.
c
⃝2010 American Mathematical Society
Reverts to public domain 28 years from publication
1819 (Communicated by Bryna Kra) Abstract. A dynamical Mertens’ theorem for ergodic toral automorphisms
with error term O(N−1) is found, and the influence of resonances among the
eigenvalues of unit modulus is examined. Examples are found with many more,
and with many fewer, periodic orbits than expected. c
⃝2010 American Mathematical Society Reverts to public domain 28 years from publicat
819 ⃝
y
Reverts to public domain 28 years from publication
1819 2. Toral automorphisms Let T : Td →Td be a toral automorphism corresponding to a matrix AT
in GLd(Z) with eigenvalues {λi | 1 ⩽i ⩽d}, arranged so that |λ1| ⩾· · · ⩾|λs| > 1 = |λs+1| = · · · = |λs+2t| > |λs+2t+1| ⩾· · · ⩾|λd|. The map T is ergodic with respect to Lebesgue measure if no eigenvalue is a root
of unity, is hyperbolic if in addition t = 0 (that is, there are no eigenvalues of unit
modulus), and is quasihyperbolic if it is ergodic and t > 0. The topological entropy
of T is given by h = h(T) = s
j=1 log |λj|. Theorem 1. Let T be a quasihyperbolic toral automorphism with topological en-
tropy h. Then there are constants C2 and m ⩾1 with
|τ|⩽N
1
eh|τ| = m log N + C2 + O
N −1
. The coefficient m in the main term is given by m =
X
t
i=1
(2 −2 cos(2πxi)) dx1 . . . dxt, where X ⊂Td is the closure of {(nθ1, . . . , nθt) | n ∈Z}, and e±2πiθ1, . . . , e±2πiθt
are the eigenvalues with unit modulus of the matrix defining T. where X ⊂Td is the closure of {(nθ1, . . . , nθt) | n ∈Z}, and e±2πiθ1, . . . , e±2πiθt
are the eigenvalues with unit modulus of the matrix defining T. As we will see in Example 3, the quantity m appearing in Theorem 1 takes on a
wide range of values. In particular, m may be much larger, or much smaller, than
its generic value 2t. Proof. Since T is ergodic, FT (n) = |{x ∈Td | T n(x) = x}| = |Zd/(An
T −I)Zd| =
d
i=1
|λn
i −1|, so OT (n) = 1
n
m|n
μ(n/m)
d
i=1
|λm
i −1|. Write Λ = s
i=1 λi (so the topological entropy of T is log |Λ|) and Write Λ = s
i=1 λi (so the topological entropy of T is log |Λ|) and κ = min{|λs|, |λs+2t+1|−1} > 1. The eigenvalues of unit modulus contribute nothing to the topological entropy, but
multiply the approximation |Λ|n to FT (n) by an almost-periodic factor bounded
above by 22t and bounded below by A/nB for some A, B > 0, by Baker’s theorem
(see [3, Ch. 1. Introduction This reveals the effect of resonances between the
eigenvalues of unit modulus, and examples show that the value of m may be very
different from the generic value given in [4]. 1820
SAWIAN JAIDEE SHAUN STEVENS AND THOMAS WARD SAWIAN JAIDEE, SHAUN STEVENS, AND THOMAS WARD 1820 2. Toral automorphisms , s + 2t}
, SAWIAN JAIDEE, SHAUN STEVENS, AND THOMAS WARD 1822 write write I(ω) = {I ⊂{s + 1, . . . , s + 2t} |
i∈I
λi = ω},
K(ω) =
I∈I(ω)
(−1)|I|, I(ω) = {I ⊂{s + 1, . . . , s + 2t} |
i∈I
λi = ω}, K(ω) =
I∈I(ω)
(−1)|I|, and let m = K(1) (notice that I(ω) = ∅unless ω ∈Ω). Then Vn =
ω∈Ω K(ω)ωn,
so, by (4), and let m = K(1) (notice that I(ω) = ∅unless ω ∈Ω). Then Vn =
ω∈Ω K(ω)ωn,
so, by (4), and let m = K(1) (notice that I(ω) = ∅unless ω ∈Ω). Then Vn =
ω∈Ω K(ω)ωn,
so, by (4), MT (N)
=
N
n=1
1
n
ω∈Ω
K(ω)ωn + C5 + O
R−N
=
m
N
n=1
1
n +
ω∈Ω\{1}
K(ω)
N
n=1
ωn
n + C5 + O
R−N
=
m log N −
ω∈Ω\{1}
K(ω) log(1 −ω) + C6 + O(N −1), since N
n=1
1
n = log N + γ + O(N −1), and N
n=1
ωn
n
= −log(1 −ω) + O(N −1)
for ω ̸= 1 by the Abel continuity theorem and partial summation. ̸
If the eigenvalues of modulus one are e±2πiθ1, . . . , e±2πiθt, then Vn =
t
i=1
(1 −e2πiθin)(1 −e−2πiθin) =
t
i=1
(2 −2 cos(2πθin)) . Let X ⊂Tt be the closure of {(nθ1, . . . , nθt) | n ∈Z}, so that by the Kronecker–
Weyl lemma we have Let X ⊂Tt be the closure of {(nθ1, . . . , nθt) | n ∈Z}, so that by the Kronecker–
Weyl lemma we have 1
N
N
n=1
t
i=1
(2 −2 cos(2πθin)) −→
X
t
i=1
(2 −2 cos(2πxi)) dx1 . . . dxt as N →∞. Then, by partial summation, as N →∞. Then, by partial summation, N
n=1
1
nVn
=
N
n=1
1
n −
1
n + 1
n
m=1
Vm +
1
N + 1
N
m=1
Vm
∼
X
t
i=1
(2 −2 cos(2πxi)) dx1 . . . dxt
log N, □ so that m has the form stated. 2. Toral automorphisms 3] for this argument). Lemma 2. FT (n) −|Λ|n
s+2t
i=s+1
|λn
i −1|
· |Λ|−n = O(κ−n). Proof. We have (2)
d
i=1
(λn
i −1) =
s
i=1
(λn
i −1)
Un
s+2t
i=s+1
(λn
i −1)
Vn
d
i=2t+s+1
(λn
i −1)
Wn
, (2) MERTENS’ THEOREM FOR TORAL AUTOMORPHISMS 1821 where Un is equal to the sum of Λn and 2s −1 terms comprising products of eigen-
values, each no larger than κ−n|Λ|n in modulus, Wn is equal to the sum of (−1)d−s
and 2d−2t−s −1 terms bounded above in absolute value by κ−n, and |Vn| ⩽22t. It
follows that d
i=1(λn
i −1) −(−1)d−sΛn s+2t
i=s+1(λn
i −1)
|Λ|n
=
Vn
UnWn −(−1)d−sΛn
|Λ|n
=
|Vn (Λn + O (Λn/κn) −Λn)|
|Λ|n
=
O(κ−n). =
O(κ−n). The statement of the lemma follows by the reverse triangle inequality. □ Now MT (N) =
N
n=1
1
n|Λ|n
⎛
⎝FT (n) +
d|n,d<n
μ
n
d
FT (d)
⎞
⎠ and ∞
n=N
1
n|Λ|n
d|n,d<n
μ
n
d
FT (d)
⩽
∞
n=N
1
n · n · O(|Λ|−n/2) = O
|Λ|−N/2
, so there is a constant C3 for which N
n=1
1
n|Λ|n
d|n,d<n
μ
n
d
FT (d) −C3
= O
|Λ|−N/2
. Therefore, by Lemma 2 and using the notation from (2), Therefore, by Lemma 2 and using the notation from (2), MT (N) =
N
n=1
1
n
Vn + O
κ−n
+ C3 + O
|Λ|−N/2
. Clearly there is a constant C4 for which (3)
N
n=1
1
n O
κ−n
−C4
= O
κ−N
, (3) so by (2) and (3), (4)
MT (N) =
N
n=1
1
nVn + C3 + C4 + O(R−N), (4) where R = min{κ, |Λ|1/2}. Since the complex eigenvalues appear in conjugate pairs
we may arrange that λi+t = ¯λi for s + 1 ⩽i ⩽s + t, and then |λi −1||λi+t −1| = (λi −1)(λi+t −1). |λi −1||λi+t −1| = (λi −1)(λi+t −1). It follows that Vn = s+2t
i=s+1(λn
i −1). Put |λi −1||λi+t −1| = (λi −1)(λi+t −1). Ω =
i∈I
λi | I ⊆{s + 1, . . . 2. Toral automorphisms The exact value of m is determined by the structure of the group X, which in
turn is governed by additive relations among the arguments of the eigenvalues of
unit modulus. Here are some illustrative examples. Example 3. (a) If all the arguments θi are independent over Q (the generic case),
then X = Tt, so m=
1
0
· · ·
1
0
t
i=1
(2−2 cos(2πxi)) dx1 . . . dxt =
1
0
(2−2 cos(2πx1)) dx1
t
= 2t. MERTENS’ THEOREM FOR TORAL AUTOMORPHISMS
1823 MERTENS’ THEOREM FOR TORAL AUTOMORPHISMS 1823 (b) A simple example with m > 2t is the following. Let T2 be the automorphism
of T8 defined by the matrix A ⊕A, where (b) A simple example with m > 2t is the following. Let T2 be the automorphism
of T8 defined by the matrix A ⊕A, where (b) A simple example with m > 2t is the following. Let T2 be the automorphism
of T8 defined by the matrix A ⊕A, where (5)
A =
⎛
⎜
⎜
⎝
0
0
0
−1
1
0
0
8
0
1
0
−6
0
0
1
8
⎞
⎟
⎟
⎠. (5) Here X is a diagonally embedded circle, and Here X is a diagonally embedded circle, and m
=
{x1=x2}
2
j=1
(2 −2 cos(2πjxj)) dx1 dx2
=
1
0
(2 −2 cos(2πx))2 dx = 6 > 22. Extending this example, let Tn be the automorphism of T4n defined by the ma-
trix A⊕· · ·⊕A (n terms). The matrix corresponding to Tn has 2n eigenvalues with
modulus one (comprising two conjugate eigenvalues with multiplicity n). Then X
is again a diagonally embedded circle, and m
=
1
0
(2 −2 cos(2πx))t dx = (2t)! (t!)2
∼
22t
√
πt by Stirling’s formula. This is much larger than 2t, reflecting the density of the
syndetic set on which the almost-periodic factor is close to 22t. Indeed, this example
shows that m
2t may be arbitrarily large. 2
(c) A simple example with m < 2t is the following. Let S be the automorphism
of T12 defined by the matrix A⊕A2 ⊕A3, with A as in (5). SAWIAN JAIDEE, SHAUN STEVENS, AND THOMAS WARD SAWIAN JAIDEE, SHAUN STEVENS, AND THOMAS WARD 1824 that the automorphism S8n of T32n has m = 24n = 2t/2. Thus m
2t may be arbitrarily
small. that the automorphism S8n of T32n has m = 24n = 2t/2. Thus m
2t may be arbitrarily
small. We close with some remarks. (1) In the quasihyperbolic case, the O(1/N) term is oscillatory, so no improve-
ment of the asymptotic in terms of a monotonic function is possible. The extent
to which the exponential dominance of the entropy term fails in this setting is
revealed by the following. Let FT (n) denote the number of points fixed by the
automorphism T n. On the one hand, Baker’s theorem implies that FT (n)1/n →eh
as n →∞. On the other hand, Dirichlet’s theorem shows that FT (n + 1)/FT (n)
does not converge (see [1, Th. 6.3]). (2) The formula for m in the statement of [4, Th. 1] is incorrect in a minor way;
as stated in [4, Rem. 2] and as illustrated in the examples above, m should be K(1),
which is not necessarily the same as 2t. (3) The proof of Theorem 1 also gives an elementary proof of the asymptotics
in the hyperbolic case: in the notation of the proof, Vn = 1 so m = 1. The
Euler-MacLaurin summation formula gives an asymptotic of the shape
|τ|⩽N
1
eh|τ| = log N + C2 +
k−1
r=0
Br+1
(r + 1)N r+1 + O
N −(k+1)
, where B1 = −1
2, B2 = 1
6, . . . are the Bernoulli numbers, for any k ⩾1. where B1 = −1
2, B2 = 1
6, . . . are the Bernoulli numbers, for any k ⩾1. are the Bernoulli numbers, for any k ⩾1. 2. Toral automorphisms Again X is a diagonally
embedded circle, and m
=
{x1=x2=x3}
3
j=1
(2 −2 cos(2πjxj)) dx1dx2 dx3 m
=
{x1=x2=x3}
j=1
(2 −2 cos(2πjxj)) dx1dx2 dx3
=
1
0
(2 −2 cos(2πx))(2 −2 cos(4πx))(2 −2 cos(6πx)) dx = 6 < 23. =
1
0
(2 −2 cos(2πx))(2 −2 cos(4πx))(2 −2 cos(6πx)) dx = 6 < 23. Extending this example, the value of m for the automorphism of T4t defined by the
matrix A ⊕A2 ⊕· · · ⊕At as t varies gives the sequence Extending this example, the value of m for the automorphism of T4t defined by the
matrix A ⊕A2 ⊕· · · ⊕At as t varies gives the sequence Extending this example, the value of m for the automorph
matrix A ⊕A2 ⊕· · · ⊕At as t varies gives the sequence 2, 4, 6, 10, 12, 20, 24, 34, 44, 64, 78, 116, 148, 208, 286, 410, 556, 808, 1120, 1620, . . . (we thank Paul Hammerton for computing these numbers). This sequence, en-
try A133871 in the Encyclopedia of Integer Sequences [7], does not seem to be
readily related to other combinatorial sequences. (d) Generalizing the example in (c), for any sequence (an) of natural numbers,
we could look at the automorphisms Sn of T4n defined by the matrices n
k=1 Aak,
with A as in (5). In order to make m small, we need a “sum-heavy” sequence,
that is, one with many three-term linear relations of the form ai + aj = ak. More
precisely, one would like many linear relations with an odd number of terms, and
few with an even number of terms. Constructing such sequences and understanding
how dense they may be seems to be difficult. Taking (an) to be the sequence whose first eight terms are 1, 2, 3, 5, 7, 8, 11, 13
and whose subsequent terms are defined by the recurrence an+8 = 100an, we find References [1] V. Chothi, G. Everest, and T. Ward. S-integer dynamical systems: periodic points. J. Reine
Angew. Math. 489, 99–132, 1997. MR1461206 (99b:11089) [1] V. Chothi, G. Everest, and T. Ward. S-integer dynamical systems: periodic points. J. Reine
Angew. Math. 489, 99–132, 1997. MR1461206 (99b:11089) [2] G. Everest, R. Miles, S. Stevens, and T. Ward. Orbit-counting in non-hyperbolic dynamical
systems. J. Reine Angew. Math. 608, 155–182, 2007. MR2339472 (2008k:37042) [2] G. Everest, R. Miles, S. Stevens, and T. Ward. Orbit-counting in non-hyperbolic dynamical
systems. J. Reine Angew. Math. 608, 155–182, 2007. MR2339472 (2008k:37042) (
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[3] G. Everest and T. Ward. Heights of polynomials and entropy in algebraic dynamics. Springer-
Verlag, London, 1999. MR1700272 (2000e:11087) (
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[3] G. Everest and T. Ward. Heights of polynomials and entropy in algebraic dynamics. Springer-
Verlag, London, 1999. MR1700272 (2000e:11087) [4] Mohd. Salmi Md. Noorani. Mertens theorem and closed orbits of ergodic toral automorphi
Bull. Malaysian Math. Soc. (2) 22(2), 127–133, 1999. MR1787646 (2001k:37036) [5] A. Pakapongpun and T. Ward. Functorial orbit counting. J. Integer Sequences 12, Article
09.2.4, 2009. MR2486259 (2010c:37044) [6] R. Sharp. An analogue of Mertens’ theorem for closed orbits of Axiom A flows. Bol. Soc. Brasil. Mat. (N.S.) 21(2), 205–229, 1991. MR1139566 (93a:58142) [7] N. J. A. Sloane. An on-line version of the encyclopedia of integer sequences. Electron. J. Combin. 1: Feature 1, approx. 5 pp., 1994. www.research.att.com/~njas/sequences/. [8] S. Waddington. The prime orbit theorem for quasihyperbolic toral automorphisms. Monatsh. Math. 112, no. 3, 235–248, 1991. MR1139101 (92k:58219) Department of Mathematics, 123 Mittraphab Road, Khon Kaen University 40002,
Thailand School of Mathematics, University of East Anglia, Norwich NR4 7TJ, United King-
dom School of Mathematics, University of East Anglia, Norwich NR4 7TJ, United King-
dom
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Characterizing Unsafe Sexual Behavior among Factory Workers in the Context of Rapid Industrialization in Northern Vietnam
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International journal of environmental research and public health/International journal of environmental research and public health
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Characterizing Unsafe Sexual Behavior among
Factory Workers in the Context of Rapid
Industrialization in Northern Vietnam Bach Xuan Tran 1,2,*
, Tracy Vo 3, Anh Kim Dang 4, Quang Nhat Nguyen 1,4,5,6
, Giang Thu Vu 7,
Linh Gia Vu 7, Khanh Nam Do 1, Carl A. Latkin 2
, Cyrus S.H. Ho 8 and Roger C.M. Ho 9,10,11 1
Institute for Preventive Medicine and Public Health, Hanoi Medical University, Hanoi 100000, Vietnam;
quang.n.nguyen@alumni.duke.edu (Q.N.N.); donamkhanh@hmu.edu.vn (K.N.D.) 1
Institute for Preventive Medicine and Public Health, Hanoi Medical University, Hanoi 100000, Vietnam;
quang.n.nguyen@alumni.duke.edu (Q.N.N.); donamkhanh@hmu.edu.vn (K.N.D.) 2
Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA;
carl.latkin@jhu.edu 2
Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA;
carl.latkin@jhu.edu 3
Department of Sociomedical Sciences, Mailman School of Public Health, Columbia University, New
NY 10027, USA; tracy.vo@columbia.edu 3
Department of Sociomedical Sciences, Mailman School of Public Health, Columbia University, New Yo
NY 10027, USA; tracy.vo@columbia.edu 4
Institute for Global Health Innovations, Duy Tan University, Da Nang 550000, Vietnam;
kimanh.ighi@gmail.com g
g
5
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD 20892-9806, USA 6
UnivLyon, Université Claude Bernard Lyon 1, 69100 Villeurbanne, France 7
Center of Excellence in Evidence-based Medicine, Nguyen Tat Thanh University, Ho Chi Minh City 700
Vietnam; giang.coentt@gmail.com (G.T.V.); linh.coentt@gmail.com (L.G.V.) Center of Excellence in Evidence-based Medicine, Nguyen Tat Thanh University, Ho Chi Minh City 700000 Center of Excellence in Evidence based Medicine, Nguyen Tat Thanh University, Ho
Vietnam; giang.coentt@gmail.com (G.T.V.); linh.coentt@gmail.com (L.G.V.) 8
Department of Psychological Medicine, National University Hospital, Singapore 119074, Singapore;
cyrushosh@gmail.com 9
Center of Excellence in Behavioral Medicine, Nguyen Tat Thanh University, Ho Chi Minh City 700000,
Vietnam; pcmrhcm@nus.edu.sg 10
Department of Psychological Medicine, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore 119228, Singapore g p
g p
11
Institute for Health Innovation and Technology (iHealthtech), National University of Singapore, Singapore
119077, Singapore *
Correspondence: bach.ipmph@gmail.com; Tel.: +84-982228662 Received: 23 September 2019; Accepted: 3 December 2019; Published: 12 December 2019 Abstract: Industrial workers or factory workers, especially migrant workers, have been found to be
vulnerable populations at risk of sexually transmitted infections (STIs). However, there has been a gap
in literature regarding health behaviors of migrant factory workers. We conducted a cross-sectional
study among 230 factory workers in Hanoi and Bac Ninh cities in Northern Vietnam from July to
September 2018 to identify sexual risk practices and related factors among migrant and nonmigrant
factory workers. International Journal of
Environmental Research
and Public Health Int. J. Environ. Res. Public Health 2019, 16, 5085; doi:10.3390/ijerph16245085 www.mdpi.com/journal/ijerph 1. Introduction The majority of migrated people in Vietnam are young adults with middle-level education status
and they migrate to more urban areas mainly for employment reasons [1]. Previous studies indicated
that moving to a new workplace may expose these migrant workers to new social networks, ideas,
behaviors, or even socially isolated situations that could alter their lifestyle choices, including engaging
in sexual risk behaviors [2]. Migrant workers are often subjected to harassment, exploitation, and
violence, which may lead to poor health outcomes, and they may also face barriers to health care
in the host country, such as limited or lack of health insurance and entitlement to statutory health
care [3]. Migration, mobility, and family separation all relate to having a greater sense of anonymity,
which might further facilitate unsafe behavioral changes, such as hazardous drinking and high-risk
sexual practices [4,5]. In fact, among mobile worker populations in selected provinces in Vietnam
in 2002, human immunodeficiency virus (HIV) prevalence was relatively low except among border
traders in An Giang and Dong Thap provinces, which saw 2.5% and 2.1% prevalence, respectively [6]. Though this study looked at a limited group of mobile workers, it is suggested that the mobility of
sex work across the border contributed to the high prevalence of HIV among border traders. In other
low-middle-income countries like Vietnam, such as India and Thailand, factory workers and male
migrant workers, respectively, are among the most vulnerable populations to HIV/AIDS (acquired
immune deficiency syndrome) and other sexually transmitted infections (STIs) [7,8]. Migrant workers
generally are at greater risk of heterosexual HIV transmission due to their demographic characteristics,
such as lack of access to HIV treatment and prevention, unstable family and work situations, and
access to sex workers [9]. Previous studies have found that risk perception and knowledge of consequences of unprotected
sex, including HIV/AIDS, are generally low among younger migrant factory workers. For instance, in
a study done in Nepal, it was reported that 90.2% of males and 41.7% of females who had nonregular
sexual partners perceived having no risk of HIV/AIDS [10]. Another study conducted among migrant
workers in manufacturing, construction, accommodation/catering, domestic service, wholesale/retail,
and entertainment fields in China revealed that the majority of them did not use condoms while
engaging in sexual intercourse, and about two-thirds of participants never or only occasionally used
condoms with their casual extramarital partners within the last 12 months [11]. Keywords: industrial worker; factory worker; sexually transmitted infections; condom use; sexual
risk behavior; Vietnam Keywords: industrial worker; factory worker; sexually transmitted infections; condom use; sexual
risk behavior; Vietnam Characterizing Unsafe Sexual Behavior among
Factory Workers in the Context of Rapid
Industrialization in Northern Vietnam Information collected regarding sexual behavior included the number of sexual
partners in the previous 12 months and whether they used condoms in their last sexual intercourse. Two-thirds of participants reported having no sexual activity in the last 12 months, and there was a
low percentage of participants using condoms in their last sexual intercourse. Being female, living
with spouses/partners, and being a nonimmigrant had a negative association with the lack of using
condoms in the last sexual intercourse with casual partners/sex workers, as opposed to having
mobility and self-care problems and identifying as a binge drinker. Therefore, workplace-based
prevention programs focusing on providing tailored sexual health education and promoting condom
use among industrial workers, especially those who are immigrant or migrant workers, in Vietnam
should be emphasized. Abstract: Industrial workers or factory workers, especially migrant workers, have been found to be
vulnerable populations at risk of sexually transmitted infections (STIs). However, there has been a gap
in literature regarding health behaviors of migrant factory workers. We conducted a cross-sectional
study among 230 factory workers in Hanoi and Bac Ninh cities in Northern Vietnam from July to
September 2018 to identify sexual risk practices and related factors among migrant and nonmigrant
factory workers. Information collected regarding sexual behavior included the number of sexual
partners in the previous 12 months and whether they used condoms in their last sexual intercourse. Int. J. Environ. Res. Public Health 2019, 16, 5085; doi:10.3390/ijerph16245085 www.mdpi.com/journal/ijerph 2 of 11 Int. J. Environ. Res. Public Health 2019, 16, 5085 1. Introduction Studies among workers
in Bangladesh, India, and Jordan also saw similar results of high levels of risky sexual practices [4,7,12]. Furthermore, a previous study conducted in Hanoi, Vietnam, found a considerable knowledge gap in
HIV/AIDS prevention among male migrant workers [13]. Lack of HIV knowledge and a variety of
essential health care resources were highlighted particularly among vulnerable groups, including those
who were mostly low-income, employed outside of labor contracts, and unregistered for residence [13]. In the context of Vietnam, the country is rapidly transitioning into an industrial country with an
increasing number of workers [14]. However, the provisions of protecting workers’ rights in using
universal health coverage and accessing health services are still limited for both those who are outside
labor contracts and within. Moreover, there is little evidence of literature on the high-risk sexual
practices among factory workers and the underlying factors of such activities in Vietnam. Therefore, we
aimed to identify the risk of sexual practices as well as related factors among factory workers in Hanoi
and Bac Ninh, which are the two major cities with an increasing rate of geographic labor mobility. These
findings may fill in the gap of HIV/AIDS and STI prevention of industrial workers and are intended to
promote the national policy to meet the health care needs of this critical working population. 3 of 11 Int. J. Environ. Res. Public Health 2019, 16, 5085 2.2.1. Socioeconomic Characteristics Respondents self-reported information regarding gender, age, educational level, marital status,
monthly income, and whether they were living with their families. Information about immigration
status, years of work experience, and the number of working hours per day was also collected. 2.2.2. Sexual Behaviors Participants were asked whether they had had sexual intercourse in the last 12 months. Those
who answered “yes” were asked to report “how many partners that they had sexual intercourse with
in the previous 12 months”, including intimate partners, sex workers, and casual partners (receiving
and without receiving money). We also asked whether or not they used condoms in their last sexual
intercourse with these partners. 2.1. Study Design and Sampling We carried out a cross-sectional study in three factories manufacturing electronics and vehicle
accessories from July to September 2018 in Hanoi and Bac Ninh. In each factory, there were
approximately 6000, 2000, and 1150 workers. We used the convenient sampling technique to select participants of the study. A total of 230
workers were recruited in the study based on the following eligibility criteria: (1) Being 18 years of age
and above; (2) having signed labor contracts; (3) having worked at the factory for at least 6 months;
and (4) having the capacity to answer the questionnaire. Workers who were not able to communicate
with the interviewers were excluded from the research. 2.2. Measure and Instruments Trained researchers conducted 20-min face-to-face interviews with participants. Briefly, we invited
participants into a private working office with restricted access in order to secure their confidentiality. Study purposes, benefits, and drawbacks were clearly introduced before asking participants to join the
study. Participants who agreed to take part in the study would sign written informed consent forms. Data collectors were researchers who had undergone research and ethics training to conduct
interviews and collect data. Staffmembers of the factories were not allowed to participate in the
data collection process. We ensured that questionnaires were anonymous by assigning participants a
randomized ID number, and any information related to a participant’s identity and contact information
was not collected during the research process. A pilot survey was conducted prior to the data collection to examine the feasibility of recruitment
and identify any needed modifications. There were 20 participants (both male and female with varied
ages) involved in the pilot stage. The final structured questionnaire included the following information: 2.3. Statistical Analysis Data were analysed using STATA 12.0 (Stata Corp. LP, College Station, TX, USA). Socioeconomic
characteristics, health risk behavior, and health status were compared between those using and not
using a condom in the last sexual intercourse with casual partners and/or sex workers. Generalized
estimating equations (GEE) binomial regression was conducted to identify the factors related to not
using a condom with casual partners and/or sex workers. GEE was used to control the potential
correlation of outcomes between respondents from the same factory, assuming an independent
correlation structure. The robust standard errors were estimated, adjusting for the clustering within
factories. A p-value less than 0.05 was statistically significant. 2.2.3. Health Risk Behaviors Drinking pattern was examined using the Alcohol Use Disorders Identification Test-Consumption
(AUDIT-C) [15]. This tool contained 3 questions with the overall score ranging from 0 to 12. The
higher score of AUDIT-C suggested a greater risk of alcohol dependence. Drinking patterns were
also determined. Participants were identified as hazardous drinkers if the total score of AUDIT-C
was higher than 4 for males and higher than 3 for females. Binge drinkers were identified as having
any positive response to the following question: “How often do you have six or more drinks on one
occasion?” We divided the smoking status of participants into 3 types: Current smokers, former smokers,
and never-smokers [16]. A current smoker was an adult who had smoked at least 100 cigarettes in his
or her lifetime and had smoked in the last 30 days. The former smoker was identified as an adult who 4 of 11 Int. J. Environ. Res. Public Health 2019, 16, 5085 had smoked at least 100 cigarettes in his or her lifetime, but had quit smoking at the time of interview. A person who had never smoked, or who had smoked less than 100 cigarettes in his or her lifetime was
defined as a never-smoker. 2.4. Ethics Approval The research protocol was approved by the Institutional Review Board of the Hanoi Medical
University (code: 01a-QD/VNCTN). 2.2.4. Health Status Health-related quality of life (HRQoL) was assessed by the Vietnamese version of the EQ-5D-5L
questionnaire [17]. There were five health aspects that were measured, including mobility, self-care,
usual activities, pain/discomfort, and anxiety and/or depression. Each domain was assessed by
a Likert scale (no problems, slight problems, moderate problems, severe problems, and extreme
problems). Respondents also self-reported their current acute and/or chronic conditions, the number
of health problems they had had, and whether they had utilized reproductive health services in the
last 12 months. 3. Results Table 1 highlights the demographic characteristics, general health status, and drinking/smoking
practices of the participants. The majority of the participants were female (81.2%) and about two-thirds
of respondents had high school education (61%). Almost all of the participants (96.5%) had a spouse or
partner. The mean monthly income was $282.40 (SD = 106.5), and the mean working time was 9.8
years (SD = 3.7). There were 16.6% and 15.7% of participants who were considered hazardous drinkers
and binge drinkers, respectively. The majority of participants were never-smokers (85.5%) and had
acute or chronic conditions (84.2%). Approximately half of participants had used reproductive health
services (45.0%). 5 of 11 Int. J. Environ. Res. Public Health 2019, 16, 5085 Table 1. Demographic characteristics, general health status, and behaviors of participants (n = 230). Characteristics
n
%
Gender (Female) (n = 230)
186
81.2
Education attainment (n = 228)
Under high school
17
7.5
High school
139
61.0
Above high school
72
31.5
Marital status (n = 230)
Single
8
3.5
Having spouse/partner
222
96.5
Currently living with (n = 228)
Parents
115
50.4
Wife/husband
187
82.0
Children
139
61.0
Brothers/sisters
18
7.9
Relatives
2
0.9
Colleague
2
0.9
Immigrants/migrant workers (n = 229)
117
51.1
Having problems in (n = 230)
Mobility
76
34.1
Self-care
7
3.2
Usual activities
73
32.7
Pain/Discomfort
132
61.4
Anxiety/Depression
135
62.8
Having acute and/or chronic conditions (n = 222)
187
84.2
Using reproductive health service in the last 12
months (n = 220)
99
45.0
Hazardous drinking (n = 223)
37
16.6
Binge drinking (n = 223)
35
15.7
Current smoking status (n = 214)
Never-smokers
183
85.5
Former smokers
14
6.6
Current smokers
17
7.9
Mean
SD
Age
31.7
4.5
Monthly income (USD)
282.4
106.5
Years of experience
9.8
3.7
Working hour per day
8.3
0.9
Number of acute and/or chronic conditions
1.8
1.7 Table 1. Demographic characteristics, general health status, and behaviors of participants (n = 230). Table 2 shows that, among the approximately two-thirds of participants who had sex in the last 12
months (67.8%), 64.8% had one sex partner. Almost half of the respondents reported using condoms
when they had sex with their spouse/intimate partners in their last sexual intercourse (42.6%). 3. Results Among
those who had sexual intercourse with sex workers, casual partners without receiving money, and
casual partners while receiving money, 38.3%, 39.3%, and 43.9%, respectively, reported using condoms
in their last sexual intercourse. 6 of 11 Int. J. Environ. Res. Public Health 2019, 16, 5085 Table 2. Sexual behaviors in the last 12 months among participants. Characteristic
Local People
(n = 113)
Migrants
(n = 117)
Total
p-value
n
%
n
%
n
%
Having sex (n = 224 )
71
64.6
77
67.5
148
66.1
0.80
Number of sex partners (all types) (n = 225)
None
20
18.2
17
14.9
38
16.5
0.83
One sex partner
69
62.7
76
66.7
145
64.8
Two sex partners or more
2
1.8
1
0.9
3
1.3
Don’t remember
19
17.3
20
17.5
39
17.4
Using condom in the last sexual intercourse with
Spouse/intimate partners (n = 214)
47
46.5
49
43.4
96
44.9
0.64
Yes
47
46.5
49
43.4
96
44.9
0.64
No
54
53.5
64
56.6
118
55.1
Sex workers (n = 177)
Yes
11
12.8
7
7.7
18
10.2
0.15
No
16
18.6
10
11.0
26
14.7
Do not remember
59
68.6
74
81.3
133
75.1
Casual partners without receiving money (n = 187)
Yes
12
13.5
8
8.2
20
10.7
0.44
No
16
18.0
16
16.3
32
17.1
Do not remember
61
68.5
74
75.5
135
72.2
Casual partners while receiving money (n = 178)
Yes
11
12.9
6
6.5
17
9.5
0.23
No
11
12.9
9
9.7
20
11.2
Do not remember
63
74.1
78
83.9
141
79.2 Table 2. Sexual behaviors in the last 12 months among participants. Table 3 assesses the factors related to not using condoms with sex workers and/or casual partners. Workers who were female, had completed high school education or above, or were living with a
spouse/partner were less likely to not use condoms with casual partners or sex workers. By contrast,
workers who were immigrants or migrants, considered to be binge drinkers and reported having
mobility and self-care problems, were more likely to not use condoms in their last sexual intercourse
with casual partners or sex workers. Table 3. Factors associated with not using a condom with casual partners or sex workers (n = 208). Table 3. Factors associated with not using a condom with casual partners or sex workers (n = 208). *** p < 0.01, ** p < 0.05, * p < 0.1. 4. Discussion This research is among the few studies that examine the sexual risk practices among factory
workers in Northern Vietnam. We reported a low percentage of condom use among the workers in
their last sexual intercourse with sex workers and/or casual partners. In addition, we found that being
a female worker, living with spouses/partners, and being a local (and not a migrant) worker were
negatively associated with no condom usage in the last sexual intercourse with casual partners or sex
workers. By contrast, having problems in mobility, having self-care problems, and being a binge drinker
were positively associated with a lack of condom usage in the last sexual intercourse with sex workers
and/or casual partners. Our results provide evidence supporting the enhanced implementation of
critical sexual health interventions for people working in industrial zones in Northern Vietnam. In this study, we found a low percentage of participants using condoms in their last sexual
intercourse with sex workers and/or casual partners. This finding is lower than the result of a previous
study in India, which found that nearly 60% of workers used condoms in their last sexual intercourse
with nonspousal partners [7]. However, when compared to the percentages of condom usage among
migrant workers and their nonspousal partners in another study in India, our study showed higher
percentages of condom usage with nonspousal, unpaid partners, but not with sex workers [18]. Another
study done in China showed that returning rural-to-urban migrant workers had an elevated risk of
risky sexual behaviors over nonmigrant workers [19], as our study also indicates. In spite of Vietnam’s
large scale “100% condom use program”, which directly promoted the use of condoms especially in
sex work as part of the National Strategy on HIV/AIDS Prevention and Control [20], our findings show
that a high percentage of factory workers engaged in high-risk sexual activities. This is concerning
given that unprotected sex is considered one of the main risks of infecting HIV via heterosexual
intercourse [21]. Further analysis of factors underlying condom use preferences among industrial
workers may provide important insight into developing more effective promotion of condom use in
this high-risk population. g
p p
Our study suggested that female workers and those who were living with spouses or partners
were less likely to not use condoms in their last sexual intercourse. 3. Results Characteristics
Not Using Condom with Casual Partners/Sex
Workers in the Last Sexual Intercourse
OR
95% CI
p-value
Gender (Female vs. Male)
0.52 ***
0.34; 0.79
0.00
Education (vs. Less than high school)
High school
0.12 ***
0.03; 0.43
0.00
Above high school
0.14 **
0.02; 0.98
0.05
Marital status (Living with spouse/partner vs. Single)
0.19 **
0.04; 0.96
0.05
Immigrants/Migrants (Yes vs. No)
2.49 ***
1.60; 3.88
0.00
Age
1.06 **
1.01; 1.11
0.03
Having problems in
Mobility problems (Yes vs. No)
2.84 ***
1.36; 5.93
0.01
Self-care problems (Yes vs. No)
4.85 ***
2.48; 9.47
0.00
Usual activities problems (Yes vs. No)
0.52 *
0.26; 1.04
0.06
Pain/Discomfort (Yes vs. No)
2.13
0.26; 17.56
0.48
Anxiety/Depression (Yes vs. No)
0.93
0.42; 2.10
0.87
Having acute or chronic conditions (Yes vs. No)
0.94 ***
0.89; 0.98
0.01
Using reproductive health service in last 12 months (Yes vs. No)
1.47
0.57; 3.77
0.42
Hazardous drinking (Yes vs. No)
0.30
0.04; 2.09
0.23
Binge drinking (Yes vs. No)
3.97 **
1.12; 4.15
0.03
Current smoking status (vs. Never-smokers)
Former smokers
1.06
0.54; 2.09
0.87
Current smokers
0.37 **
0.15; 0.91
0.03
*** p < 0.01, ** p < 0.05, * p < 0.1. 7 of 11 Int. J. Environ. Res. Public Health 2019, 16, 5085 7 of 11 4. Discussion First, in terms of marital status,
a previous study done in Chiang Mai, Thailand, showed that factory workers without a cohabiting
partner were more likely to engage in higher-risk sexual activity, as compared to those with a cohabiting
partner [21]. Moreover, migrant workers living alone were at greater risk of sexual behaviors potentially
exposing them to HIV transmission [22,23]. Regarding the association between being a female worker
and condom use, it should be noted that most of our study participants were female, which could
have impacted our results [24,25]. In fact, a woman’s judgment of her own ability to negotiate and
practice a given sexual behavior has been found to be a strong predictor for condom use in the last
sexual intercourse with her husband [26]. Similarly, another study found that the more a female
student perceived that women were subordinate to men, the lower a female student’s self-efficacy
related to sexual communication, which involves communicating preference for condom use with
their partners [27]. Moreover, a study conducted among factory workers in Chiang Mai, Thailand,
revealed that sexual behaviors are normalized among men, but not women among Chiang Mai factory
workers [21]. Lastly, a high proportion of female migrant workers in the industrial parks in Vietnam
considered condoms as a contraceptive method as well as preventing sexually transmitted diseases [28]. In contrast, we found that migrant and immigrant workers had higher odds of not using condoms
in the last sexual intercourse with sex workers and/or casual partners. Sexual behaviors of migrants and
immigrants often changes upon moving, and the feeling of anonymity in a new region or country may
increase the likelihood of participating in risky sexual activities, such as alcohol abuse, multiple sex
partners, or having sexual intercourse with commercial sex workers [11,29]. Studies in the past further
indicated that such high-risk sexual activities might be the social-psychological characteristics of some
migrant workers. Specifically, migrant workers, who are often forced into physically demanding jobs
with poor living conditions and little to no benefits while having to live apart from their spouses
and families, may develop new sexual relationships and engage in high-risk behaviors that may 8 of 11 Int. J. Environ. Res. Public Health 2019, 16, 5085 increase the probability of HIV infection [30,31]. For example, Chen et al. 4. Discussion emphasized the relationship
between alcohol use risk and a series of sexual risk behaviors (early sexual debut, not utilizing
condoms, and having sex under the impact of alcohol) in China, but saw this among female sex
workers [32]. Furthermore, as found in our study, the co-occurrence of alcohol use and unprotected
sex has been reported in other settings [33–36]. Aside from sexual risk behaviors and other risky
behaviors, migration status remained significant even when we accounted for certain mental health
status indicators, such as anxiety and depression and having self-care problems. This suggests that
factors related to a worker’s identity as a migrant or immigrant affects condom use and other unsafe
sexual behaviors. In addition, health status was another factor we saw associated with low condom use. Agreeing
with previous findings [37], we found that those who reported having mobility issues or self-care
problems had higher odds of not using condoms with casual partners and/or sex workers. Our study’s
results with health status indicators were similar to those of Nguyen et al. where drug use and sexual
risk behavior among male laborers were associated with psychological factors, social integration,
and social barriers [13]. Having physical and mental health problems, in fact, have been found to be
associated with unsafe sexual activity [38,39], which may cause psychosocial and cognitive impairment
and reduce one’s capacity to avoid engaging in risky behaviors [40]. Our findings emphasized the importance of implementing workplace-based health programs that
focus on providing tailored education to prevent risky sexual behaviors. These programs should be
free and targeted to promote condom use among industrial workers in Vietnam. As the majority of
workers are low-wage and have limited formal education, these prevention programs should keep in
mind the health literacy of workers and should approach workers and their families through broader
public health messages. Additionally, an enhanced alcohol abuse screening program may help facilitate
safe sex practices among workers. Such programs should be targeted at populations that are at higher
risk of engaging in unprotected sexual activities, such as single male workers, migrant workers, and
workers with mobility issues and/or self-care problems. A preliminary study evaluated the behavioral
outcomes of an HIV counseling and testing service that integrated sexual and reproductive health
services for young people living in industrial zones in Vietnam [41]. 4. Discussion This study found that willingness
to obtain an HIV test increased significantly and saw changes towards more positive knowledge,
attitudes, and risk perception towards HIV/AIDS [41]. Results from our study also provided critical
evidence for assessing sexual health risk behavior of industrial workers, which may help to promote
national policies to meet the health care needs of this critical working population. It should also be noted that our study has several limitations. First, the cross-sectional design
constrained our ability to establish causal relationships regarding the associated factors and sexual
risk behaviors. Second, convenience sampling may have reduced the generalizability of the study
results to a larger factory worker population. In addition, our sample size of 230 factory workers from
three different factories in Northern Vietnam may not have fully represented all factory workers in the
country. Finally, self-reporting sensitive behaviors, such as alcohol use, self-care issues, mental illness,
and risky sexual habits, may have been underreported due to the participants’ social desirability and
recall bias. These behaviors may be underreported due to a worker’s fear of job loss or potential
consequences about one’s behaviors, despite this study’s confidentiality and anonymity. References 1. Coxhead, I.; Cuong, N.V.; Vu, L.H. Migration in Vietnam: New Evidence from Recent Surveys; Vietnam
Development Economics Discussion Papers; World Bank: Hanoi, Vietnam, 2015. 1. Coxhead, I.; Cuong, N.V.; Vu, L.H. Migration in Vietnam: New Evidence from Recent Surveys; Vietnam
Development Economics Discussion Papers; World Bank: Hanoi, Vietnam, 2015. 2. Huang, W.; Dong, Y.; Chen, L.; Song, D.; Wang, J.; Tao, H.; Zaller, N.; Zhang, H.; Operario, D. Hiv r
sexual health among female migrants in china. Health Promot. Int. 2016, 31, 595–605. [CrossRef] [Pu 3. Hargreaves, S.; Rustage, K.; Nellums, L.B.; McAlpine, A.; Pocock, N.; Devakumar, D.; Aldridge, R.W.;
Abubakar, I.; Kristensen, K.L.; Himmels, J.W.; et al. Occupational health outcomes among international
migrant workers: A systematic review and meta-analysis. Lancet Glob. Health 2019, 7, e872–e882. [CrossRef] 3. Hargreaves, S.; Rustage, K.; Nellums, L.B.; McAlpine, A.; Pocock, N.; Devakumar, D.; Aldridge, R.W.;
Abubakar, I.; Kristensen, K.L.; Himmels, J.W.; et al. Occupational health outcomes among international
migrant workers: A systematic review and meta-analysis. Lancet Glob. Health 2019, 7, e872–e882. [CrossRef] 4. Al Rifai, R.; Nakamura, K.; Seino, K.; Kizuki, M.; Morita, A. Unsafe sexual behaviour in domestic and
foreign migrant male workers in multinational workplaces in Jordan: Occupational-based and behavioural
assessment survey. BMJ Open 2015, 5, e007703. [CrossRef] [PubMed] 4. Al Rifai, R.; Nakamura, K.; Seino, K.; Kizuki, M.; Morita, A. Unsafe sexual behaviour in domestic and
foreign migrant male workers in multinational workplaces in Jordan: Occupational-based and behavioural
assessment survey. BMJ Open 2015, 5, e007703. [CrossRef] [PubMed] 5. Pham, K.T.H.; Nguyen, L.H.; Vuong, Q.H.; Ho, M.T.; Vuong, T.T.; Nguyen, H.T.; Vu, G.T.; Nguyen, H.L.T.;
Tran, B.X.; Latkin, C.A.; et al. Health inequality between migrant and non-migrant workers in an industrial
zone of vietnam. Int. J. Environ. Res. Public Health 2019, 16, 1502. [CrossRef] [PubMed] 5. Pham, K.T.H.; Nguyen, L.H.; Vuong, Q.H.; Ho, M.T.; Vuong, T.T.; Nguyen, H.T.; Vu, G.T.; Nguyen, H.L.T.;
Tran, B.X.; Latkin, C.A.; et al. Health inequality between migrant and non-migrant workers in an industrial
zone of vietnam. Int. J. Environ. Res. Public Health 2019, 16, 1502. [CrossRef] [PubMed] 6. Tuan, N.A.; Fylkesnes, K.; Thang, B.D.; Hien, N.T.; Long, N.T.; Kinh, N.V.; Thang, P.H.; Manh, P.D.;
O’Farrell, N. Human immunodeficiency virus (hiv) infection patterns and risk behaviours in different
population groups and provinces in viet nam. Bull. World Health Organ. 2007, 85, 35–41. [CrossRef] 7. Abdulkader, R.S.; Goswami, K.; Rai, S.K.; Misra, P.; Kant, S. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: We would like to thank all workers in the three factories manufacturing electronics
vehicle accessories in Hanoi and Bac Ninh for supporting us in this study. Acknowledgments: We would like to thank all workers in the three factories manufacturing electronics and
vehicle accessories in Hanoi and Bac Ninh for supporting us in this study. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions Our findings highlight the high percentage of workers in the sampled factories in Northern
Vietnam who did not use condoms during their last sexual intercourse with sex workers and/or casual
partners. Several characteristics that were associated with such risky sexual behavior included being
immigrant and migrant workers, identifying as a binge drinker, and having mobility and self-care
problems. Future workplace-based sexually transmitted infection prevention programs should focus
on enhanced sexual education and promotion of safe sex practices among these high-risk populations. 9 of 11 Int. J. Environ. Res. Public Health 2019, 16, 5085 Author Contributions: Conceptualization, B.X.T., T.V., A.K.D., Q.N.N., C.A.L., and C.S.H.H.; Data curation, B.X.T.,
A.K.D., G.T.V., L.G.V., K.N.D., C.S.H.H., and R.C.M.H.; Formal analysis, Q.N.N. and G.T.V.; Investigation, T.V.,
A.K.D., and G.T.V.; Methodology, B.X.T., T.V., A.K.D., L.G.V., K.N.D., C.A.L., and R.C.M.H.; Project administration,
B.X.T.; Software, L.G.V.; Supervision, B.X.T. and C.A.L.; Validation, Q.N.N., K.N.D., C.S.H.H., and R.C.M.H.;
Visualization, G.T.V.; Writing—original draft, T.V., A.K.D., and Q.N.N.; Writing—review and editing, B.X.T.,
Q.N.N., C.A.L., C.S.H.H., and R.C.M.H. Author Contributions: Conceptualization, B.X.T., T.V., A.K.D., Q.N.N., C.A.L., and C.S.H.H.; Data curation, B.X.T.,
A.K.D., G.T.V., L.G.V., K.N.D., C.S.H.H., and R.C.M.H.; Formal analysis, Q.N.N. and G.T.V.; Investigation, T.V.,
A.K.D., and G.T.V.; Methodology, B.X.T., T.V., A.K.D., L.G.V., K.N.D., C.A.L., and R.C.M.H.; Project administration,
B.X.T.; Software, L.G.V.; Supervision, B.X.T. and C.A.L.; Validation, Q.N.N., K.N.D., C.S.H.H., and R.C.M.H.;
Visualization, G.T.V.; Writing—original draft, T.V., A.K.D., and Q.N.N.; Writing—review and editing, B.X.T.,
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Khan, M.R.; Kaufman, J.S.; Pence, B.W.; Gaynes, B.N.; Adimora, A.A.; Weir, S.S.; Miller, W.C. Depression,
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Synthesis, crystal structure and thermal properties of di-μ-iodido-bis[bis(2-chloropyrazine-κ<i>N</i>)copper(I)]
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Acta crystallographica. Section E, Crystallographic communications
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cc-by
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Synthesis, crystal structure and thermal properties
of di-l-iodido-bis[bis(2-chloropyrazine-jN)-
copper(I)] ISSN 2056-9890 Christian Na¨ther* and Inke Jess Institut fu¨r Anorganische Chemie, Universita¨t Kiel, Max-Eyth.-Str. 2, 24118 Kiel, Germany. *Correspondence e-mail:
cnaether@ac.uni-kiel.de Received 16 January 2023
Accepted 10 February 2023 Received 16 January 2023
Accepted 10 February 2023 Received 16 January 2023
Accepted 10 February 2023 Reaction of copper(I) iodide in pure 2-chloropyrazine leads to the formation of
a few crystals of the title compound, [Cu2I2(C4H3ClN2)4] or (CuI)2(2-chloro-
pyrazine)4, which was characterized by single-crystal X-ray diffraction. In its
crystal structure, the CuI cations are each tetrahedrally coordinated by two
iodide anions and two 2-chloropyrazine ligands and are linked into binuclear
complexes consisting of (CuI)2 rings located on centers of inversion. PXRD
investigations of a few crystals obtained from the suspension indicate that the
title compound is contaminated with a small amount of the 2-chloropyrazine-
deficient compound CuI(2-chloropyrazine) already reported in the literature. PXRD investigations prove that the title compound immediately decomposes at
room temperature into CuI(2-chloropyrazine) and this might be the reason why
no pure samples can be obtained. TDA–TG–MS investigations shows two mass
losses, the first of which corresponds to the formation of CuI(2-chloropyrazine),
whereas in the second mass loss CuI is formed. Edited by V. Jancik, Universidad Nacional
Auto´noma de Me´xico, Me´xico Keywords: synthesis; crystal structure; binuclear
complex; thermal properties. CCDC reference: 2241117 research communications research communications research communications The cations are linked into double chains via
bridging iodide anions (Fig. S1). If the chloride and the
bromide compounds are heated, all 2-chloropyrazine ligands
are removed in a single step, leading to the formation of CuX
(X = Cl, Br). In contrast, the iodide compound decomposes in
two discrete steps, where in the first step only half of the
coligands are removed, leading to the formation of (CuI)2(2-
chloropyrazine), which decomposes into CuI upon further
heating (Na¨ther, Greve & Jess, 2002). To check if such a compound can be synthesized, all three
copper(I) halides were reacted in different solvents with a
very large excess of 2-chloropyrazine, but no new crystalline
phases were observed. On the contrary, if CuI is reacted as a
suspension in pure 2-chloropyrazine, yellow-colored crystals
of a new crystalline phase are obtained. In contrast, with CuCl
or CuBr only the known compounds CuX(2-chloropyrazine)
with X = Cl, Br are obtained. Single-crystal structure analysis
proved that a new compound with the composition (CuI)2(2-
chloropyrazine)4 has been obtained. 1. Chemical context 1. Chemical context Coordination compounds based on transition-metal halides
show a versatile structural behavior, which is observed parti-
cularly in compounds that contain CuI cations (Kromp &
Sheldrick, 1999; Peng et al., 2010; Li et al., 2005; Na¨ther & Jess,
2004). These compounds are also of interest because of their
luminescence behavior (Gibbons et al., 2017; Mensah et al.,
2022). For one given metal halide CuX (X = Cl, Br, I) and one
specific neutral coligand, several compounds are usually
observed that differ in the ratio between the metal halide and
the coligand – this is the reason why so many compounds with
different CuX (X = Cl, Br, I) substructures (such as, for
example, dimers, single and double chains or layers) are
observed that can be further connected into more condensed
networks if bridging neutral coligands are used in the synth-
esis. In general, it is observed that with decreasing amounts of
the coligand, the synthesis leads to the formation of
compounds with more condensed CuX substructures. In this
context, it is noted that upon heating, the most coligand-rich
compounds usually lose their coligands stepwise and trans-
form into coligand-deficient phases and that this is not limited
to CuI, but can also be expanded to CdII and ZnII compounds
(Na¨ther et al., 2001, 2007, 2017; Na¨ther & Jess, 2001). This can
easily be investigated by thermogravimetry of the most
coligand-rich compounds, where each mass loss corresponds
to the formation of a new coligand-deficient phase with a
more condensed CuX substructure. Surprisingly, even for Published under a CC BY 4.0 licence Published under a CC BY 4.0 licence Ac
Pub 167 https://doi.org/10.1107/S2056989023001238 Acta Cryst. (2023). E79, 167–171 research communications research communications pyrazine)4 (Refcode: EMELEN; Na¨ther et al., 2003), (CuI)2-
(methylsulfanylpyrazine)4 (Refcode: QOWYOT; Artem’ev et
al., 2019) and (CuI)2(2,20-biquinoxaline) (Refcode: RIXGEL;
Fitchett & Steel, 2008), all with iodide as counter-anion. compounds with the same CuX:ligand ratio, sometimes a
different thermal reactivity is observed. This is the case, for
example, for compounds based on CuX (X = Cl, Br, I) and
2-chloropyrazine as ligands with the general composition
CuX(2-chloropyrazine) (X = Cl, Br, I; Na¨ther, Wriedt & Jess,
2002; Na¨ther, Greve & Jess, 2002). In the isotypic chloride and
bromide compounds, the copper cations are tetrahedrally
coordinated by two bridging 2-chloropyrazine ligands and two
halide anions. The cations are linked by single -1,1-bridging
halide anions into chains that are further connected into layers
by -1,4-bridging 2-chloropyrazine ligands (Fig. S1 in the
supporting
information). In
contrast,
in
CuI(2-chloro-
pyrazine), each copper cation is tetrahedrally coordinated by
three iodide anions and only one terminal N-bonding
2-chloropyrazine ligand that is coordinated to the copper
center by the N atom that is not adjacent to the chloro
substituent. The cations are linked into double chains via
bridging iodide anions (Fig. S1). If the chloride and the
bromide compounds are heated, all 2-chloropyrazine ligands
are removed in a single step, leading to the formation of CuX
(X = Cl, Br). In contrast, the iodide compound decomposes in
two discrete steps, where in the first step only half of the
coligands are removed, leading to the formation of (CuI)2(2-
chloropyrazine), which decomposes into CuI upon further
heating (Na¨ther, Greve & Jess, 2002). compounds with the same CuX:ligand ratio, sometimes a
different thermal reactivity is observed. This is the case, for
example, for compounds based on CuX (X = Cl, Br, I) and
2-chloropyrazine as ligands with the general composition
CuX(2-chloropyrazine) (X = Cl, Br, I; Na¨ther, Wriedt & Jess,
2002; Na¨ther, Greve & Jess, 2002). In the isotypic chloride and
bromide compounds, the copper cations are tetrahedrally
coordinated by two bridging 2-chloropyrazine ligands and two
halide anions. The cations are linked by single -1,1-bridging
halide anions into chains that are further connected into layers
by -1,4-bridging 2-chloropyrazine ligands (Fig. S1 in the
supporting
information). In
contrast,
in
CuI(2-chloro-
pyrazine), each copper cation is tetrahedrally coordinated by
three iodide anions and only one terminal N-bonding
2-chloropyrazine ligand that is coordinated to the copper
center by the N atom that is not adjacent to the chloro
substituent. 2. Structural commentary The asymmetric unit of the title compound (CuI)2(2-chloro-
pyrazine)4 consists of one copper(I) cation, one iodide anion
and two 2-chloropyrazine ligands that are located in general
positions. The copper(I) cations are tetrahedrally coordinated
by two symmetry-related iodide anions and two crystal-
lographically independent 2-chloropyrazine ligands (Fig. 1). Each two copper(I) cations are linked by pairs of -1,1-brid-
ging iodide anions into binuclear complexes consisting of four-
membered (CuI)2 rings located on centers of inversion. The
Cu—Cu distance within these rings amounts to 2.5643 (10) A˚
(Table 1). Bond lengths and angles are similar to those in
related compounds and show that the tetrahedra are strongly
distorted (Table 1). This structure is similar to those of (CuI)2(2-cyano-
pyrazine)4 (Rossenbeck & Sheldrick, 1999; Jana et al., 2016), Figure 1
Crystal structure of the title compound with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Symmetry
codes: (i) x + 2, y + 1, z + 2. Concerning the composition of all of these compounds, in
principle, more 2-chloropyrazine-rich compounds with the
composition CuX(2-chloropyrazine)2 might exist, in which,
according to simple chemical considerations, each two copper
cations would be tetrahedrally coordinated by two halide
anions and two N-terminal 2-chloropyrazine ligands and
linked into binuclear complexes by pairs of -1,1-bridging
halide anions. One might argue that this arrangement is less
stable compared to that with bridging 2-chloropyrazine
ligands, but one should keep in mind that both N atoms of this
ligand are not equivalent, because coordination to the N atom
that is adjacent to the chloro substituent is sterically hindered. That this coordination exists is obvious from the crystal
structure of (CuI)(2-chloropyrazine) mentioned above, even if
this
CuX
substructure
is
different. Moreover,
a
few
compounds with such a structure have already been reported
in the literature, including, for example, (CuI)2(2-cyano-
pyrazine)4 (Refcodes: DINQIA and DINQIA01; Rossenbeck
& Sheldrick, 1999 and Jana et al., 2016), (CuI)2(2-ethyl- Figure 1 Figure 1
Crystal structure of the title compound with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Symmetry
codes: (i) x + 2, y + 1, z + 2. g
Crystal structure of the title compound with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Symmetry
codes: (i) x + 2, y + 1, z + 2. g
Crystal structure of the title compound with atom labeling and
displacement ellipsoids drawn at the 50% probability level. 3. Supramolecular features 3. Supramolecular features In the crystal structure of the title compound, the binuclear
complexes are arranged in columns that propagate along the
crystallographic a-axis direction (Fig. 2). No directional
intermolecular interactions occur between the complexes. One
C—H N and one C—H I contact is observed, but their
distances and angles indicate that they do not correspond to
significant interactions (Table 2). Figure 3
DTG, TG, DTA and MS trend scan curves for the title compound
measured with a heating rate of 4C min1. research communications Table 1
Selected geometric parameters (A˚ , ). Cu1—N2
2.070 (3)
Cu1—I1
2.6093 (5)
Cu1—N12
2.078 (3)
Cu1—I1i
2.6476 (6)
Cu1—Cu1i
2.5643 (10)
N2—Cu1—N12
103.01 (12)
N2—Cu1—I1i
107.58 (9)
N2—Cu1—I1
109.42 (9)
N12—Cu1—I1i
105.80 (9)
N12—Cu1—I1
107.83 (9)
I1—Cu1—I1i
121.61 (2)
Symmetry code: (i) x þ 2; y þ 1; z þ 2. Table 2
Hydrogen-bond geometry (A˚ , ). D—H A
D—H
H A
D A
D—H A
C3—H3 N11ii
0.94
2.68
3.241 (5)
119
C13—H13 I1
0.94
3.24
3.872 (4)
127
Symmetry code: (ii) x þ 1; y þ 1; z þ 1. that this sample is contaminated at least with CuI(2-chloro-
pyrazine) reported in the literature (Na¨ther, Greve & Jess,
2002). This indicates that the title compound has already
decomposed into this compound at room temperature. To
prove this assumption, freshly prepared crystals were stored at
room temperature overnight and were afterwards investigated
by PXRD, confirming that the title compound has been
completely transformed into the ligand-deficient compound
CuI(2-chloropyrazine) (Fig. S3). These observations indicate
that CuI(2-chloropyrazine) with a bridging coordination of the
2-chloropyrazine
ligand
is
more
stable
than
the
title
compound, in which the 2-chloropyrazine acts as a terminal
ligand. Additional DTA–TG–MS investigations reveal that
the title compound loses two 2-chloropyrazine ligands in two
subsequent steps, in which 2-chloropyrazine is always removed
(m/z = 114, Fig. 3). The experimental mass loss in the first step
(mexp . = 37.5%) is much larger than that expected for the Symmetry code: (i) x þ 2; y þ 1; z þ 2. (CuI)2(2-ethylpyrazine)4
(Na¨ther
et
al.,
2003),
(CuI)2-
(methylsulfanylpyrazine)4
(Artem’ev
et
al.,
2019)
and
(CuI)2(2,20-biquinoxaline) (Fitchett & Steel, 2008) already
reported
in
the
literature,
which
also
form
binuclear
complexes with (CuI)2 rings as the main structural motif. 2. Structural commentary Symmetry
codes: (i) x + 2, y + 1, z + 2. 168
Na¨ther and Jess
[Cu2I2(C4H3ClN2)4] Acta Cryst. (2023). E79, 167–171 Acta Cryst. (2023). E79, 167–171 4. Powder X-ray diffraction and thermoanalytical
investigations Further investigations prove that the unreacted 2-chloro-
pyrazine cannot be removed by filtration and washing because
immediate decomposition is observed. Nevertheless, XRPD
investigations reveal that most of the sample consists of
crystals of the title compound, even if all of the powder
patterns are of very low quality, which can be traced back to
the instability of this compound and to the fact that only very
small amounts of crystals were obtained and these were
embedded in pure 2-chloropyrazine and that grinding of such
samples leads to the formation of an amorphous phase
(Fig. S2). Careful inspection of the powder pattern indicates Figure 2
Crystal structure of the title compound in a view along the crystal-
lographic a-axis. Figure 2
Crystal structure of the title compound in a view along the crystal-
lographic a-axis. Figure 3
DTG, TG, DTA and MS trend scan curves for the title compound
measured with a heating rate of 4C min1. Na¨ther and Jess
[Cu2I2(C4H3ClN2)4]
169 169 Acta Cryst. (2023). E79, 167–171 research communications Figure 4
Experimental PXRD pattern of the residue obtained after the first mass
loss in a DTA–TG–MS measurement of the title compound (top) and
PXRD pattern calculated for CuI(2-chloropyrazine) (bottom). Some related compounds can also be found with 2-meth-
ylpyrazine as coligand because the exchange of a chloro atom
by a methyl group sometimes leads to compounds with similar
crystal structures as the van der Waals radius of a chlorine
atom is comparable to that of a methyl group (Desiraju &
Sarma, 1986). This is obvious from CuX(2-methylpyrazine)
with X = Cl, Br (Refcodes: XEBMOG and XEBMIA;
Rossenbeck & Sheldrick, 2000), in which the copper(I) cations
are linked by -1,1-bridging halide anions into chains that are
further connected into layers by bridging 2-methylpyrazine
ligands. This structure is identical to that of CuX(2-chloro-
pyrazine) (X = Cl, Br). Moreover, both the 2-methylpyrazine
and the 2-chloropyrazine compounds crystallize in the
monoclinic space group P21/c with very similar lengths of the
unit-cell axes, but with a significantly different angle. In this
context it is noted that with 2-methylpyrazine, two coligand-
deficient compounds with the composition (CuX)2(2-methyl-
pyrazine) with X = Br, I (Refcodes: XEBMUM and XEBNAT;
Rossenbeck & Sheldrick, 2000) were observed that could not
be prepared with 2-chloropyrazine. Synthesis CuI and 2-chloropyrazine were purchased from Sigma-
Aldrich and used as received. Yellow-colored single crystals suitable for single-crystal
X-ray analysis were obtained within three days by the reaction
of 0.5 mmol (95.23 mg) of CuI and 2 mL of 2-chloropyrazine. No stoichiometric ratios can be used as an excess of
2-chloropyrazine is needed because it acts as reactant and
solvent. The additional 2-chloropyrazine cannot be removed
by filtration and washing, because this leads immediately to
the transformation of the title compound into CuI(2-chloro-
pyrazine). A further search for compounds based on copper halides
and 2-chloropyrazine as ligand lead to only a very few
compounds. They include the three compounds with the
composition CuX(2-chloropyrazine) with X = Cl, Br, I
mentioned
above
(Refcodes:
ODOFES,
ODOFIW and
ODOFOC; Na¨ther, Wriedt & Jess 2002) as well as two isotypic
discrete complexes with the composition CuX2(2-chloro-
pyrazine)2 with X = Cl, Br that contain CuII cations (Refcodes:
FULYIV and FULYOB; Herringer et al., 2010). 4. Powder X-ray diffraction and thermoanalytical
investigations Figure 4 g
Experimental PXRD pattern of the residue obtained after the first mass
loss in a DTA–TG–MS measurement of the title compound (top) and
PXRD pattern calculated for CuI(2-chloropyrazine) (bottom). Furthermore, the isotypic compounds (CuX)2(2-methyl-
pyrazine)(triphenylphosphine)2 acetonitrile solvate with X =
Br, I [Refcodes: AKOPOI (Kuwahara et al., 2020) and
RAYXAT (Liu et al., 2017)], in which each copper cation is
tetrahedrally coordinated by one 2-methylpyrazine and one
trimethylphoshine ligand as well as two halide anions, are
known. Similarly to the title compound, both copper cations
are linked by two bridging halide anions into (CuI)2 rings, but
strikingly the binuclear units are linked by the 2-methyl-
pyrazine ligands into chains. Moreover, (CuI)2(2-methyl-
pyrazine)2-2-methylpyrazine solvate (XEBMEW; Rossenbeck
& Sheldrick, 2000) also contains (CuI)2 rings. removal of all of the 2-chloropyrazine ligands from the title
compound (mcalc. = 19.1%), which originates from the fact
that the 2-chloropyrazine coating the crystals cannot be
removed. However, PXRD of the residue obtained after the
first mass loss confirms that CuI(2-chloropyrazine) is formed
as an intermediate (Fig. 4). It is noted that no additional step is
observed that would correspond to the formation of the most
2-chloropyrazine-deficient
compound,
(CuI)2(2-chloro-
pyrazine), because this event would happen at a much lower
temperature, whereas our measurements indicate an excess of
2-chloropyrazine is still present in the gas phase. Finally, the
product formed after the second mass loss was also investi-
gated py PXRD, which proves that CuI (Hull, & Keen, 1994) is
formed in this step (Fig. S4). Finally, two compounds with the composition CuX(2-
methylpyrazine)(triphenylphosphine)2
with
X
=
Cl,
I
[Refcodes: KAMKER (Ohara, et al., 2017) and NAKYIL
(Kondo et al., 2020)] are reported, which form discrete
complexes. This structural motif is unknown for compounds
based on CuX and 2-chloropyrazine. 5. Database survey A search in the CCDC database (Groom et al., 2016, CSD
Version 5.43, March 2022) for compounds with a structure
similar to that of the title compound revealed several hits,
including (CuI)2(2-cyanopyrazine)4 (Refcodes: DINQIA and
DINQIA01; Rossenbeck & Sheldrick, 1999; Jana et al., 2016),
(CuI)2(2-ethylpyrazine)4
(Refcode:
EMELEN;
Na¨ther
et
al.,
2003),
(CuI)2-(methylsulfanylpyrazine)4
(Refcode:
QOWYOT; Artem’ev et al., 2019) and (CuI)2(2,20-biquinoxa-
line) (Refcode: RIXGEL; Fitchett & Steel, 2008). References Computer programs: X-AREA (Stoe, 2008), SHELXT2014/5 (Sheldrick, 2015a),
SHELXL2016/6 (Sheldrick, 2015b), DIAMOND (Brandenburg, 1999) and publCIF
(Westrip, 2010). Artem’ev, A. V., Beresin, A. S. & Bagryanskaya, I. Y. (2019). J. Struct. Chem. 60, 967–971. Brandenburg, K. (1999). DIAMOND. Crystal Impact GbR, Bonn,
Germany. Na¨ther, C., Bhosekar, G. & Jess, I. (2007). Inorg. Chem. 46, 8079–
8087. Desiraju, G. R. & Sarma, J. P. (1986). Proc. - Indian Acad. Sci. Chem. Sci. 96, 599–605. Na¨ther, C., Greve, J. & Jess, I. (2002). Solid State Sci. 4, 813–820. Fitchett, C. M. & Steel, P. J. (2008). Polyhedron, 27, 1527–1537. Na¨ther, C. & Jess, I. (2001). Monatsh. Chem. 132, 897–910. Gibbons, S. K., Hughes, R. P., Glueck, D. S., Royappa, A. T.,
Rheingold, A. L., Arthur, R. B., Nicholas, A. D. & Patterson, H. H. (2017). Inorg. Chem. 56, 12809–12820. Na¨ther, C. & Jess, I. (2004). Eur. J. Inorg. Chem. pp. 2868–2876. Na¨ther, C., Jess, I. & Greve, J. (2001). Polyhedron, 20, 1017–1022 Na¨ther, C., Jess, I., Lehnert, N. & Hinz-Hu¨bner, D. (2003). Solid State
Sci. 5, 1343–1357. (
)
g
Groom, C. R., Bruno, I. J., Lightfoot, M. P. & Ward, S. C. (2016). Acta
Cryst. B72, 171–179. Na¨ther, C., Jess, I., Germann, L. S., Dinnebier, R. E., Braun, M. &
Terraschke, H. (2017). Eur. J. Inorg. Chem. pp. 1245–1255. y
Herringer, S. N., Longendyke, A. J., Turnbull, M. M., Landee, C. P., Herringer, S. N., Longendyke, A. J., Turnbull, M. M., Landee, C. P.,
Wikaira, J. L., Jameson, G. B. & Telfer, S. G. (2010). Dalton Trans. 39, 2785–2797. Wikaira, J. L., Jameson, G. B. & Telfer, S. G. (2010). Dalton Trans. 39, 2785–2797. Na¨ther, C., Wriedt, M. & Jess, I. (2002). Z. Anorg. Allg. Chem. 628,
394–400. ,
Hull, S. & Keen, D. A. (1994). Phys. Rev. B, 50, 5868–5885. Ohara, H., Ogawa, T., Yoshida, M., Kobayashi, A. & Kato, M. (2017). Dalton Trans. 46, 3755–3760. Jana, S., Harms, K. & Chattopadhyay, S. (2016). J. Iran. Chem. Soc. 13,
1713–1721. R., Li, M. & Li, D. (2010). Coord. Chem. Rev. 254, 1– Kondo, S., Yoshimura, N., Yoshida, M., Kobayashi, A. & Kato, M. (2020). Dalton Trans. 49, 16946–16953. Rossenbeck, B. & Sheldrick, W. S. (1999). Z. Naturforsch. B, 54, 1510–
1516. (
)
Kromp, T. & Sheldrick, W. S. (1999). Z. Naturforsch. B, 54, 1175–1180. Rossenbeck, B. & Sheldrick, W. S. (2000). Z. Naturforsch. B, 55, 467–
472. Financial support by the State of Schleswig-Holstein is
gratefully acknowledged. Financial support by the State of Schleswig-Holstein is
gratefully acknowledged. research communications The PXRD measurements were performed with Cu K1
radiation ( = 1.540598 A˚ ) using a Stoe Transmission Powder
Diffraction System (STADI P) equipped with a MYTHEN 1K
detector and a Johansson-type Ge(111) monochromator. [Cu2I2(C4H3ClN2)4]
839.02
Triclinic, P1
220
7.5220 (6), 10.1067 (8), 10.1973 (9)
108.932 (9), 101.922 (10),
111.088 (9)
636.62 (11)
1
Mo K
4.54
0.25 0.20 0.18 Differential thermoanalysis and thermogravimetry coupled
to mass spectrometry (DTA–TG–MS) investigations were
performed with a STA-429 thermobalance from Netzsch with
skimmer coupling to a quadrupole mass spectrometer from
Balzers. The measurements were performed in a dynamic
nitrogen atmosphere in Al2O3 crucibles with a heating rate of
4C min1. The instrument was calibrated using standard
reference materials. Stoe IPDS1
Numerical (X-SHAPE and X-RED
32; Stoe, 2008)
0.556, 0.667
6865, 3018, 2670 7. Refinement Crystal data, data collection and structure refinement details
are summarized in Table 3. The C—H hydrogen atoms were
positioned in an idealized geometry and refined isotropically
with Uiso(H) = 1.2Ueq(C). Refinement
R[F 2 > 2(F 2)], wR(F 2), S
No. of reflections
No. of parameters
H-atom treatment
max, min (e A˚ 3) Experimental details The data collection for single crystal structure analysis was
performed using an Imaging Plate Diffraction System (IPDS-
1) from Stoe with Mo K radiation. 170
Na¨ther and Jess
[Cu2I2(C4H3ClN2)4] Acta Cryst. (2023). E79, 167–171 research communications Computer programs: X-AREA (Stoe, 2008), SHELXT2014/5 (Sheldrick, 2015a),
SHELXL2016/6 (Sheldrick, 2015b), DIAMOND (Brandenburg, 1999) and publCIF
(Westrip, 2010). References Kuwahara, T., Ohtsu, H. & Tsuge, K. (2020). Inorg. Chem. 60, 1299–
1304. Sheldrick, G. M. (2015a). Acta Cryst. A71, 3–8. Li, D., Shi, W. J. & Hou, L. (2005). Inorg. Chem. 44, 3907–3913. Sheldrick, G. M. (2015b). Acta Cryst. C71, 3–8. Liu, W., Zhu, K., Teat, S. J., Deibert, B. J., Yuan, W. & Li, J. (2017). Chem. Mater. 5, 5962–5969. Stoe (2008). X-AREA, X-RED32 and X-SHAPE. Stoe & Cie,
Darmstadt, Germany. Westrip, S. P. (2010). J. Appl. Cryst. 43, 920–925. Mensah, A., Shao, J. J., Ni, J. L., Li, G. J., Wang, F. M. & Chen, L. Z. (2022). Front. Chem. 9, 816363. Westrip, S. P. (2010). J. Appl. Cryst. 43, 920–925. Na¨ther and Jess
[Cu2I2(C4H3ClN2)4]
171 Acta Cryst. (2023). E79, 167–171 supporting information supporting information Acta Cryst. (2023). E79, 167-171 [https://doi.org/10.1107/S2056989023001238]
Synthesis, crystal structure and thermal properties of di-µ-iodido-bis[bis(2-
chloropyrazine-κN)copper(I)] supporting information Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq
Cu1
0.83699 (6)
0.44607 (6)
0.88784 (5)
0.02577 (15)
I1
1.13274 (3)
0.38430 (3)
0.84123 (3)
0.02918 (13)
N1
0.1826 (5)
−0.0235 (4)
0.6635 (5)
0.0382 (8)
C1
0.2875 (6)
0.0483 (5)
0.8075 (5)
0.0310 (8)
C2
0.4784 (6)
0.1784 (5)
0.8761 (5)
0.0273 (7)
H2
0.545096
0.224589
0.980055
0.033*
N2
0.5673 (4)
0.2377 (4)
0.7947 (4)
0.0240 (6)
C3
0.4652 (6)
0.1671 (5)
0.6476 (5)
0.0352 (9)
H3
0.524713
0.206273
0.587078
0.042*
C4
0.2745 (7)
0.0380 (6)
0.5828 (5)
0.0400 (10)
H4
0.206856
−0.008357
0.478949
0.048*
Cl1
0.1760 (2)
−0.02696 (16)
0.91711 (16)
0.0573 (4)
N11
0.6729 (5)
0.7005 (4)
0.5844 (4)
0.0312 (7)
C11
0.5985 (6)
0.6885 (4)
0.6870 (4)
0.0271 (8)
C12
0.6478 (6)
0.6211 (5)
0.7776 (4)
0.0270 (7)
H12
0.589696
0.617235
0.850384
0.032*
N12
0.7785 (4)
0.5614 (4)
0.7617 (3)
0.0238 (6)
C13
0.8562 (5)
0.5723 (5)
0.6574 (4)
0.0267 (7)
H13
0.948559
0.531426
0.643161
0.032*
C14
0.8049 (6)
0.6421 (5)
0.5694 (5)
0.0311 (8)
H14
0.864111
0.648491
0.497602
0.037*
Cl11
0.42309 (19)
0.76067 (15)
0.70711 (14)
0.0455 (3)
Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
Cu1
0.0206 (2)
0.0312 (3)
0.0281 (3)
0.01328 (19)
0.00699 (17)
0.0157 (2)
I1
0.02846 (17)
0.03876 (19)
0.02799 (18)
0.02343 (12)
0.01185 (11)
0.01347 (12)
N1
0.0280 (16)
0.0314 (19)
0.043 (2)
0.0099 (14)
0.0061 (15)
0.0109 (16)
C1
0.0267 (17)
0.0208 (17)
0.043 (2)
0.0098 (14)
0.0161 (16)
0.0099 (16)
C2
0.0249 (17)
0.0229 (17)
0.0288 (19)
0.0099 (14)
0.0075 (14)
0.0080 (15)
N2
0.0185 (13)
0.0233 (15)
0.0279 (16)
0.0083 (11)
0.0045 (11)
0.0124 (13)
C3
0.0318 (19)
0.039 (2)
0.032 (2)
0.0127 (17)
0.0053 (16)
0.0214 (19)
C4
0.035 (2)
0.038 (2)
0.030 (2)
0.0087 (18)
−0.0033 (16)
0.0141 (19)
Cl1
0.0531 (7)
0.0426 (7)
0.0509 (7)
−0.0019 (5)
0.0312 (6)
0.0111 (6)
N11
0.0345 (17)
0.0280 (17)
0.0299 (17)
0.0132 (14)
0.0054 (13)
0.0166 (14)
C11
0.0267 (17)
0.0237 (17)
0.0264 (19)
0.0141 (14)
0.0013 (14)
0.0082 (15)
C12
0.0266 (17)
0.035 (2)
0.0263 (18)
0.0188 (15)
0.0113 (14)
0.0151 (16)
N12
0.0230 (14)
0.0247 (15)
0.0257 (15)
0.0123 (12)
0.0065 (11)
0.0135 (13) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq
Cu1
0.83699 (6)
0.44607 (6)
0.88784 (5)
0.02577 (15)
I1
1.13274 (3)
0.38430 (3)
0.84123 (3)
0.02918 (13)
N1
0.1826 (5)
−0.0235 (4)
0.6635 (5)
0.0382 (8)
C1
0.2875 (6)
0.0483 (5)
0.8075 (5)
0.0310 (8)
C2
0.4784 (6)
0.1784 (5)
0.8761 (5)
0.0273 (7)
H2
0.545096
0.224589
0.980055
0.033*
N2
0.5673 (4)
0.2377 (4)
0.7947 (4)
0.0240 (6)
C3
0.4652 (6)
0.1671 (5)
0.6476 (5)
0.0352 (9)
H3
0.524713
0.206273
0.587078
0.042*
C4
0.2745 (7)
0.0380 (6)
0.5828 (5)
0.0400 (10)
H4
0.206856
−0.008357
0.478949
0.048*
Cl1
0.1760 (2)
−0.02696 (16)
0.91711 (16)
0.0573 (4)
N11
0.6729 (5)
0.7005 (4)
0.5844 (4)
0.0312 (7)
C11
0.5985 (6)
0.6885 (4)
0.6870 (4)
0.0271 (8)
C12
0.6478 (6)
0.6211 (5)
0.7776 (4)
0.0270 (7)
H12
0.589696
0.617235
0.850384
0.032*
N12
0.7785 (4)
0.5614 (4)
0.7617 (3)
0.0238 (6)
C13
0.8562 (5)
0.5723 (5)
0.6574 (4)
0.0267 (7)
H13
0.948559
0.531426
0.643161
0.032*
C14
0.8049 (6)
0.6421 (5)
0.5694 (5)
0.0311 (8)
H14
0.864111
0.648491
0.497602
0.037*
Cl11
0.42309 (19)
0.76067 (15)
0.70711 (14)
0.0455 (3) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) omic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
Cu1
0.0206 (2)
0.0312 (3)
0.0281 (3)
0.01328 (19)
0.00699 (17)
0.0157 (2)
I1
0.02846 (17)
0.03876 (19)
0.02799 (18)
0.02343 (12)
0.01185 (11)
0.01347 (12)
N1
0.0280 (16)
0.0314 (19)
0.043 (2)
0.0099 (14)
0.0061 (15)
0.0109 (16)
C1
0.0267 (17)
0.0208 (17)
0.043 (2)
0.0098 (14)
0.0161 (16)
0.0099 (16)
C2
0.0249 (17)
0.0229 (17)
0.0288 (19)
0.0099 (14)
0.0075 (14)
0.0080 (15)
N2
0.0185 (13)
0.0233 (15)
0.0279 (16)
0.0083 (11)
0.0045 (11)
0.0124 (13)
C3
0.0318 (19)
0.039 (2)
0.032 (2)
0.0127 (17)
0.0053 (16)
0.0214 (19)
C4
0.035 (2)
0.038 (2)
0.030 (2)
0.0087 (18)
−0.0033 (16)
0.0141 (19)
Cl1
0.0531 (7)
0.0426 (7)
0.0509 (7)
−0.0019 (5)
0.0312 (6)
0.0111 (6)
N11
0.0345 (17)
0.0280 (17)
0.0299 (17)
0.0132 (14)
0.0054 (13)
0.0166 (14)
C11
0.0267 (17)
0.0237 (17)
0.0264 (19)
0.0141 (14)
0.0013 (14)
0.0082 (15)
C12
0.0266 (17)
0.035 (2)
0.0263 (18)
0.0188 (15)
0.0113 (14)
0.0151 (16)
N12
0.0230 (14)
0.0247 (15)
0.0257 (15)
0.0123 (12)
0.0065 (11)
0.0135 (13) Atomic displacement parameters (Å2) Acta Cryst. supporting information supporting information p
Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance
matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles;
correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate
(isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles;
correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate
(isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Christian Näther and Inke Jess Data collection: X-AREA (Stoe, 2008); cell refinement: X-AREA (Stoe, 2008); data reduction: X-AREA (Stoe, 2008);
program(s) used to solve structure: SHELXT2014/5 (Sheldrick, 2015a); program(s) used to refine structure:
SHELXL2016/6 (Sheldrick, 2015b); molecular graphics: DIAMOND (Brandenburg, 1999); software used to prepare
material for publication: publCIF (Westrip, 2010). H-atom parameters constrained H-atom parameters constrained Acta Cryst. (2023). E79, 167-171 sup-1 Symmetry code: (i) −x+2, −y+1, −z+2. supporting information (2023). E79, 167-171 sup-2 supporting information
C13
0.0230 (16)
0.0296 (19)
0.0288 (19)
0.0128 (14)
0.0079 (14)
0.0143 (16)
C14
0.0314 (18)
0.031 (2)
0.030 (2)
0.0118 (16)
0.0097 (15)
0.0162 (17)
Cl11
0.0526 (6)
0.0525 (7)
0.0470 (6)
0.0421 (6)
0.0143 (5)
0.0220 (5)
Geometric parameters (Å, º)
Cu1—N2
2.070 (3)
C3—H3
0.9400
Cu1—N12
2.078 (3)
C4—H4
0.9400
Cu1—Cu1i
2.5643 (10)
N11—C11
1.302 (6)
Cu1—I1
2.6093 (5)
N11—C14
1.336 (6)
Cu1—I1i
2.6476 (6)
C11—C12
1.380 (5)
N1—C1
1.313 (6)
C11—Cl11
1.739 (4)
N1—C4
1.342 (6)
C12—N12
1.336 (5)
C1—C2
1.382 (5)
C12—H12
0.9400
C1—Cl1
1.735 (4)
N12—C13
1.330 (5)
C2—N2
1.327 (5)
C13—C14
1.384 (6)
C2—H2
0.9400
C13—H13
0.9400
N2—C3
1.335 (5)
C14—H14
0.9400
C3—C4
1.378 (6)
N2—Cu1—N12
103.01 (12)
N2—C3—H3
119.3
N2—Cu1—Cu1i
130.55 (9)
C4—C3—H3
119.3
N12—Cu1—Cu1i
126.35 (9)
N1—C4—C3
121.9 (4)
N2—Cu1—I1
109.42 (9)
N1—C4—H4
119.0
N12—Cu1—I1
107.83 (9)
C3—C4—H4
119.0
Cu1i—Cu1—I1
61.554 (19)
C11—N11—C14
115.8 (3)
N2—Cu1—I1i
107.58 (9)
N11—C11—C12
124.4 (4)
N12—Cu1—I1i
105.80 (9)
N11—C11—Cl11
116.8 (3)
Cu1i—Cu1—I1i
60.06 (2)
C12—C11—Cl11
118.7 (3)
I1—Cu1—I1i
121.61 (2)
N12—C12—C11
119.8 (3)
Cu1—I1—Cu1i
58.39 (2)
N12—C12—H12
120.1
C1—N1—C4
115.1 (4)
C11—C12—H12
120.1
N1—C1—C2
124.4 (4)
C13—N12—C12
116.9 (3)
N1—C1—Cl1
117.0 (3)
C13—N12—Cu1
123.3 (3)
C2—C1—Cl1
118.6 (3)
C12—N12—Cu1
119.7 (3)
N2—C2—C1
119.7 (4)
N12—C13—C14
121.8 (4)
N2—C2—H2
120.1
N12—C13—H13
119.1
C1—C2—H2
120.1
C14—C13—H13
119.1
C2—N2—C3
117.4 (3)
N11—C14—C13
121.3 (4)
C2—N2—Cu1
122.7 (3)
N11—C14—H14
119.3
C3—N2—Cu1
119.5 (3)
C13—C14—H14
119.3
N2—C3—C4
121.4 (4)
Symmetry code: (i) −x+2, −y+1, −z+2. H d
b
d
(Å º) supporting information
C13—H13···I1
0.94
3.24
3.872 (4)
127
Symmetry code: (ii) −x+1, −y+1, −z+1. C13—H13···I1 Symmetry code: (ii) −x+1, −y+1, −z+1. Acta Cryst. (2023). E79, 167-171 sup-4
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The Exotic Materia Medica of Chinese Medicine: A Survey of Its Resources and Medical Information in 1029 BC–1999 AD
|
Research Square (Research Square)
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cc-by
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Qingqing Cai Chengdu University of Traditional Chinese Medicine Wenjiang Campus: Chengdu University of
Traditional Chinese Medicine ty of Traditional Chinese Medicine
https://orcid.org/0000-0003-2501-3652 The Exotic Materia Medica of Chinese Medicine: A
Survey of Its Resources and Medical Information in
1029 BC–1999 AD Qingqing Cai
Chengdu University of Traditional Chinese Medicine Wenjiang Campus: Chengdu University of
Traditional Chinese Medicine
Zhu-yun Yan
(
yanzhuyun@cdutcm.edu.cn
)
Chengdu University of Traditional Chinese Medicine
https://orcid.org/0000-0003-2501-3652
Research
Keywords: Drug Communication, Exotic Chinese Medicine, Medicinal Resources, Traditional Chinese
Medicine, Chinese Materia Medica
Posted Date: October 12th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-900422/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Conclusion Chinese doctors are more concerned with the efficacy of the drug when using it in clinical settings than
whether the drug comes from afar. The record of origin and source was to ensure the supply and quality
of the drugs. The use of foreign resources as Chinese medicine is the fact that Chinese medicine
objectively exists. The existence of ECM has enriched the resources of TCM. Furthermore, both foreign
medicine and local medicine currently protect people's lives and health. Background Traditional Chinese medicine (TCM) gives people an image of its medicinal resources all from within
China. Nevertheless, the fact is that the Exotic Materia Medica(ECM) has been used in Chinese medicine
from Zhou Dynasty (1029 BC-221 AD). However, no extensive studies have focused on it. Now, the high
exchange speed of information and material brings TCM opportunities to apply exotic resources in
clinics. So, surveying the historical ECM’s resources and medical information is valuable. Results Consequently, we found information on 788 ECM and their clinical application. A total of 99% of ECM
were found to be botanicals mainly belonging to Solanaceae, Amaranthaceae, Malvaceae, Myrtaceae,
and Cruciferae. The exchange speed of these medicines between China and other regions peaked in the
Han (206 BC–220 AD), Tang (618–907 AD), and Ming dynasties (1368–1644 AD), and increased sharply
from 1912 onwards. The main registered efficacies of the ECM were "clear heat" and diuresis. The most
efficient way to convert foreign medicine to TCM is to absorb drugs from foreign regions directly. Last but
not least, about One-fifth of commonly used TCM in the clinic is ECM. Research Page 1/15 Page 1/15 Methods We investigated the origin of 8980 Chinese Materia Medica’s resources included in Chinese Materia
Medica and compiled a species list of exotic Chinese medicines (ECM) used in China between 1029 BC–
1999 AD and their medical information, introduction time, introduction routes, and place of origin. 1. Background Traditional Chinese medicine (TCM) or Traditional Chinese Materia Medica has provided medical
knowledge and resources in China. While exploring and utilizing local medical resources, the Chinese
doctor also applies effective foreign medicines to the clinical. The history of using exotic medicines in
China can trace back to the Zhou dynasty(1029-221 BC). The Eastern Han philosopher Chong Wang's
Lun Heng volume eight "Ruzeng Pian" said: "During the Zhou Dynasty, the world was peaceful, Vietnam
sent white pheasants, and Japanese sent tulip grass[1]. " And then, in the Middle Ages, exotic medicines
were introduced by the Persians from the Silk Road. Moreover, during the Tang dynasty(618-907 AD), Page 2/15 Page 2/15 Page 2/15 overseas medicines were introduced to China via the Maritime Silk Road due to the development of
maritime trade[2]. In the Five Dynasties and Ten Kingdoms period(907-979 AD), the Persian businessman
Xun Li's Marine Herbal Medicine systematically recorded the foreign medicines. Exotic medicines have
been introduced in medical practice in China during each historical period. overseas medicines were introduced to China via the Maritime Silk Road due to the development of
maritime trade[2]. In the Five Dynasties and Ten Kingdoms period(907-979 AD), the Persian businessman
Xun Li's Marine Herbal Medicine systematically recorded the foreign medicines. Exotic medicines have
been introduced in medical practice in China during each historical period. Although the history of using exotic medicines in China can trace back to the Zhou dynasty, modern
research on exotic medicines began with the Chinese pharmacist Professor Ling Yikui. He and his student
first defined exotic Chinese medicine (ECM) in 1980. ECM refers to imported medicines or foreign
medicines introduced in China. ECM should: 1) be produced outside the territory, relying solely on
importers, such as frankincense, myrrh, benzoin, and storax; 2) be produced outside and inside the
territory, imported from abroad, such as Korean ginseng, Dongyang ginseng, American ginseng; 3) firstly
produced outside the territory but later introduced in China, such as white cardamom, pepper, cinchona,
and coix seed[3].Chinese pharmacists complied with this definition and inclusion criteria while exploring
the ECM[2,4–8].In this research, we followed the definition of Yikui Ling, except we emphasized that the
original resources came from abroad. ECM covers plants, animals, and minerals or their processed
products that have entered China through various methods, and have been used in the Chinese medicine. 1. Background Which includes animals and plants originally distributed in China, but the initial resources were from
abroad or naturalized in China, such as turmeric, coix seed, American ginseng, patchouli, winter melon
seeds. Now, the high exchange speed of information and material brings TCM opportunities to apply exotic
resources in the clinic. So, surveying the historical ECM’s resources and medical information is valuable. However, no extensive studies have focused on it. The existing research on ECM have focused on a
classic pharmacy book or a certain dynasty[7–16]. Most of them focusing on a classic pharmacy book or
a certain dynasty. Therefore, we compiled a list of ECM from the 8980 traditional Chinese medicines
included in the Chinese Materia Medica [17].which covers the drugs that are recorded in all the classic
pharmacy books of TCM and still being used today. We then analyzed their place of origin, time of
application, time of introduction, characteristics of the resources, drug efficacy, and clinical application. This study represents the first attempt at a compilation of the basic general list of ECM with
comprehensive analysis of its efficacy, indications, and resources. 3. Results A total of 788 ECM among 8980 TCM were retrieved from the Chinese Materia Medica. The ECM’s drug
names, plant names, sources, effects, indications, medicinal properties, medicinal taste, meridian,
medicinal parts, toxicity, introduction route, life type, family, genus, introduction time, first medical
literature of loading, the first application time, and the place of origin, can be seen in Supplement 1. 2. Methods In this research, the definition of ECM refers to the part of TCM that its initial resources were from abroad,
which covers plants, animals, and minerals or their processed products that have entered China through
various methods and have been used in Chinese medicine. ECM should: 1) be produced outside the
territory, relying solely on importers, such as frankincense, myrrh, benzoin, and storax; 2) be produced
outside and inside the territory, the initial resources were from abroad, such as turmeric, coix seed,
American ginseng, patchouli, winter melon seeds; 3) firstly produced outside the territory but later
introduced in China, such as white cardamom, pepper, cinchona, and coix seed. Under the guidance of the Page 3/15 Page 3/15 above three standards, we have assessed 8980 Chinese medicines recorded in Chinese Materia
Medica[17], Withing the help of Guangyao's Xu research[18]. The scientific name of plants was determined according to The Plant List database
(www.theplantlist.org) and the Chinese Flora database (www.iplant.cn/frps). The scientific name of the
animal resources was determined using www.marinespecies.org/index.php and a-z-animals.com. According to the relevant appendix data of the "Exotic Plants from China" and the Plant Introduction and
Conservation Database of the Xishuangbanna Tropical Botanical Garden of the Chinese Academy of
Sciences (http://sdb.xtbg.ac.cn), we analyzes the introduction pathway, time, and origin. Additional
information was collected from journal literature. A database of ECM based on the aforementioned, including scientific name, taxonomy information,
traditional efficacy, introduction route, introduction time, place of origin, the first application time (based
on history literature), and first medical literature. The drug application time was mainly based on the first
recording time of traditional Chinese medical literature. We have also referred to Chinese clinical
pharmacies and diagnostics to determine the traditional efficacy of ECM. Due to the complex relationship
between TCM and biological resources, this study used two standards for statistical analysis of
medicinal resources and clinical information: 1) ECM‘s data of medicinal resource of the original place,
family, introduction time, and introduction way were considered as statistic objects; 2) clinical
information on efficacy, indication, application time, and the total number of ECM, were set as statistics
objects. 3.1.1 The dominant family of ECM and its introduction route The percentage of botanical ECM was 98.70%. The botanical ECM was distributed among 94 families,
316 genera, and 469 species (including subspecies, varieties, and hybrids). The top 10 families were
Fabaceae, Asteraceae, Euphorbiaceae, Solanaceae, Amaranthaceae, Malvaceae, Lamiaceae, Myrtaceae,
Brassicaceae, and Liliaceae. The percentage of animal ECM was 0.84%: Corallium rubrum of Coralliidae,
Cairina moschata of Anatidae, Capra aegagrus hircus of Bovidae, and Castor canadensis of Castoridae. The percentage of mineral ECM was 0.42%: Agate of Oxide Quartz Group and the Manganite of oxide Page 4/15 Page 4/15 Page 4/15 rutile group. The most dominant family and its introduction route can be seen in Fig.1. The overall view
on families and their introduction route in China can be seen in Supplement 2. Fig.1: The most dominant family and its introduction route in China. Fig.1: The most dominant family and its introduction route in China. Fig.1: The most dominant family and its introduction route in China. Notes: Introduction Way: Af, afforestation; Co, compound; CR, cotton ramie; Dy, dye; FAV, fruits and
vegetables; FI, Food intake; Fo, forge; GM, green manure; Gr, grain; He, hedge; Me, medicinal; Na, natural;
Oi, oil; Or, ornamental; Pe, perfume; SC, sugar crop; ST, street trees; UI, unintentional; Wo, wood. 3.1.2 The origin place of ECM To assess the origin place of ECM, we used the continental division system of Biodiversity Information
Standards (www.tdwg.org). For species already described in Guangyao's Xu research on naturalized
plants in China, we have used these source information. The results are standardized according to the
Biodiversity Information Standards. The place of origin for the ECM were: 144 from South America, 96
from tropical Asia, 69 from temperate Asia, 67 from Oceania, 62 from Africa, 56 from North America, 42
from Europe, six unknown, five from the Pacific, two Paleotropic, two with hybrid origin (especially refers
to the hybrid plants of Chinese native plants and naturalized plants), one with origin of cultivation, and
six non-verified specific origins. 3.1.3 Introduction time and routes of ECM We confirmed the introduction time of the 410 species, and 65 species had not been verified. The annual
introduction rate of each dynasty is shown in Fig.2. A total of 33 species were introduced during Zhou
dynasty (1029–221BC) to Han dynasty (206 BC–220 AD), 24 species were introduced during Wei, Jin,
Southern, and Northern Dynasties (220–581 AD), 25 species introduced during Sui and Tang Dynasties
(581–907 AD), 26 species introduced during Song Jinyuan period (960–1368 AD), 35 species introduced
during Ming dynasty (1368–1644 AD), 95 species introduced during Qing dynasty (1636–1911 AD), 80
species introduced during the Republic period, and 172 species introduced during 1912–1999 AD. Fig.2: Annual average number of introduced species per dynasty. A total of 29 routes for the introduction of ECM in China were found. The main ones(Fig.3) were
ornamental (23.05%), fruit and vegetable (22.07%), unintentional introduction (13.41%), medicinal
(10.61%), and spice (5.61%). The ECM introduced by these routes accounted for 74.76% of the total. A
few species were found to be introduced in China in multiple ways (e.g., nutmeg through medicinal and
spice routes). Fig.3: Introduction routes of exotic Chinese medicines in China. Fig.3: Introduction routes of exotic Chinese medicines in China. 3.2.1 The efficacy of ECM The efficacy of TCM is recorded with a summary of diagnosis and treatment. The current Chinese clinical
medicine usually divides the efficacy of Chinese medicines into 22 major categories. We classified the
ECM based on efficacy and indication, Chinese medical diagnostics, and basic medical knowledge. And
then, we get the frequency of exotic Chinese medicines with various functions in each dynasty (Table 1),
which shows that the most introduced categories of ECM were "clear heat" and diuresis, suggesting that
local ECM may lack resources in these two areas. The efficacy of the 788 ECM here studied is distributed throughout the major of Chinese clinical medicine
The main efficacies found for the ECM were: "clearing heat," "promoting urination and draining,"
"promoting blood circulation and removing stasis," "hemostasis," "regulating Qi," "relieve cough and
asthma," "resolving phlegm," "counteracting toxic substances and insecticides, remove rotting skin and
muscle and convergence wet sores," "removing rheumatism," and "tonic." Table 1: Frequency of exotic Chinese medicines with various functions in each dynasty. 3.2.2 The indication of ECM The indications of TCM refer to the main clinical adaptation of the drug, also called the main adaptation
range. Ancient Chinese doctors obtain indications of Chinese medicine through clinical practice. The
descriptions of ECM indications come from different documents, thus we have analyzed and
standardized the treatment of the main treatment of each medicine. The detailed treatment process and
results are shown in Supplement 3. A total of 826 indications were reported for ECM. The top 50 were:
sore, carbuncle, fall injury, cough, dysentery, diarrhea, edema, rheumatic arthralgia, sore, boil, vomiting
blood, eczema, respite, jaundice, vomiting, hot eyes, sore throat, metrorrhagia and metro taxis, scald,
blood in stool, boil, difficulty in urination, stomach ache, hemoptysis, scabies, gangrene, headache,
traumatic bleeding, malaria, irregular menstruation, stomachache, diarrhea, hemorrhoids, leukorrhea,
sputum, leucorrhea, toothache, dysmenorrhea, lung fever, bloating, beriberi, blood, food accumulation,
constipation, hypertension, scrofula, snakebite, fracture, amenorrhea, and malnutrition. The sum of the
frequency of the top 50 accounts for 59% of all attending diseases. The top 10 frequencies were: 162,
151, 142, 129, 110, 80, 76, 63, 59, and 53. Half of the scope of the treatment of foreign Chinese medicine
belongs to skin diseases, urinary system diseases, trauma, respiratory diseases, digestive system
diseases, and gynecological diseases. This result is consistent with its main effects of clearing heat,
diuresis, activating blood to remove blood stasis, hemostasis, relieving cough, relieving cough and
asthma, and resolving phlegm. 4. Discussion
4.1 The dominant families of ECM Page 6/15 To clarify the unique superior families of ECM relative to TCM, we collected the family of 7812 non-
animal and mineral medicine (i.e., algae, lichens, and plant) included in the Chinese Materia Medica
(Supplement 4). The 7,812 non-animal Chinese medicine resources came from 8,394 species distributed
between 353 families. The top 20 families of TCM were: Fabaceae, Asteraceae, Lamiaceae, Rosaceae,
Ranunculaceae, Apiaceae, Liliaceae, Orchidaceae, Euphorbiaceae, Scrophulariaceae, Gramineae,
Rubiaceae, Asclepiadaceae, Urticaceae, Polygonaceae, Saxifragaceae, Rutaceae, Araliaceae, Lauraceae,
Polypodiaceae. The top 10 families of ECM were: Fabaceae, Asteraceae, Euphorbiaceae, Solanaceae,
Amaranthaceae, Malvaceae, Lamiaceae, Myrtaceae, Brassicaceae, Liliaceae. Thus, the common
dominant families of ECM and TCM were Fabaceae, Asteraceae, Euphorbiaceae, Lamiaceae, and
Liliaceae; The unique dominant families of ECM were Solanaceae, Amaranthaceae, Malvaceae,
Myrtaceae, and Cruciferae(Fig.4). Fig.4: The common and unique dominant families of exotic Chinese medicines compared with traditional
Chinese medicine. 4.2 Directly absorb and convert natural medicines from
foreign regions into TCM is Commendable. The top four introductory routes found were the ornamental route, the fruit and vegetable route, the
medicinal route, and the spice route. The number of ECM introduced through the ornamental route was
the highest. However, just 11 (pittosporum bark, elderberry, calendula, cassia, trumpet creeper, morning
glory, green box, opium, digitalis, poppy, and poppy husk) had high-quality clinical efficacy and
pharmacological effects. Although the number of ECM introduced through the fruit and vegetable route is
relatively high, they all appear as fruits and vegetables in supermarkets and vegetable markets, rather
than medicines in hospitals and pharmacies. The number of ECM introduced through medicinal routes
ranks third. More than half of ECM introduced through medicinal routes is commonly used in clinics of
Chinese medicine. They are used in hospitals and pharmacies as medicines. The 16 (asafoetida, benzoin,
citrus sinensis, aloes, cloves, dried ginger, pepper, horseradish, myrrh, ginger, nutmeg, frankincense, ginger,
sesame seeds, cumin, and cumin)items introduced through the spice route in Chinese clinical medicine
have the effect of "warming the spleen" and "regulating the stomach, " mainly treating digestive system
diseases. This feature is consistent with the current application of spices. To sum up, directly absorb and
convert natural medicines from foreign regions into TCM is Commendable(Fig.5). Fig.5: Directly absorb and convert natural medicines from foreign Regions into traditional Chinese
medicine is Commendable. 4.4 About One-fifth of commonly used TCM in the clinic is
ECM According to Chinese Materia Medica, the total resources of TCM is 8980. Based on our result, the total
resources of ECM are 788. So the percentage of ECM accounts for TCM in its total resources is 8.8%. But
the key point is the percentage of commonly used exotic Materia Medica in clinics (CECM) accounts for
the commonly used traditional Chinese medicine (CTCM). Now, the current official standards of CTCM
have not yet been established[19–23]. To obtain the percentage of the CECM accounts for the CTCM, we
chose the drugs recorded in The Identification of Traditional Chinese Medicine as the CTCM. The
Identification of Traditional Chinese Medicine is a book of great reference value, which recorded 240
botanical CTCM[24]. With the help of this book, we analyzed the characteristic of different medicinal
parts that the CECM accounts for the CTCM(Table 2). Among them, the more prominent is the resin
medicinal herbs that account for 100%, which means all of the resin TCM clinically commonly used in
clinics belongs to ECM. They are storax, frankincense, myrrh, Asafoetida, benzoin, and dragon's blood. Then it is worth noting that the most frequently used medicinal sites are fruits and seeds. The frequency
of clinically commonly used in clinic ECM in fruits and seeds is as high as 13(nutmeg, semen cassiae,
boat-fruited scaphium seed, medicine terminalia fruit, fennel fruit, semen strychni, Semen Pharbitidis,
henbane, common carpesium fruit, Areca catechu, fructus amomi, fructus amomi rotundus, galanga
galangal seed). Last but not least, the percentage of the CECM accounts for the CTCM is 17.1%. Table 2: The characteristic of different medicinal parts that the CECM accounts for the CTCM 4.3 Trends in the exchange of medical information between
China and other regions Page 7/15 Because the time of different dynasties existed and the number of ECM that were introduced during that
dynasty were different, we have use 1000 times OF the annual average number of ECM in each dynasty
to analyze the tendency (Fig.6). The introduction rate of ECM with different efficacies generally peaked
during the Han (206 BC–220 AD), Tang (618–907 AD), and Ming dynasties (1368–1644 AD). In the late
Qing dynasty, most types of ECM began to accelerate sharply, in line with the rapid acceleration of the
material and information exchange between the East and the West. The exchange speed of medical
resources and information between China and other regions increased sharply from 1912 onwards. Fig.6: The annual average*1000 of exotic Chinese medicines introduced with different efficacies in each
dynasty. Fig.6: The annual average*1000 of exotic Chinese medicines introduced with different efficacies in each
dynasty. 5. Conclusion Funding
Not applicable
Authors' Contributions
Zhuyun Yan: Conceptualization, Methodology, Writing- Review & Editing, and Sup
Qingqing Cai: Investigation, Writing-Original Draft. 5. Conclusion Although the percentage of ECM accounts for TCM in its total resources is 8.8%, the percentage of the
CECM accounts for the CTCM is 17.1%.This suggests that Chinese doctors are more concerned with the
efficacy of the drug when using it in clinical settings than whether the drug comes from afar. Although in
the past, the use of distant drugs meant more money to spend. The famous American Orientalist Laufer
said: "The Chinese are a well-thought-out, sensible, and open-minded nation. They have always been Page 8/15 Page 8/15 willing to accept good things from outsiders[25]" Chinese medicine uses foreign medicine resources for
its effectiveness in treatment. Therefore, proactive absorption is the main attitude of Chinese
pharmacists when facing effective foreign drugs. The record of origin and source was to ensure the
supply and quality of the drugs. Both the accumulation of effective drugs and edible food play an
important role in evolution. The use of foreign resources as Chinese medicine is the fact that Chinese
medicine objectively exists. The existence of ECM has enriched the resources of TCM. Furthermore, both
foreign medicine and local medicine currently protect people's lives and health. Page 9/15
important role in evolution. The use of foreign resources as Chinese medicine is the fac
medicine objectively exists. The existence of ECM has enriched the resources of TCM. F
foreign medicine and local medicine currently protect people's lives and health. Abbreviations
ECM Exotic Chinese medicine. TCM Traditional Chinese medicine. CECM Commonly used Exotic Materia Medica in clinics. CTCM Commonly used Traditional Chinese medicine in clinics. Declarations
Ethics approval and consent to participate
Not applicable
Consent to publish
Not applicable
Availability of data and materials
All data generated or analyzed during this study are included in this published article. Competing interests
The authors declare that they have no competing interests. Funding
Not applicable
Authors' Contributions
Zhuyun Yan: Conceptualization, Methodology, Writing- Review & Editing, and Supervisio
Qingqing Cai: Investigation, Writing-Original Draft. Abbreviations
ECM Exotic Chinese medicine. TCM Traditional Chinese medicine. CECM Commonly used Exotic Materia Medica in clinics. CTCM Commonly used Traditional Chinese medicine in clinics. Declarations
Ethics approval and consent to participate
Not applicable
Consent to publish
Not applicable
Availability of data and materials
All data generated or analyzed during this study are included in this published art
Competing interests
The authors declare that they have no competing interests. Abbreviations Zhuyun Yan: Conceptualization, Methodology, Writing- Review & Editing, and Supervision. Qingqing Cai: Investigation, Writing-Original Draft. Qingqing Cai: Investigation, Writing-Original Draft. Page 9/15 Correspondence to Zhuyun Yan. Correspondence to Zhuyun Yan. Acknowledgements We are very grateful to all the works of predecessors that had done. We are very grateful to all the
databases of which help us greatly. Finally, we wish to acknowledge our family and friends. References 1. Hongzhu L. A brief history of the importation of aromatic medicines from Arab countries into China. Chinese J. Ethn. Med. China; 1999. p. 165. 2. Menghua W, Zhongzhen Z, Hui C. Transmission and development of foreign medicinal materials in the
Tang and Song Dynasties. Chinese J. Chinese Mater. Medica. China; 2016. p. 4076–82. 3. Cheng Z, Yikui L. Examination of Foreign Medicines in Past Dynasties. J Chengdu Univ TCM. China;
1980;06:1–5. 4. Xiaoyan S, Lili X, Wang X. Study on foreign drugs in herbal medicines in “Compendium of Materia
Medica.” J Tradit Chinese Med Lit. China; 2013;31:7–8. 5. Xiaolei T, Yong P, Lu W. Study on the collation of foreign drugs included in “Compendium of Materia
Medica.” J Chinese Med Mater. China; 2014;37:2099–102. 6. Linrong X, Shenchu Y. A Brief Discussion on Importation of Foreign Drugs and the Development of
Traditional Chinese Medicine. Fujian Tradit Chinese Med. China; 2020;05:557–86. 7. Zhang J, Chun W, Wenting F, Yingli Z, Jingxia W, Shaohong C, et al. Strategies and approaches on
introducing foreign medicine to TCM. China J. Tradit. Chinese Med. Pharm. China; 2017. p. 1425–30. 8. Xiong X. The description and use of foreign medicines in Ming and Qing China: centered on
Supplements to Compendium of Materia Medica". Res Med Soc Hist. China; 2020;5:53–63. 9. Jianjun Z, Wenting F, Chun W. Concept and research range of overseas Chinese medicine. China J
Tradit Chinese Med Pharm. China; 2019;34:61–4. 10. Huasheng P, Luqi H. Exotic drugs in “The Sketch Book.” Chinese J Med Hist . China; 2021;51:12–23. 11. Rong W, Xiaoyang H, Bin Y, Guangsong W. Discussion on the Concept of Fragrant Medicinal. Inf Tradit
Chinese Med. China; 2020;37:100–2. 12. Wang L, Wang C, Hou Y, Zhang M, Zhang J. Construction and application of modern research
paradigm of new foreign traditional Chinese medicine. China J Chinese Mater Medica. China;
2020;45:967–77. Page 10/15 13. Zhiyong L, Hongjun Y, Shihuan T, Bin Y, Weike J, Luqi H. Study strategies on medicinal properties of
foreign medicine introduced to traditional Chinese medicine. China J Chinese Mater Medica. China;
2019;44:2972–9. 14. Minru J, Tiantian W, Xiaolin L, Yuying M. Exploration on an overview of history (from Chunqiu to Ming
and Qing Dynasties) and Chinese exotic traditional medicine species. China J Chinese Mater Medica. China; 2017;42:1659–67. 15. Wanping R, Fuxiang G, Bingchen H. The material and culture exchanges between China and foreign
regions in the 17th and 18th centuries. References Xiamen Univ. Press. Xiamen, China; 2017. 16. Cuifang W. Research on Exotic Fragrant Medicine in Medieval China, Science Press. Beijing, China;
2016. 17. Medicine SA of TC. Chinese Materia Medica. Shanghai Sci. Technol. Press. Shanghai, China; 1999. 18. Guangyao X, Hongyuan L, Mo XQ. Composition and spatial-temporal distribution of Chinese
naturalized plants. Chinese J Plant Ecol. China; 2019;43:601–10. 19. Xinzhong Q. Colored Illustrated Book of Chinese Materia Medica. Common Chinese Medicines. Beijing, China: People’s Health Press; 1996. 20. Yuefeng L, Xingke Y. Hand-drawn color atlas of commonly used Chinese medicines. Lanzhou, China:
Science and Technology Press; 2018. 21. Guijun Z, RenWei L, GuoLian L. Identification of physical constants of commonly used Chinese
medicines. Beijing, China: Chemical Industry Press; 2005. 22. Huiguang X, Yarong H. Commonly used Chinese medicine. China; 1994. 23. Chunshan Z, Hua Z, LianWu X. Handbook of commonly used Chinese medicines and their active
ingredients. China; 2017. 24. Yingfang W. Chinese Medicine Identification. Shanghai, China: Shanghai Science and Technology
Press; 2010. 25. Laufer B. SINO-IRANICA: Chinese Contributions to the History of Civilization in Ancient Iran-With
Special Reference to the History of Cultivated Plants and Products,. Chicago; 1964. Tables Due to technical limitations, table 1 and 2 is only available as a download in the Supplemental Files
section. Page 11/15 Page 11/15 Figures
Figure 1 Figures
Figure 1
The most dominant family and its introduction route in China. Figure 2 Figures Figures Figures
Figure 1 Figure 1
The most dominant family and its introduction route in China. Figure 1 Page 12/15
The most dominant family and its introduction route in China. Figure 2 The most dominant family and its introduction route in China. The most dominant family and its introduction route in China. Page 12/15
The most dominant family and its introduction route in China. Figure 2 Figure 2 Annual average number of introduced species per dynasty. Figure 3
Introduction routes of exotic Chinese medicines in China. Annual average number of introduced species per dynasty. Annual average number of introduced species per dynasty. Figure 3 Introduction routes of exotic Chinese medicines in China. Introduction routes of exotic Chinese medicines in China. Introduction routes of exotic Chinese medicines in China. Introduction routes of exotic Chinese medicines in China. Introduction routes of exotic Chine Page 13/15 Figure 4 The common and unique dominant families of exotic Chinese medicines compared with traditional
Chinese medicine. The common and unique dominant families of exotic Chinese medicines compared with traditional
Chinese medicine. gure 5
rectly absorb and convert natural medicines from foreign Regions into traditional Chinese medicine Figure 5 Directly absorb and convert natural medicines from foreign Regions into traditional Chinese medicine is
Commendable. Page 14/15 Figure 6 Figure 6 The annual average*1000 of exotic Chinese medicines introduced with different efficacies in each
dynasty. The annual average*1000 of exotic Chinese medicines introduced with different efficacies in each
dynasty. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Table1.xlsx Table2.xlsx supplement1.xlsx supplement2.xlsx supplement3.xlsx supplement4.xlsx Graphicalabstract.tif Page 15/15
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Intradiscal treatment of the cartilage endplate for improving solute transport and disc nutrition
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Frontiers in bioengineering and biotechnology
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UCSF
UC San Francisco Previously Published Works
Title
Intradiscal treatment of the cartilage endplate for improving solute transport and dis
nutrition
Permalink
https://escholarship.org/uc/item/7rg6q8nt
Authors
Habib, Mohamed
Hussien, Shayan
Jeon, Oju
et al.
Publication Date
2023
DOI
10.3389/fbioe.2023.1111356
Peer reviewed UCSF
UC San Francisco Previously Published Works
Title
Intradiscal treatment of the cartilage endplate for improving solute transport and dis
nutrition
Permalink
https://escholarship.org/uc/item/7rg6q8nt
Authors
Habib, Mohamed
Hussien, Shayan
Jeon, Oju
et al. Publication Date
2023
DOI
10.3389/fbioe.2023.1111356
Peer reviewed UCSF
UC San Francisco Previously Pub
Title
Intradiscal treatment of the cartilage endplate
nutrition
Permalink
https://escholarship.org/uc/item/7rg6q8nt
Authors
Habib, Mohamed
Hussien, Shayan
Jeon, Oju
et al. Publication Date
2023
DOI
10.3389/fbioe.2023.1111356
Peer reviewed OPEN ACCESS Mohamed Habib 1,2, Shayan Hussien1, Oju Jeon3, Jeffrey C. Lotz1,
Peter I-Kung Wu1, Eben Alsberg3 and Aaron J. Fields1* 1Department of Orthopaedic Surgery, University of California, San Francisco, San Francisco, CA,
United States, 2Department of Mechanical Engineering, Al Azhar University, Cairo, Egypt, 3Department of
Biomedical Engineering, University of Illinois, Chicago, IL, United States Poor nutrient transport through the cartilage endplate (CEP) is a key factor in the
etiology of intervertebral disc degeneration and may hinder the efficacy of
biologic strategies for disc regeneration. Yet, there are currently no treatments
for improving nutrient transport through the CEP. In this study we tested whether
intradiscal delivery of a matrix-modifying enzyme to the CEP improves solute
transport into whole human and bovine discs. Ten human lumbar motion
segments harvested from five fresh cadaveric spines (38–66 years old) and
nine bovine coccygeal motion segments harvested from three adult steers
were treated intradiscally either with collagenase enzyme or control buffer that
was loaded in alginate carrier. Motion segments were then incubated for 18 h at
37 °C, the bony endplates removed, and the isolated discs were compressed under
static (0.2 MPa) and cyclic (0.4–0.8 MPa, 0.2 Hz) loads while submerged in
fluorescein tracer solution (376 Da; 0.1 mg/ml). Fluorescein concentrations
from site-matched nucleus pulposus (NP) samples were compared between
discs. CEP samples from each disc were digested and assayed for sulfated
glycosaminoglycan
(sGAG)
and
collagen
contents. Results
showed
that
enzymatic treatment of the CEP dramatically enhanced small solute transport
into the disc. Discs with enzyme-treated CEPs had up to 10.8-fold (human) and
14.0-fold (bovine) higher fluorescein concentration in the NP compared to site-
matched locations in discs with buffer-treated CEPs (p < 0.0001). Increases in
solute transport were consistent with the effects of enzymatic treatment on CEP
composition, which included reductions in sGAG content of 33.5% (human) and
40% (bovine). Whole disc biomechanical behavior—namely, creep strain and disc
modulus—was similar between discs with enzyme- and buffer-treated CEPs. Taken
together,
these
findings
demonstrate
the
potential
for
matrix
modification of the CEP to improve the transport of small solutes into whole
intact discs. Front. Bioeng. Biotechnol. 11:1111356. doi: 10.3389/fbioe.2023.1111356 COPYRIGHT
© 2023 Habib, Hussien, Jeon, Lotz, Wu,
Alsberg and Fields. This is an open-access
article distributed under the terms of the
Creative Commons Attribution License
(CC BY). KEYWORDS cartilage endplate, intervertebral disc degeneration, biotransport, spine biomechanics
low back pain, tissue engineering Powered by the California Digital Library
University of California Powered by the California Digital Library
University of California eScholarship.org TYPE Original Research
PUBLISHED 27 February 2023
DOI 10.3389/fbioe.2023.1111356 TYPE Original Research
PUBLISHED 27 February 2023
DOI 10.3389/fbioe.2023.1111356 cartilage endplate, intervertebral disc degeneration, biotransport, spine biomechanics,
low back pain, tissue engineering OPEN ACCESS The use, distribution or
reproduction in other forums is
permitted, provided the original author(s)
and the copyright owner(s) 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. frontiersin.org Intradiscal treatment of the
cartilage endplate for improving
solute transport and disc nutrition OPEN ACCESS
EDITED BY
Graciosa Q. Teixeira,
Universität Ulm, Germany
REVIEWED BY
Yong-Can Huang,
Peking University, China
Ryan Michael Porter,
University of Arkansas for Medical
Sciences, United States
*CORRESPONDENCE
Aaron J. Fields,
aaron.fields@ucsf.edu
SPECIALTY SECTION
This article was submitted to
Biomaterials, a section of the journal
Frontiers in Bioengineering and
Biotechnology
RECEIVED 29 November 2022
ACCEPTED 09 February 2023
PUBLISHED 27 February 2023
CITATION
Habib M, Hussien S, Jeon O, Lotz JC,
Wu PI-K, Alsberg E and Fields AJ (2023),
Intradiscal treatment of the cartilage
endplate for improving solute transport
and disc nutrition. Front. Bioeng. Biotechnol. 11:1111356. doi: 10.3389/fbioe.2023.1111356
COPYRIGHT
© 2023 Habib Hussien Jeon Lotz Wu OPEN ACCESS
EDITED BY
Graciosa Q. Teixeira,
Universität Ulm, Germany
REVIEWED BY
Yong-Can Huang,
Peking University, China
Ryan Michael Porter,
University of Arkansas for Medical
Sciences, United States
*CORRESPONDENCE
Aaron J. Fields,
aaron.fields@ucsf.edu
SPECIALTY SECTION
This article was submitted to
Biomaterials, a section of the journal
Frontiers in Bioengineering and
Biotechnology
RECEIVED 29 November 2022
ACCEPTED 09 February 2023
PUBLISHED 27 February 2023
CITATION
Habib M, Hussien S, Jeon O, Lotz JC,
Wu PI-K, Alsberg E and Fields AJ (2023),
Intradiscal treatment of the cartilage
endplate for improving solute transport
and disc nutrition. Front. Bioeng. Biotechnol. 11:1111356. doi: 10.3389/fbioe.2023.1111356 Frontiers in Bioengineering and Biotechnology 2.2 Human tissues Five human cadaveric lumbar spines (age range: 38–66 years old;
mean age: 56 ± 10 years) were obtained from donors with no medical
history of musculoskeletal disorders (UCSF Willed Body Program). From these spines, ten intact motion segments (2 motion segments
per spine) were harvested and randomly assigned to two groups (n =
5 motion segments/group; Table 2); collagenase – 0.15 U in 50 µl of
alginate carrier; or control buffer–HBSS in 50 µL of alginate carrier). Prior to spine harvest all cadavers underwent MR imaging (GE
Discovery MR 750 W 3T; GE Healthcare). Imaging consisted of a
sagittal fast spin-echo T2-weighted sequence and a 3D multi-echo
UTE cones mapping sequence. Sequence parameters were identical
to those reported previously (Wang et al., 2021). To ensure that both
motion segments from the same donor were structurally intact and
of a similar degree of degeneration, we selected motion segments
from levels with intact CEPs and similar Pfirrmann degeneration
grades (Supplementary Materials). Motivated by those findings, we explored a matrix modification
strategy for improving disc nutrition that involves enzymatically
reducing the amounts of collagen and aggrecan in the impermeable
and fibrotic CEPs (Dolor et al., 2019). Treatment of human
cadaveric CEPs with recombinant human MMP-8 enzyme, which
is one of the matrix metalloproteinases that has a high specificity for
type II collagen and aggrecan, reduced sGAG content in a dose-
dependent manner. Importantly, reductions in CEP matrix content
with MMP-8 treatment improved the NP cell viability inside
diffusion chambers, indicating improved CEP permeability to
nutrients (Dolor et al., 2019). Although the diffusion chambers
represent a useful model system for studying the effects of CEP
permeability enhancement, the chambers do not mimic the complex
microenvironment or loading of an intact intervertebral disc, and
thus, the effects of CEP permeability enhancement on solute
transport into whole, intact discs remain unclear. The objectives
of this study were to: 1) develop a model for the controlled,
intradiscal delivery of a matrix-modifying enzyme to the CEP;
and 2) measure solute transport into whole human and bovine 2.1 Bovine tissues Nine intact bovine coccygeal motion segments were harvested
from three oxtails (3 segments per oxtail) acquired within 2 h of
slaughter from a local abattoir (approximate age range of the steers:
18–22 months old). After harvesting the motion segments, a 17G
Crawford
needle
with
a
plasma-mediated
coblation
wand
(ArthroCare
2000)
was
inserted
through
the
posteriolateral
annulus directed toward one CEP in each disc, the wand was
energized on a low-power setting (2 W) for 10 s as the needle
was advanced to the distal annulus, and the coblated tissue was
debrided with PBS. This coblation step was necessary for
overcoming the high back pressure in the otherwise healthy
bovine discs. Following coblation, the wand was removed with
the needle in place, and the motion segments were randomly
assigned
to
three
groups
(n
=
3
motion
segments/group;
Table 1): collagenase—0.15 U in 50 µl of slow-release alginate
carrier; control buffer–HBSS in 50 µl of alginate carrier; or
sham–needle insertion only. g
p
Proper disc nutrition involves nutrient and metabolite exchange
between the nucleus pulposus (NP) cells and vertebral capillaries
(Maroudas et al., 1975; Nachemson et al., 1970; Ohshima et al., 1989;
Urban et al., 1977), and several factors can impair the normal
patterns of solute exchange. For example, nutrients entering the
disc and exiting metabolites must pass through the cartilage
endplates (CEPs), and solute passage could be slowed or blocked
by changes to the composition of the CEP matrix, including
dehydration (Antoniou et al., 1996), mineralization (Wong et al.,
2019; Benneker et al., 2005; Grant et al., 2016) and fibrosis
(Antoniou et al., 1996; Wong et al., 2019). For example, we
found that CEPs with higher amounts of collagen, sulfated
glycosaminoglycan
(sGAG),
and
mineral
hindered
nutrient
diffusion, thereby impairing NP cell survival and function inside
diffusion chambers (Wong et al., 2019). Moreover, those same
deficits
in
CEP
composition
were
also
associated
with
significantly worse disc degeneration in low back pain patients
(Bonnheim et al., 2022). 1 Introduction Back pain is the leading cause of disability and is a major public health problem
worldwide (Haralson & Zuckerman, 2009; Vassilaki & Hurwitz Dc, 2014; Matta et al.,
2017; Fernandez-Moure et al., 2018). In certain subgroups of back pain patients,
intervertebral disc degeneration may cause pain (Chou et al., 2011). Current medical Frontiers in Bioengineering and Biotechnology 01 frontiersin.org Habib et al. 10.3389/fbioe.2023.1111356 interventions for disc degeneration are surgical in nature and are
often unsuccessful, which motivates development of non-
operative alternatives. Non-operative approaches to regenerate
the disc and alleviate pain include delivering genes (Woods et al.,
2011), growth factors (Bae & Masuda, 2011; Ju et al., 2022), cells
(Velikonja et al., 2014; Ju et al., 2022), or other small molecules
(Gawri et al., 2013; Wang et al., 2021) to promote cell
proliferation,
stimulate
anabolic
activities,
and
reduce
catabolism and inflammation. Importantly, these approaches
all require a rich nutrient supply to sustain higher cell
numbers
or
metabolic
rates
(Horner
&
Urban,
2001;
Fernandez-Moure et al., 2018). However, disc degeneration in
humans is typically accompanied by a restricted nutrient supply
(Fields et al., 2018; Huang et al., 2014; Urban et al., 2004), which
may limit the clinical utility and effectiveness of these therapies
(Huang et al., 2014; Zhu et al., 2016; Binch et al., 2021; Ju et al.,
2022). Yet, there are no existing treatments to improve disc
nutrient supply. Developing treatment strategies to improve
disc nutrition may therefore aid in slowing or reversing
degeneration, and could enhance the regenerative potential of
biologic therapies that increase nutrient demands inside the disc. discs following intradiscal treatment of the CEP with a matrix-
modifying enzyme. discs following intradiscal treatment of the CEP with a matrix-
modifying enzyme. discs following intradiscal treatment of the CEP with a matrix-
modifying enzyme. Frontiers in Bioengineering and Biotechnology frontiersin.org 2.4 Intradiscal CEP treatment TABLE 1 Bovine coccygeal (CC) disc allocation to study groups. TABLE 1 Bovine coccygeal (CC) disc allocation to study groups. Steer #
Sham
Control buffer
Collagenase
Bovine 1
CC3
CC1
CC2
Bovine 2
CC1
CC2
CC3
Bovine 3
CC2
CC3
CC1 Enzymatic treatment or control buffer was delivered to the
CEP under fluoroscopic guidance. Briefly, a 17G Tuohy needle
(Medline,
item:
PAIN8001)
was
inserted
through
the
posterolateral annulus fibrosus and advanced to the central
region of the superior CEP in each disc (Figure 1). After
needle placement, 50 µL of either collagenase or control
buffer that was suspended in the alginate carrier was injected
to
the
CEP,
and
then
fibrin
sealant
(TISSEEL,
Baxter
International Inc., CA, United States) was applied to the
annulus during needle retraction. All motion segments were
then incubated in protease inhibitor cocktail dissolved in PBS
buffer according to the manufacturer’s protocol (Sigma-Aldrich,
catalog number P2714) for 18 h at 37 °C. method (Jeon et al., 2017). Briefly, sodium alginate (10 g) was
dissolved
in
ultrapure
deionized
water
(diH2O,
900 ml)
overnight. Sodium periodate (543 and 1,085 mg) was dissolved
in 100 ml diH2O and added to separate alginate solutions to
achieve different degrees of theoretical alginate oxidation (5%
and 10%, respectively) under stirring in the dark at room
temperature for 24 h. The OAs were purified by dialysis
against diH2O (MWCO 3500; Spectrum Laboratories Inc.) for
3
days,
treated
with
activated
charcoal
(0.5
mg/100ml,
50–200 mesh, Fisher) for 30 min, filtered (0.22 μm filter) and
lyophilized. The actual oxidation (4.2% ± 0.5% and 8.5% ± 0.3%)
was determined by using the AmpliteTM Colorimetric Aldehyde
Quantitation
Kit
(AAT
Bioquest
Inc.)
according
to
the
manufacturer’s instructions. 2.3 Alginate preparation For alginate purification, sodium alginate (10 g, Protanal LF 20/
40) was dissolved in 1,000 ml ultrapure deionized water (diH2O),
dialyzed against diH2O (MWCO 3500; Spectrum Laboratories Inc.)
for 3 days, treated with activated charcoal (0.5 mg/100mL,
50–200 mesh, Fisher) for 30 min, filtered (0.22 μm filter) and
lyophilized. Oxidized alginates (OA) were prepared by reacting sodium
alginate (Protanal LF 20/40, 196,000 g/mol, FMC Biopolymer)
with sodium periodate (Sigma) using a previously described 02 frontiersin.org Habib et al. 10.3389/fbioe.2023.1111356 2.5 Transport experiment A custom setup was used to evaluate the effects of CEP
permeability enhancement on solute transport into the disc
(Figure 2). To eliminate any potentially confounding effects of
differences in bony endplate structure within and between
species, the inferior and superior bony endplates were carefully
removed from the motion segments with a burring tool (Figure 3A). Several x-ray images (Faxitron Cabinet X-ray Systems model
43855A) were taken during the bone removal process to ensure We compared the release kinetics of the different alginate
formulations (0% oxidation, 5% oxidation, 10% oxidation) in an
ex-situ pilot study (Supplementary Materials), and based on the
observed release kinetics, we used the 0% oxidation alginate for all
intradiscal injections. FIGURE 1
Intradiscal delivery of collagenolytic enzyme treatment via 17G Tuohy needle to superior CEP of (A) bovine coccygeal and (B) human lumbar
intervertebral discs under fluoroscopic guidance. FIGURE 1
Intradiscal delivery of collagenolytic enzyme treatment via 17G Tuohy needle to superior CEP of (A) bovine coccygeal and (B) human lumbar
intervertebral discs under fluoroscopic guidance. TABLE 2 Human cadaver demographic data and lumbar disc allocation to study groups. TABLE 2 Human cadaver demographic data and lumbar disc allocation to study groups. Control buffer
Collagenase
Donor #
Age (yrs)
Sex (F/M)
BMI (kg/m^2)
level
Pfirrmann Grade
level
Pfirrmann Grade
Donor 1
56
F
18.3
L2-L3
III
L3-L4
III
Donor 2
57
F
24.8
L4-L5
III
L3-L4
III
Donor 3
25
M
23.7
L3-L4
II
L4-L5
II
Donor 4
61
M
27.4
L3-L4
III
L4-L5
III
Donor 5
63
F
21.1
L4-L5
III
L3-L4
III 03 frontiersin.org Habib et al. 10.3389/fbioe.2023.1111356 FIGURE 2
Experimental setup (A) and the compression testing protocol (B) of the whole intact disc while being immersed in fluorescein tracer solution. FIGURE 2
Experimental setup (A) and the compression testing protocol (B) of the whole intact disc while being immersed in fluorescein tracer solution. pressure range measured during various static postures (0.2 MPa)
(Nachemson, 1966; Wilke, et al., 1999), followed by 1.6 h of cyclic
compressive loads resulting in a pressure range (0.4–0.8 MPa) and
frequency (0.2 Hz) that is similar to those measured in the disc
during walking (Wilke, et al., 1999). 2.7 Fluorescein concentration NP tissue digests were assayed for fluorescence on a
SpectraMax iD5 microplate reader (Molecular Devices, San
Jose, CA), and the fluorescence readings (ex. 485 nm, em. 525 nm) referenced to a standard curve of known fluorescein
concentrations. Fluorescein
concentrations
inside
the
NP
samples were calculated based on the measured water content
from lyophilization and were compared between site-matched
locations in the discs having collagenase-treated vs buffer-
treated CEPs. the complete removal of all bone tissues and to calculate the cross-
sectional area of the isolated discs (ImageJ, NIH). The isolated discs were then sandwiched between porous
stainless-steel platens (0.8 mm hole diameter, 1.8 mm thickness
and 17.9% hole coverage) and open-cell porous aluminum foam
blocks with similar porosity and stiffness of vertebral trabecular
bone (Duocel® aluminum foam disc, 7%–9% relative density, ERG
Aerospace Corp, Oakland, CA) while submerged in 200 mL of
fluorescein
tracer
solution
(376 Da;
0.1 mg/mL
in
PBS)
(Figure 2). A servo-hydraulic load frame (MTS Bionix 858; MTS
Systems Corp., Eden Prairie, MN) was used to apply loads resulting
in physiologic disc pressures. Specifically, we applied 6 h of static
compression loads that represent the lower end of intradiscal FIGURE 3 (A) Prior to compression testing, whole discs were isolated from
the surrounding soft tissue, and the bony endplate was removed with a
burring tool. Circle indicates alginate gel placement underneath the
superior CEP as in Figure 1B. (B) After compression testing, discs
were snap-frozen in liquid nitrogen and two 5 mm-wide mid-sagittal
slabs were removed for further biochemical and fluorescein
measurement. Circles indicate the locations of CEP punches
harvested for biochemical measurements. (C) Mid-sagittal slab
showing NP tissue sample positions (three 2 mm punches per inferior-
superior positions) relative to treated and un-treated CEPs. (D) Two
4 mm-diameter punches were harvested from the central region of
each CEP in the mid-sagittal slab (B). Frontiers in Bioengineering and Biotechnology frontiersin.org 2.6 NP and CEP tissue sampling Immediately after the transport experiments, discs were snap
frozen in liquid nitrogen to prevent any further fluorescein
dispersion. Next, two 5-mm-thick-coronal slabs were cut with a
low-speed diamond saw (IsoMet, Buehler, IL) (Figure 3B). From
each slab, three NP samples were collected using a 2-mm biopsy
punch at four superior-inferior locations (Figure 3C). The superior
and inferior CEP layers were then carefully removed from the discs
and trimmed of any residual NP tissue (Figure 3D). Two CEP
samples were collected from each intact CEP using a 4-mm biopsy
punch (Figure 3D). All NP and CEP samples were massed on
microbalance, dried by lyophilization, and completely digested in
papain for subsequent assays. 2.8 CEP biochemical composition FIGURE 5
(A) In the bovine discs that underwent collagenase treatment,
sGAG content in the treated (superior) CEP was significantly lower
than the sGAG content in the untreated (inferior) CEPs (p = 0.03,
inferior versus superior paired t-test; mean ± SD; n = 3 discs/
group) (B) In the human discs that underwent collagenase treatment,
sGAG content in the treated (superior) CEPs was significantly lower
than the sGAG content in the untreated (inferior) CEPs (p < 0.01,
inferior versus superior paired t-test; mean ± SD; n = 5 discs/group). FIGURE 4
(A) The average fluorescein concentration (mean ± SD; n =
3 discs/group) was significantly higher in the bovine NP proximal to
the collagenase-treated superior CEP, position A (a p < 0.0001 vs
control buffer; b p < 0.0001 vs sham; p > 0.10 all other
comparisons). (B) The average fluorescein concentration (mean ± SD;
n = 5 discs/group) was significantly higher in the human NP proximal
to the collagenase-treated superior CEP, position A (a p < 0.0001). FIGURE 5
(A) In the bovine discs that underwent collagenase treatment,
sGAG content in the treated (superior) CEP was significantly lower
than the sGAG content in the untreated (inferior) CEPs (p = 0.03,
inferior versus superior paired t-test; mean ± SD; n = 3 discs/
group) (B) In the human discs that underwent collagenase treatment,
sGAG content in the treated (superior) CEPs was significantly lower
than the sGAG content in the untreated (inferior) CEPs (p < 0.01,
inferior versus superior paired t-test; mean ± SD; n = 5 discs/group). FIGURE 4
(A) The average fluorescein concentration (mean ± SD; n =
3 discs/group) was significantly higher in the bovine NP proximal to
the collagenase-treated superior CEP, position A (a p < 0.0001 vs
control buffer; b p < 0.0001 vs sham; p > 0.10 all other
comparisons). (B) The average fluorescein concentration (mean ± SD;
n = 5 discs/group) was significantly higher in the human NP proximal
to the collagenase-treated superior CEP, position A (a p < 0.0001). 3 Results a microplate reader at 595 nm and referenced to a standard curve of
known chondroitin sulfate concentrations. 2.10 Statistics Mixed effects models accounting for multiple measurements per
subject via random intercept were used to estimate the least-squares
mean fluorescein concentration at each NP location and the least-
squares mean sGAG and collagen content in the CEP, and Tukey
HSD post hoc tests were used to test for differences between groups. One-way ANOVA was used to compare the whole-disc compressive
creep strain and modulus between groups. Statistical analyses were
performed in JMP Pro 15; p < 0.05 was considered statistically
significant. 2.9 Whole disc biomechanical properties The axial force, crosshead displacement and time data were
collected during the transport experiment and processed with a
custom script (MATLAB, Mathworks; Natwick, MA) that calculates
the disc modulus and the total creep strain. Disc modulus was
considered as the slope of the stress-strain curve at maximum value
of the stress in the linear part of the static compression phase. The
total creep strain was considered as the ratio between total
displacement at the end of the static compression phase and the
initial height of the disc. Similarly, intradiscal enzymatic treatment of the CEP improved
solute transport into human lumbar discs (Figure 4B). The
fluorescein concentration in the NP near to the superior treated
CEP, at position A, was 10.8-fold higher inside the discs that had
CEPs treated with collagenase than in the discs that had CEPs
treated with the control buffer (55.27 ± 31.11 μg/mL vs 5.14 ±
3.54 μg/mL; p < 0.0001). In the discs with buffer-treated CEPs,
fluorescein concentration varied with position; concentrations were
generally highest adjacent to the CEPs, positions A (5.14 ± 3.54 μg/
ml) and D (7.14 ± 2.26 μg/ml), and lowest in the center of the discs,
at positions B (0.58 ± 0.18 μg/ml) and C (0.62 ± 0.19 μg/ml). Frontiers in Bioengineering and Biotechnology 3.1 Effects of enzymatic treatment on solute
transport Aliquots of CEP digests were also hydrolyzed in 6 N HCL for
18 h at 110°C. Neutralized acid hydrolysates were then assayed
for total collagen content using a chloramine-T colorimetric
assay
(Fields
et
al.,
2014),
referencing
the
absorbance
measurements to a standard curve of known hydroxyproline
concentrations. Intradiscal enzymatic treatment of the CEP significantly
improved
solute
transport
into
the
bovine
coccygeal
discs
(Figure 4A). Specifically, in the NP near to the superior treated
CEP, fluorescein tracer concentration was 14-fold higher in the discs
that had CEPs treated with collagenase than in the discs that had
CEPs treated with control buffer (19.72 ± 5.17 μg/mL vs 1.41 ±
1.11 μg/mL; p < 0.0001) and 7-fold higher than discs with untreated
CEPs (2.81 ± 1.79 μg/mL; sham). In discs with buffer-treated and
sham CEPs, fluorescein concentrations were generally highest near
the CEPs, at positions A (buffer: 1.41 ± 1.11; sham: 2.81 ± 1.79 μg/
mL) and D (buffer: 1.48 ± 1.66; sham: 1.35 ± 0.52 μg/ml), and lowest
in the center of the discs, at positions B (buffer: 0.33 ± 0.14; sham:
0.81 ± 0.34 μg/ml) and C (buffer: 0.43 ± 0.27; sham: 0.71 ±
0.25 μg/ml). The total sGAG and collagen contents in the CEP tissues were
used to evaluate the effect of the enzymatic treatment on the CEP
matrix composition. 2.8 CEP biochemical composition CEP tissue digests were assayed for sGAG content using a
dimethylmethylene blue assay (DMMB) (Farndale et al., 1986;
Sampson et al., 2019). The digest absorbance was measured with 04 frontiersin.org 10.3389/fbioe.2023.1111356 Habib et al. FIGURE 4
(A) The average fluorescein concentration (mean ± SD; n =
3 discs/group) was significantly higher in the bovine NP proximal to
the collagenase-treated superior CEP, position A (a p < 0.0001 vs
control buffer; b p < 0.0001 vs sham; p > 0.10 all other
comparisons). (B) The average fluorescein concentration (mean ± SD;
n = 5 discs/group) was significantly higher in the human NP proximal
to the collagenase-treated superior CEP, position A (a p < 0.0001). FIGURE 5
(A) In the bovine discs that underwent collagenase treatment,
sGAG content in the treated (superior) CEP was significantly lower
than the sGAG content in the untreated (inferior) CEPs (p = 0.03,
inferior versus superior paired t-test; mean ± SD; n = 3 discs/
group) (B) In the human discs that underwent collagenase treatment,
sGAG content in the treated (superior) CEPs was significantly lower
than the sGAG content in the untreated (inferior) CEPs (p < 0.01,
inferior versus superior paired t-test; mean ± SD; n = 5 discs/group). FIGURE 4
(A) The average fluorescein concentration (mean ± SD; n =
3 discs/group) was significantly higher in the bovine NP proximal to
the collagenase-treated superior CEP, position A (a p < 0.0001 vs
control buffer; b p < 0.0001 vs sham; p > 0.10 all other
comparisons). (B) The average fluorescein concentration (mean ± SD;
n = 5 discs/group) was significantly higher in the human NP proximal
to the collagenase-treated superior CEP, position A (a p < 0.0001). FIGURE 5
(A) In the bovine discs that underwent collagenase treatment,
sGAG content in the treated (superior) CEP was significantly lower
than the sGAG content in the untreated (inferior) CEPs (p = 0.03,
inferior versus superior paired t-test; mean ± SD; n = 3 discs/
group) (B) In the human discs that underwent collagenase treatment,
sGAG content in the treated (superior) CEPs was significantly lower
than the sGAG content in the untreated (inferior) CEPs (p < 0.01,
inferior versus superior paired t-test; mean ± SD; n = 5 discs/group). frontiersin.org 3.2 Effects of enzymatic treatment on CEP
matrix composition Treatment
Position A
Position B
Position C
Position
D
Control
231.9 ± 107.4
222.9 ± 133.2
225.3 ± 107.6
223.1 ± 95.4
Collagenase
207.9 ± 133.8
241.3 ± 132.9a
218.8 ± 105.4
245.2 ± 98.2a
ap < 0.05 vs Position A by paired t-test. All other comparisons p > 0.05. TABLE 3 Effects of intradiscal treatment on sGAG content (µg/mg dry) at the NP
different positions. FIGURE 7
Whole-disc compressive creep strain (top) and modulus (bottom
was similar in (A) bovine and (B) human discs with collagenase-treated
CEPs vs control CEPs. p-values are from one-way ANOVA. FIGURE 7
Whole-disc compressive creep strain (top) and modulus (bottom
was similar in (A) bovine and (B) human discs with collagenase-treated
CEPs vs control CEPs. p-values are from one-way ANOVA. fluorescein (Table 3). Collagenase treatment had a small, localized
effect on the sGAG content of the adjacent NP tissue. Specifically,
mean sGAG of NP samples adjacent to the collagenase-treated CEP
at position A was 15.2% lower than the NP tissue samples adjacent to
the untreated CEP at position D in the same discs (p = 0.027) and
13.8% lower than the tissue NP samples at position B in the same
discs (p = 0.026). Comparisons of mean sGAG between the NP
tissue samples at the other positions were not significant. and human CEPs. Specifically, collagenase treatment reduced sGAG
content, with treated bovine CEPs showing, on average, 40% lower
sGAG content than their paired untreated CEPs (p = 0.03;
Figure 5A). The CEPs that were untreated as well as those that
were treated with the control buffer had similar sGAG contents. Likewise, the same enzyme dose delivered intradiscally to human
CEPs significantly reduced sGAG content, with the superior CEPs
showing 33.5% lower sGAG content, on average, than their paired
inferior CEPs (p = 0.01; Figure 5B). 3.4 Effects of enzymatic treatment on
whole-disc biomechanical behavior To determine the biomechanical effects of CEP matrix
modification
at
the
whole-disc
level,
we
compared
the
compressive modulus and total creep strain between groups by
ANOVA. In the bovine discs, total creep strain and compressive
modulus were similar between discs with the collagenase-treated
CEPs, buffer-treated CEPs, and untreated CEPs (Figure 7A). Likewise, in the human discs, total creep strain and compressive
modulus were similar between discs with collagenase-treated and
buffer-treated CEPs (Figure 7B). Collagenase treatment tended to associate with lower collagen
content in the treated CEPs, but the differences were not statistically
significant. Specifically, mean collagen content was lower in the
superior treated CEPs (156.79 ± 84.55 μg/mg dry wt) than their
paired inferior CEPs (233.39 ± 85.73 μg/mg dry wt; p = 0.21;
Figure
6A),
but
the
difference
did
not
reach
statistical
significance. In the human discs that underwent collagenase
treatment,
collagen
content
in
the
treated
superior
CEPs
(327.75 ± 188.65 μg/mg dry wt) tended to be lower than their
paired untreated CEPs (334.7186 ± 208.01 μg/mg dry wt; p =
0.07; Figure 6B). Frontiers in Bioengineering and Biotechnology frontiersin.org 3.2 Effects of enzymatic treatment on CEP
matrix composition Intradiscal enzymatic treatment significantly reduced the
amount of solute-blocking matrix constituents in both bovine 05 frontiersin.org Habib et al. 10.3389/fbioe.2023.1111356 FIGURE 6
(A) In the bovine discs that underwent collagenase treatment,
collagen content in the treated (superior) CEPs was similar to that of
the untreated (inferior) CEPs (p = 0.21, paired t-test; mean ± SD; n =
3 discs/group) (B) In the human discs that underwent
collagenase treatment, collagen content in the treated (superior) CEPs
was similar to that in the untreated (inferior) CEPs (p = 0.07, paired
t-test; mean ± SD; n = 5 discs/group). TABLE 3 Effects of intradiscal treatment on sGAG content (µg/mg dry) at the NP
different positions. Treatment
Position A
Position B
Position C
Position
D
Control
231.9 ± 107.4
222.9 ± 133.2
225.3 ± 107.6
223.1 ± 95.4
Collagenase
207.9 ± 133.8
241.3 ± 132.9a
218.8 ± 105.4
245.2 ± 98.2a
ap < 0.05 vs Position A by paired t-test. All other comparisons p > 0.05. FIGURE 7
Whole-disc compressive creep strain (top) and modulus (bottom
was similar in (A) bovine and (B) human discs with collagenase-treated
CEPs vs control CEPs. p-values are from one-way ANOVA. FIGURE 7
Whole-disc compressive creep strain (top) and modulus (bottom
was similar in (A) bovine and (B) human discs with collagenase-treated
CEPs vs control CEPs. p-values are from one-way ANOVA. FIGURE 6
(A) In the bovine discs that underwent collagenase treatment,
collagen content in the treated (superior) CEPs was similar to that of
the untreated (inferior) CEPs (p = 0.21, paired t-test; mean ± SD; n =
3 discs/group) (B) In the human discs that underwent
collagenase treatment, collagen content in the treated (superior) CEPs
was similar to that in the untreated (inferior) CEPs (p = 0.07, paired
t-test; mean ± SD; n = 5 discs/group). FIGURE 6 FIGURE 6
(A) In the bovine discs that underwent collagenase treatment,
collagen content in the treated (superior) CEPs was similar to that of
the untreated (inferior) CEPs (p = 0.21, paired t-test; mean ± SD; n =
3 discs/group) (B) In the human discs that underwent
collagenase treatment, collagen content in the treated (superior) CEPs
was similar to that in the untreated (inferior) CEPs (p = 0.07, paired
t-test; mean ± SD; n = 5 discs/group). TABLE 3 Effects of intradiscal treatment on sGAG content (µg/mg dry) at the NP
different positions. 3.3 Effects of enzymatic treatment on NP
tissue composition Enzymatic treatment also led to reductions
in collagen content, but the treatment effects were heterogeneous,
and the differences in CEP collagen content between treated and
untreated CEPs of the same disc were not statistically significant. Importantly, enzymatic treatment of the CEP coincided with greater
transport of 376-Da fluorescein into the nucleus pulposus under
static and cyclic compressive loads. Specifically, NP tissue adjacent
to collagenase-treated human and bovine CEPs had 10.8-fold
(human) and 14-fold (bovine) higher fluorescein concentrations
compared to the NP tissue adjacent to buffer-treated CEPs after
loading (Figure 4). The similar pattern of the findings in bovine discs
with relatively healthy CEP and NP tissues and human discs with
more degenerated CEP and NP tissue lends confidence in the
generalizability of the treatment mechanism and the overall
magnitude of the treatment effect. In our previous study of
human CEPs, we observed that ex situ treatment of CEPs with
truncated human recombinant MMP-8 reduced sGAG content in a
dose-dependent manner, increased fluorescein uptake into unloaded
CEPs, and enhanced nutrient transport and NP cell viability (Dolor
et al., 2019). Our new findings demonstrate for the first time the
concept of CEP matrix modification to whole, compressively loaded
discs, and taken together, these findings support the potential for
matrix modification of the CEP to improve the transport of small
solutes in the size range of essential nutrients, e.g., glucose (180 Da). The goal of CEP matrix modification is to reduce solute-
blocking matrix constituents, improve the CEP’s permeability to
nutrients, and enhance disc cell viability/function (Dolor et al., 2019;
Wong et al., 2019). In our previous study, reducing the amount of
sGAG in the CEP by 6%–22% coincided with increases in passive
fluorescein uptake by 16%–24% (Dolor et al., 2019). In vivo, the disc
is subjected to a combination of static and cyclic loads, which
improves
solute
transport
through
the
CEP
by
inducing
convection (Sampson et al., 2019). The effects of fluid flow on
solute convection may explain why a similar enzyme dose used in
the current study produced a larger increase in fluorescein transport
through the CEP than observed with passive diffusion. g
g
Targeted delivery of matrix-modifying enzymes to the CEP
presents several technical challenges. We chose a 50 µl volume of
injectate based on preliminary intradiscal pressure testing; injecting
larger volumes increased the back pressure and made it difficult to
prevent leakage. 3.3 Effects of enzymatic treatment on NP
tissue composition Choice of the hydrogel carrier was made to control
the release of the enzymatic treatment (Supplementary Materials),
and intradiscal delivery was performed using a 17G Tuohy epidural
needle with a curved end at the needle’s tip that allows the hydrogel
to exit laterally at a 45-degree angle. Combined with the oblique
trajectory of the needle, it was possible to deliver the treatment to the
center of the desired CEP (Figure 1). We treated one CEP from each
disc to demonstrate proof-of-principle and to ensure the non-
treated CEP would act as an intra-disc control. At this point, it is
not clear if treating one CEP is sufficient to improve disc nutrition or
if treating both CEPs is necessary. More extensive matrix
modification
of one or both CEPs could be achieved by
redirecting the needle to reach different areas of the target CEP;
however, redirecting the needle within the disc to treat multiple
locations
is
challenging
and
may
require
more
specialized
instrumentation. A related challenge is localizing the enzyme and
minimizing the effects on the nucleus pulposus tissue. For example,
we found that NP tissues adjacent to these enzyme-treated CEPs had
15% lower sGAG content compared to the NP tissues adjacent to the
untreated CEP of the same disc (Table 3). This suggests that other
strategies may be needed to further reduce off-target treatment
effects. For example, attaching the enzyme to large liposomal
nanoparticles
may
limit
enzyme
mobility
and
help
reduce
unwanted enzyme migration in the NP (Dolor et al., 2019). The goal of CEP matrix modification is to reduce solute-
blocking matrix constituents, improve the CEP’s permeability to
nutrients, and enhance disc cell viability/function (Dolor et al., 2019;
Wong et al., 2019). In our previous study, reducing the amount of
sGAG in the CEP by 6%–22% coincided with increases in passive
fluorescein uptake by 16%–24% (Dolor et al., 2019). In vivo, the disc
is subjected to a combination of static and cyclic loads, which
improves
solute
transport
through
the
CEP
by
inducing
convection (Sampson et al., 2019). The effects of fluid flow on
solute convection may explain why a similar enzyme dose used in
the current study produced a larger increase in fluorescein transport
through the CEP than observed with passive diffusion. Proper CEP function requires balancing conflicting biotransport
and
biomechanical
demands,
and
thus,
successful
matrix
modification
strategies
for
improving
biotransport
must
be
sensitive to any biomechanical effects. 3.3 Effects of enzymatic treatment on NP
tissue composition Specifically, excessive
cleavage and removal of collagen fibrils or proteoglycans in the
CEP could inadvertently lower disc modulus and risk clinically
significant disc injury. The collagen network helps resist transverse
CEP stretching that arises during disc compression (Fields et al.,
2010; Fields et al., 2014; DeLucca et al., 2016), and proteoglycans in
the CEP are believed to help prevent the loss of proteoglycan
aggregates from the nucleus pulposus (Roberts et al., 1996). We
found that discs with enzyme-treated CEPs had similar whole-disc
modulus and total creep strain as buffer-treated discs (Figure 7),
which suggests that the modest dose used here was sufficient to
improve solute transport without impacting whole-disc behavior
under static and cyclic compressive loading. Additional mechanical
testing with more complex loading protocols is needed to further
understand the biomechanical effects of CEP matrix modification. The CEP is rich in type II collagen (Schollmeier et al., 1976;
Roberts et al., 1991; Moore, 2006; Nosikova et al., 2012) and
aggrecan. We used a commercial form of collagenase p from
Clostridium
histolyticum
(C. histolyticum)
for
matrix
modification, as this enzyme can break down the peptide bonds
in four types of collagens (I, II, III, and IV) (Alipour et al., 2016) and
has proteolytic activity on proteoglycans. Matrix metalloproteinases
(MMPs) also have a high specificity for type II collagen and aggrecan
(Nagase and Kashiwag, 2003; Dolor et al., 2019). In the current
study, we treated both human and bovine CEPs, so we chose a
generic
collagenase
enzyme
rather
than
a
specific
human
recombinant MMP. Nevertheless, the effects of collagenase p on
human CEP composition appeared similar to those observed with
truncated human MMP-8 (Dolor et al., 2019), and together our
findings suggest that both enzymes reduce sGAG content in the CEP
and have more subtle effects on the collagen. Although collagen
content in the collagenase-treated CEPs was typically lower than
buffer-treated
CEPs,
these
differences
were
not
statistically
significant (Figure 6). One possible explanation is that cleaved
collagen fragments remained intertwined in the CEP matrix, and This study had several limitations. First, we removed the bony
endplates before the transport experiments in order to isolate the
effects of CEP matrix composition on solute transport into the discs
and to facilitate comparison between species. For example, bovine
spinal units have vertebral epiphyses and growth plates that persist
through adulthood, whereas adult human discs do not. Frontiers in Bioengineering and Biotechnology frontiersin.org 3.3 Effects of enzymatic treatment on NP
tissue composition Here we investigated intradiscal treatment to modify the matrix
of the CEP to enhance the transport of small solutes into whole
intervertebral discs. Collagenase enzyme was loaded into a
degradable hydrogel carrier and then delivered via needle to the
superior CEP under fluoroscopic guidance. Results show that
collagenase treatment (0.15 U) significantly reduced the amount To understand how the enzymatic treatment delivered to the
CEP affected the composition of the NP tissue, we measured the
sGAG content in the human NP tissue samples that were assayed for 06 frontiersin.org 10.3389/fbioe.2023.1111356 Habib et al. greater exposure times are required for the removal of these
fragments (Jiang et al., 2020). of sGAG in the treated CEP: enzymatic treatment of human lumbar
and bovine coccygeal CEPs reduced sGAG by 33.5% and 40%,
respectively (Figure 5). Enzymatic treatment also led to reductions
in collagen content, but the treatment effects were heterogeneous,
and the differences in CEP collagen content between treated and
untreated CEPs of the same disc were not statistically significant. Importantly, enzymatic treatment of the CEP coincided with greater
transport of 376-Da fluorescein into the nucleus pulposus under
static and cyclic compressive loads. Specifically, NP tissue adjacent
to collagenase-treated human and bovine CEPs had 10.8-fold
(human) and 14-fold (bovine) higher fluorescein concentrations
compared to the NP tissue adjacent to buffer-treated CEPs after
loading (Figure 4). The similar pattern of the findings in bovine discs
with relatively healthy CEP and NP tissues and human discs with
more degenerated CEP and NP tissue lends confidence in the
generalizability of the treatment mechanism and the overall
magnitude of the treatment effect. In our previous study of
human CEPs, we observed that ex situ treatment of CEPs with
truncated human recombinant MMP-8 reduced sGAG content in a
dose-dependent manner, increased fluorescein uptake into unloaded
CEPs, and enhanced nutrient transport and NP cell viability (Dolor
et al., 2019). Our new findings demonstrate for the first time the
concept of CEP matrix modification to whole, compressively loaded
discs, and taken together, these findings support the potential for
matrix modification of the CEP to improve the transport of small
solutes in the size range of essential nutrients, e.g., glucose (180 Da). of sGAG in the treated CEP: enzymatic treatment of human lumbar
and bovine coccygeal CEPs reduced sGAG by 33.5% and 40%,
respectively (Figure 5). Data availability statement The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be directed
to the corresponding author. Author contributions Conceptualization: MH, AF Data curation: MH, SH, and AF
Formal analysis: MH, AF Funding acquisition: AF Methodology:
MH, PI-KW, JL, and EA Resources: JL, EA, OJ, PI-KW, and AF
Supervision: MH, EA, and AF Writing – original draft: MH, AF
Writing – review & editing: MH, SH, OJ, JL, PI-KW, EA, and AF. 5 Conclusion We found that intradiscal delivery of a matrix-modifying
enzyme to the CEP significantly reduced the amount of sGAG. Treating the CEP in this manner dramatically enhanced small-solute
transport into the disc. This is an important step towards in vivo pre-
clinical studies. Future studies should focus on optimizing enzyme
delivery and release, as well as on discovering the native cells’
response to matrix-modifying enzymes and the by-products from
matrix proteolysis. Publisher’s note All claims expressed in this article are solely those of the authors
and
do
not
necessarily
represent
those
of
their
affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. Funding This research was supported by grants from the North American
Spine Society (AF), the Research Evaluation and Allocation
Committee at UCSF (AF), the Core Center for Musculoskeletal
Biology and Medicine at UCSF (AF), the Center for Disruptive
Musculoskeletal Innovation at UCSF (AF), the National Institutes of
Health (AF: R01 AR070198 and P30 AR066262). Conflict of interest Strategies for enhancing nutrient transport across the CEP may
have the potential to slow or reverse disc degeneration. Our new
results are significant because they suggest that intradiscal delivery
of a matrix-modifying enzyme to the CEP can effectively reduce
solute-blocking matrix constituents and thereby improve solute
transport. This treatment strategy could stand alone or possibly
be combined with biologic regenerative therapies that target the NP
and increase nutrient demands. The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. Acknowledgments The authors thank the UCSF Willed Body Program for assisting
with cadaver procurement and screening. 3.3 Effects of enzymatic treatment on NP
tissue composition Also, the
bony endplate is much thicker in bovine coccygeal vertebrae than
human lumber vertebrae. Given the total permeability of the bony
and cartilaginous endplates is more strongly correlated with CEP
permeability than with bony endplate permeability (Rodriguez et al., 07 frontiersin.org Habib et al. 10.3389/fbioe.2023.1111356 obtained from the UCSF Willed Body Program. The UCSF Willed
Body Program obtains consent from the donor, and cadaveric
tissues were provided to the study team without any personal
identifiers. Since the study did not involve human participants or
medical data, the study was exempt from IRB review. 2011), removing the bony endplates is unlikely to have affected our
conclusions about the relative increase in solute transport into the
disc with enzymatic treatment. Further, the bony endplate removal
was verified by imaging to not disrupt the CEP. A second limitation
is that we used one solute size in the transport experiments, and
permeability enhancement may be size-dependent. We focused on
376 Da-sized tracer because of its similarity to glucose (180 Da),
which is vital to NP cell survival. The effects of enzymatic treatments
on large solute filtration by the CEP are also important and remain
unknown. A third limitation is that we did not measure localized
mechanical
properties
of
the
CEP
tissue,
which
are
also
physiologically important (Lotz et al., 2013; Fields et al., 2014;
Fields et al., 2018). Finally, we did not assess the biologic effects
of matrix modification in this cadaveric study. To address this,
ongoing work is using cell and organ culture models to assess
treatment safety and efficacy, including characterizing the cellular
response to the by-products from CEP matrix proteolysis. Frontiers in Bioengineering and Biotechnology References Alipour, H., Raz, A., Zakeri, S., and Dinparast Djadid, N. (2016). Therapeutic
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A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment
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© The Author(s). 2021 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
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https://doi.org/10.1186/s13395-021-00261-w Benedetti et al. Skeletal Muscle (2021) 11:7
https://doi.org/10.1186/s13395-021-00261-w A novel approach for the isolation and
long-term expansion of pure satellite cells
based on ice-cold treatment A novel approach for the isolation and
long-term expansion of pure satellite cells
based on ice-cold treatment Anna Benedetti1, Gianluca Cera2,3, Daniele De Meo2,3, Ciro Villani2,3, Marina Bouche1† and
Biliana Lozanoska-Ochser1*† Anna Benedetti1, Gianluca Cera2,3, Daniele De Meo2,3, Ciro Villani2,3, Marina Bouche1† and
Biliana Lozanoska-Ochser1*† Abstract Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional
potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved
myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a
simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human
SCs. It involves a brief (15–30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent
muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior
purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging
technique, allowing SC expansion over extended periods of time without compromising their proliferation and
differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating
injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or
expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human
biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical
simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational
research bringing their therapeutic use closer to the clinic. Keywords: Satellite cell isolation, Satellite cells in vitro expansion, Skeletal muscle regeneration Background 5]. As SCs become activated, a proportion of them iden-
tified as Pax7+MyoD- replenish the stem cell pool, while
others acquire the expression of MyoD (Pax7+MyoD+),
differentiate into myoblasts and enter the myogenic pro-
gram. After several rounds of division myoblasts give
rise to Myogenin+ myocytes, which fuse together to form
new myofibers [1, 4, 6–9]. The muscle is endowed with an exceptional regenerative
ability primarily due to a resident population of stem
cells called satellite cells (SCs) [1–3]. Ensconced between
the basal lamina and the plasma membrane of muscle fi-
bers, SCs respond to injury or various stress stimuli by
becoming activated, and undergoing proliferation, self-
renewal, and differentiation to form new myofibers [4, A major stumbling block in the study of the functional
potential of SCs has been the lack of isolation methods
involving minimal cell manipulation and allowing the
isolation and expansion of highly pure SCs with pre-
served myogenic properties after serial passages in vitro
[10–12]. Moreover, the success of SC transplantation
therapy depends on having an efficient method to isolate * Correspondence: biliana.lozanoska-ochser@uniroma1.it
†Bouche Marina Lozanoska-Ochser Biliana are co-senior authors. 1Department of Anatomical, Histological, Forensic and Orthopedic Sciences,
Section of Histology and Embryology, Sapienza University of Rome, Rome,
Italy
F ll li
f
h
i f
i
i
il bl
h
d
f h
i l y
l list of author information is available at the end of the article Italy
Full list of author information is available at the end of the article * Correspondence: biliana.lozanoska-ochser@uniroma1.it
†Bouche Marina Lozanoska-Ochser Biliana are co-senior authors.
1Department of Anatomical, Histological, Forensic and Orthopedic Sciences,
Section of Histology and Embryology, Sapienza University of Rome, Rome,
Italy
Full list of author information is available at the end of the article Human muscle sourcing Muscle biopsies (gluteus maximus) were obtained from
patients (8 males and 7 females, age range 50–90 years)
undergoing surgery at the Department of Orthopaedics
and Traumatology, Umberto I Hospital in Rome, Italy. According to the Italian law, the authors are not re-
quired to ask for approval from an institutional review
board or ethical committee for this type of study. In any
case, all patients gave their approval to undergo intraop-
erative muscle biopsy and to publish the clinical and la-
boratory data obtained. The FACS sorting method sorts muscle mononuclear
cells pre-labelled with SC specific antibodies. Following
digestion of muscle with various enzymes the resulting
mixture of cells is labelled with specific antibodies to fa-
cilitate the identification of SCs, which are then sorted
using a FACS sorter instrument [16–21]. At present, the
FACS sorting method represents the gold standard for
the isolation and study of SCs. Nevertheless, there are
several disadvantages to this method including high cost
and the requirement for a FACS sorter instrument. Moreover, this method is time consuming, requires ex-
pertise to perform and cell purity can be variable. The
cell labelling step followed by the sorting procedure can
potentially stress or damage the cells, decrease their via-
bility, or alter their functional properties in vitro [12]. The third method is based on magnetic cell separation
(MACS) and uses magnetic columns and SC specific
magnetic bead kits [22]. It is based on negative selec-
tion of SCs by magnetically labelling and removing
other cell lineages. Because this method assumes that
all the other cell types are successfully removed from
the muscle cell preparation, it is less precise than the
FACS sorting method. This method is expensive to
perform, time consuming, and stressful for the cells. As for the other two methods, cell purity is variable
and often the SC cultures become overgrown by fi-
broblasts by day 7 [10, 12]. Satellite cell isolation with the ice-cold treatment method
SCs were isolated from hind-limb muscles of 4–8-week-
old mice or from human biopsies. Muscles were dis-
sected with scissors and finely diced with a scalpel in a
dish containing DMEM (Sigma-Aldrich, St. Louis, MO,
USA, D5671). This was followed by enzymatic digestion
with 10 ml/g of muscle of Collagenase type II (Sigma-Al-
drich, SCR103) at a concentration of 0.4 mg/ml in PBS
(Sigma-Aldrich), for 45 min in a water bath at 37 °C
with agitation. Benedetti et al. Skeletal Muscle (2021) 11:7 Page 2 of 12 and expand these cells in vitro in undifferentiated state
and in sufficient numbers [13]. There is therefore a need for new and improved
methods for the isolation, expansion and culture of SCs. Here, we describe a simple, inexpensive, and efficient
method for the isolation of highly pure mouse and hu-
man SCs that can be serially expanded in vitro to obtain
sufficient number of SCs with preserved proliferation
potential, capable of regenerating injured muscle in vivo. Presently, there are three main methods commonly
used for the isolation of SCs: the pre-plating method,
fluorescence activated cell sorting (FACS), and magnetic
bead isolation method. The pre-plating method is based on the differing
adhesive properties of muscle cells, with SCs being
the least adherent. Following enzymatic digestion, a
heterogeneous mix of skeletal muscle cells is plated
onto uncoated culture dishes and after a 1–24-h incu-
bation at 37 ○C, the non-adherent cells are collected
and plated onto new collagen coated dishes [10–12,
14]. The resulting cell culture contains both SCs and
fibroblasts in variable proportion. To improve SC
purity, the pre-plating step can be repeated every 24
h over 6 days [10]. Although cheap and straightfor-
ward to perform, this method’s main disadvantage is
that it is time consuming and gives rise to cultures of
variable
purity,
with
fibroblast
contamination
and
overgrowth by day 7 of culture, leading to early sen-
escence and detachment of myotubes [10]. A recently
described version of the pre-plating method intro-
duces a re-plating step whereby after a 2-day expan-
sion, the adhered cells are detached with trypsin and
replated onto Matrigel coated dishes, giving rise to
SC cultures of much improved purity [15]. Methods
Mice C57BL/10ScSn-Dmdmdx, C57BL/10ScSn, and C57BL/6J
mice were purchased from the Jackson laboratory (Bar
Harbor, ME, USA). Both male and female mice were
used. The mice were housed in the Histology Depart-
ment–accredited animal facility at the University of
Sapienza. All the procedures were approved by the Ital-
ian Ministry for Health and were conducted according
to the EU regulations and the Italian Law on Animal
Research. Human muscle sourcing Digestion was blocked with DMEM 10%
FBS and after centrifuging the muscle preparation and
removing the supernatant, a second digestion was per-
formed with 10 ml/g of muscle of Collagenase/Dispase
at a concentration of 1 mg/ml (Roche, Basel, CH,
11097113001) in PBS Calcium-Magnesium free (Sigma-
Aldrich), for 30 min at 37 °C in a water bath with agita-
tion. The digested muscle was then passed first through
a 70-μm cell strainer followed by 40-μm cell strainer to
obtain single cell suspension. Next, the cells were centri-
fuged, resuspended in DMEM 10% FBS (Sigma-Aldrich,
F2442), counted and plated at 2 × 106 cells/100 mm dish
(uncoated) (Corning, NY, USA, 430167), and incubated Page 3 of 12 Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle (2021) 11:7 Page 3 of 12 Page 3 of 12 at 37 °C for 1 h. Non-adherent cells were collected, cen-
trifuged, and the cell pellet was resuspended in DMEM
10% FBS, plated again, and incubated for another 1 h at
37 °C. After the second pre-plating, non-adherent cells
were collected, centrifuged, counted, resuspended in SC
Growth Medium (GM) DMEM, 20% Horse Serum
(Thermo
Fisher
Scientific,
Waltham,
MA,
USA,
26050088), 3% Chicken Embryo Extract (Seralab, CE-
650-J), and plated into 100 mm dishes coated with 0.1%
gelatin (Stem Cell Technologies, Vancouver, BC, CAN,
07903), at 106 cells/dish. The next day, the dishes con-
taining a heterogeneous mix of adhered muscle cells
were washed 3 times with PBS, and 10 ml of ice-cold
PBS was added into each dish. The dishes were then
placed on ice (0 °C) for 15–30 min with occasional gen-
tle manual shaking (swirling motion). The detached cells
were collected, centrifuged, resuspended in GM, and
plated into 0.1% gelatin-coated 35-mm dishes (Corning,
353001) at a density of 103 cells/dish. To induce differ-
entiation, proliferating cells (day 3 after adding GM)
were cultured in differentiating medium (DM) contain-
ing DMEM 5% Horse Serum, 1% Chicken Embryo
Extract. Satellite cell isolation with magnetic bead labelling Satellite cell isolation with magnetic bead labelling
Mouse SC isolation by magnetic bead labelling was per-
formed by using a SC Isolation Kit (Miltenyi Biotech,
Bergisch Gladbach, DE, REF: 130-104-268) as previously
described [23]. Briefly, minced muscle was digested as
described
above. The
digested
muscle
was
passed
through 70 μm and 40 μm cell strainers, and the result-
ing single cell suspension was centrifuged, resuspended
in 80 μl buffer (PBS pH 7.2, 0.5% FBS, 2 mM EDTA),
and incubated with 20 μl of Satellite Cell Isolation Kit
per gram of muscle, for 15 min at 4 °C. Next, the cell
suspension was passed through a LS column (Miltenyi
Biotech, 130-042-401) placed in a magnetic field of a
MACS Separator (Miltenyi Biotech). Unlabeled SCs were
collected in the flow-through, counted, washed, resus-
pended in growth medium (GM), and plated into 35-
mm dishes at a density of 103 cells/dish. For immunofluorescence analysis of mdx TA muscle
transplanted with WT SCs, 8-μm-thick muscle cryosec-
tions were fixed in 4% PFA for 10 min at room
temperature
(RT),
and
then
permeabilized
in
cold
methanol for 6 min at – 20 °C. Sections were then
blocked in 5% Goat Serum (Sigma-Aldrich) in PBS for
30 min at RT. Next, sections were incubated with pri-
mary rabbit anti-dystrophin antibody (1:200, Abcam,
Cambridge, UK) overnight at 4 °C. The next day, sec-
tions were washed and incubated with a secondary anti-
body goat anti rabbit Alexa Fluor 488 (1:1000 Abcam). Nuclei were counterstained with Hoechst. Samples were analyzed under an epifluorescence Zeiss
Axioskop 2 Plus microscope (Carl Zeiss, Oberkochen,
DE). Mouse and human SCs were cultured either in GM
containing DMEM, 20% Horse Serum (Sigma-Aldrich),
3% Chicken Embryo Extract, or in DM containing
DMEM 5% Horse Serum, 1% Chicken Embryo Extract. Bright field images were acquired with an inverted
phase-contrast microscope (Nikon Eclipse, TS100). Im-
ages were acquired with a Nikon DS-Fi2 camera and
NIS Elements version 4.0 Imaging System. SC transplantation Acute muscle injury was induced the day before SC
transplantation. To induce muscle injury tibialis muscles
were injected with 0.01 ml of Cardiotoxin from Naja Pal-
lida (10 μM) (Latoxan ZA, Les Auréats, Fr), using a 30
Gauge micro-syringe [23–26]. For cell transplantation, 15,000 SCs were resuspended
in 20 μl of DMEM 2% FBS (Sigma-Aldrich) and injected
into the TA muscle of one leg with a single injection by
using a 30 Gauge micro-syringe. Contralateral TA
muscle was injected with only PBS and used as control. SC detachment with trypsin SCs were rinsed once with PBS and then incubated with
trypsin-EDTA solution (Sigma-Aldrich, T3924) for 5
min at 37 °C. SCs were then collected, centrifuged, re-
suspended in GM, and plated at a density of 103 cells/
dish. Immunofluorescence and microscopy For immunofluorescence analysis cultured SCs were
fixed in PFA 4% for 10 min RT, permeabilized in cold
methanol at – 20 °C for 6 min, blocked in 5% Goat
Serum in PBS for 30 min RT, and incubated overnight
at 4 °C in 4% BSA in PBS with the following primary
antibodies: mouse anti-Pax7-c (1:10 DSHB, Iowa City,
IA, USA), rabbit anti-MyoD (1:50 Santa Cruz C20: sc-
304, Dallas, TX, USA), mouse anti-myogenin (1:20,
DSHB), mouse anti-Myosin Heavy Chain (1:20 DSHB),
and mouse anti-desmin (1:20, DSHB). The next day, SCs
were washed 3 times in PBS for 15 min, and then incu-
bated with secondary antibodies goat anti-rabbit Alexa
Fluor 488 (1:1000, Abcam) and goat anti-mouse Alexa
Fluor 555 (1:1000, Thermo Fisher Scientific) diluted in
1% BSA in PBS, for 1 h RT. Nuclei were counterstained
with Hoechst. CFSE staining Isolated SCs were stained with CFSE (ThermoFisher Sci-
entific) at a concentration of 1 μM for 10 min at 37 °C
in the dark prior to culture. After 4 days of culture, the
cells were detached with Accutase Solution (Sigma- Page 4 of 12 Page 4 of 12 Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle as well as the notion that like all stem cells, SCs are sen-
sitive to stress signals and are among the first muscle
resident cells to respond to injury [4, 5], we hypothe-
sized that subjecting a heterogeneous culture of muscle
cells
to
a
mild
stress
stimulus
such
as
ice-cold
temperature will lead to the detachment of only the SCs. To test this hypothesis, we obtained a mix of muscle
cells following enzymatic digestion, and after 2 h of pre-
plating on uncoated dishes, cultured them overnight on
gelatin-coated dishes. The next day, after washing and
removing the non-adhered cells and debris, we placed
the dishes of heterogeneous muscle cells on ice for 30’
(Fig. 1a, b). This time point was chosen based on the
purity and number of SCs obtained following 15, 30, 45,
and 60’ on ice. Although the number of SCs obtained in-
creased with longer incubations, the purity of SCs de-
creased from 100% at 15–30’ to 95 and 90% at 45 and
60’ on ice, respectively (data not shown). The ice-cold
treatment (ICT) method can be used to harvest SCs
from the original dish containing the heterogeneous
muscle cells for at least the first 3 days of culture. Pla-
cing the heterogeneous muscle culture dish on ice for
30’ resulted in the detachment of only the SCs giving
rise to a highly pure culture of SCs that proliferated and
differentiated into myotubes upon culture in differentiat-
ing medium (Fig. 1b–d). Satellite cells isolated using the
ICT method were 100% pure as determined by the ex-
pression of the SC markers Pax7 and MyoD (Fig. 1c). At
day 3 of proliferation, 100% of the cells were positive for
the satellite cell marker Pax7 and of these 97% were acti-
vated and expressed MyoD (Pax7+MyoD+) (Fig. 1c). To
examine the myogenic capacity of ICT-isolated SCs we
performed a clonal myogenicity assay, by plating a single
cell per well and analyzing the formation of myogenic
colonies. CFSE staining Satellite cells isolated with the ICT method dis-
played a similar clonal myogenicity of 40 % and a doub-
ling time of 17 h to SCs isolated using the magnetic
beads method (Figure S1A and B). The myogenic iden-
tity of the cultured cells was further confirmed by the
expression of myosin heavy chain (MHC) and the forma-
tion of myotubes. After 3 days in differentiating medium
the SCs differentiated into myoblasts that fused into
MHC expressing myotubes with a fusion index of 90%
(Fig. 1d). The purity of the isolated SCs at the begin-
ning of culture, as well as the fusion index and num-
ber of myonuclei per myotubes were similar between
SCs isolated with the ICT and magnetic cell separ-
ation (MACS) method (Figure S1C–E). However, by
day
5–7,
the
cultures
obtained
with
the
MACS
method became
overgrown
by
non-myogenic
cells Aldrich). Samples were acquired with a CyAn ADP
(DAKO) flow cytometer and acquired data were ana-
lyzed using FlowJo software version 10 (FlowJo LLC,
Ashland, OR, USA). Clonal myogenicity assay For the clonal myogenicity assay, SCs were plated into
0.1% gelatin coated 96-well plates, (excluding the outer
wells of the plate) at 1 cell per well, in growth medium. Colony formation and number of cells were assessed at
24, 48, and 72 h of culture. RNA isolation and quantitative real-time PCR For RNA preparation, cells were lyzed with TRI reagent
(Sigma-Aldrich) and processed as previously described
[23]. Reverse transcription was performed with Sensi-
FAST™cDNA Synthesis Kit (Bioline, Memphis, TN,
USA). Quantitative real-time PCR assays were per-
formed according to the MIQE criteria, using Sensi-
FAST™
SYBR
No-ROX
Kit
(Bioline)
following
manufacturer’s protocol. All reactions were performed
in duplicate. Data were collected and analyzed using ABI
PRISM 7500 Sequence Detection System (Life Technolo-
gies, Carlsbad, CA, USA). Quantitative RT–PCR values
were normalized to the expression of GAPDH mRNA. The relative gene expression level was calculated using
the 2−ΔΔCT method and reported as mean fold change
in gene expression. The following primers were used for amplification:
Pax7 (FW: 5′ GTCCCAGTCTTACTGCCCAC 3′, RV:
5′ TGTGGACAGGCTCACGTTTT 3′), Myogenin (FW:
5′ GCATGGAGTTCGGTCCCAA 3′, RV: 5′ TATCCT
CCACCGTGATGCTG 3′), GAPDH (FW: 5′ ACCCAG
AAGACTGTGGATGG
3′,
RV:
5′
CACATTGGGG
GTAGGAACAC 3′). Statistical analysis All statistical analyses were performed using Graph-
Pad Prism software version 8 (La Jolla, CA, USA). Data are presented as mean ± SEM. Statistical signifi-
cance was determined using unpaired 2-tailed Stu-
dent’s t test with Welch’s correction for unequal
variances. A P value of ≤0.05 was considered statisti-
cally significant. Isolation and characterisation of muscle SCs using the ice-
cold treatment method Previous
studies
have
demonstrated
that
cold
temperature causes a reduction in cell adhesion, likely
due to the downregulation of adhesion receptors [27,
28]. Compared to other cells, such as fibroblasts, which
typically contaminate SC cultures, SC are considered to
be less adherent [11, 14]. Taking this into consideration, Benedetti et al. Skeletal Muscle (2021) 11:7 Page 5 of 12 Benedetti et al. Skeletal Muscle Fig. 1 (See legend on next page.) Fig. 1 (See legend on next page.) Fig. 1 (See legend on next page.) Fig. 1 (See legend on next page.) Page 6 of 12 Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle (See figure on previous page.) g
p
p g
Fig. 1 Isolation and characterization of muscle SCs using the ICT method. a Schematic representation of the ICT method. b Representative bright
field images of the heterogeneous muscle mononuclear cell culture from which SCs were isolated by ICT, and representative images of the ICT-
isolated SCs at D2 and D4 in growth medium (GM) and at D3 after adding differentiation medium (DM) (n = 15 independent experiments). c
Representative immunofluorescence images of ICT-isolated SCs stained for Pax7 (red), MyoD (green), and nuclei (blue) and a graph showing the
percentage of cells positive for Pax7 and/or MyoD at day 3 of culture in GM (n = 3 independent experiments). d Representative
immunofluorescence images of ICT-isolated SCs stained for myosin heavy chain (MHC) (red) and nuclei (blue) and a graph showing percent
fusion after differentiation (4 days in GM followed by 3 days in differentiating medium (DM)) (n = 3 independent experiments). e Representative
immunofluorescence images of ICT-isolated SCs and MB-isolated SCs stained for Myogenin (red) PDGFR⍺(green) and nuclei (blue) and a graph
showing percent PDGFR⍺+ cells at day 5 of culture in GM. f Representative bright field images of ICT- and magnetic beads (MB)-isolated SCs and
a quantification graph showing percentage of myogenic cells in ICT- and MB-isolated SCs after differentiation at day 7 of culture (n = 3
independent experiments). Non-myogenic cells were identified as Pax7-MyoD-nuclei outside the myotubes. Scale bar = 100 μm. Error bars
represent mean ± sem. *p < 0.05 by Student’s t test. Overall, these results confirm our hypothesis that ICT
leads to the preferential detachment of SCs and using
this method allows the isolation of 99–100% pure SCs. [31]. The ICT approach can also be used for serial passaging
and long-term expansion of SCs g
p
A major obstacle in SC research has been that cultured
SCs lose their proliferation potential after a couple of
passages
and
begin
to
differentiate
into
myotubes
thereby limiting the number of SCs that can be serially
expanded in vitro. Loss of differentiation potential has
also been noted with increasing number of passages
[10–13, 29]. We reasoned that, since the ICT leads to
SC detachment, it can also be used to passage growing
cultures of proliferating SCs. Indeed, placing the dishes
of proliferating SCs on ice for 30 min led to the detach-
ment of around 30% of the proliferating cells and we
were able to serially passage and expand the proliferating
SCs without compromising their proliferation and differ-
entiation capacity (Fig. 2a). We used the ICT approach
on proliferating SCs and then successively on each
established culture of proliferating SCs (for more than
10 passages). This approach yielded on average 2.5 × 106
cells/g of muscle (Fig. 2b). The SCs passaged using the
ICT method did not lose their proliferation and differen-
tiation potential and displayed a minimally altered Pax7
and myogenin gene expression (Fig. 2c, d). While ICT-
passaged SCs retained their proliferation and differenti-
ation capacity even after 10 passages, SCs passaged with
the most commonly used passaging reagent trypsin, lost
the ability to form new myogenic colonies after just 2
passages and instead begun to differentiate into myo-
tubes (Fig. 2e–g). Similar to SCs detached by trypsin,
SCs detached using a gentler detachment solution such
as Accutase, exhibited accelerated differentiation after
more than 3 passages (data not shown). Next, we examined the longevity of differentiated ICT-
isolated SCs in culture. The highly pure cultures of SCs
isolated using the ICT method gave rise to myotubes
that could be maintained in culture for up to 1 month,
compared to just 7 days when isolated using the mag-
netic beads (Figure S2A–C) or the pre-plating method
(Figure S2D). Overall, these data show that the ICT method can be
used for the serial expansion of SCs with preserved pro-
liferation and myogenic potential over an extended
period of time compared to other methods. To examine the potential of ICT-isolated and ex-
panded SCs to regenerate injured muscle, we injected
15,000 SCs isolated from wild-type mice into the tibialis
anterior of mdx mice (lacking dystrophin) CTX injured
24 h previously. Isolation and characterisation of muscle SCs using the ice-
cold treatment method Unlike the fast-dividing SCs, which have a limited
ability to form secondary myogenic colonies after pas-
sage and instead undergo differentiation, the slow divid-
ing
SCs
form
secondary
myogenic
colonies
when
passaged [30, 31]. To examine the prevalence of fast and
slow dividing SCs within our population, we labelled the
ICT-passaged SCs with CFSE prior to plating and ana-
lyzed the rate of proliferation 3 days later. We compared
the proliferation rate of ICT-isolated and passaged SCs
with that of SCs isolated using magnetic beads and pas-
saged with trypsin. In line with previous observations,
we found that most of the activated SCs isolated using
the commercial kit and passaged with trypsin were fast-
dividing cells (CFSElo), with slow-dividing cells (CFSEhi)
representing less than 10% of the total. Interestingly,
while similarly heterogeneous, the SCs isolated and pas-
saged using the ICT method were enriched in the slow
dividing SC population (Fig. 2h, i). The ICT approach can also be used for serial passaging
and long-term expansion of SCs SCs were either injected immediately
after ICT isolation, or after 3-day in vitro expansion fol-
lowing ICT. Transplantation of both SCs immediately
after ICT-isolation and after in vitro expansion contrib-
uted to the regeneration process to a similar extent as
evidenced
by
the
appearance
of
newly
formed
dystrophin-positive fibers (Figure S3A and B). There was
no difference in the ability of transplanted ICT-isolated Previous studies have demonstrated that based on
their rate of proliferation, SCs can be divided into two
subpopulations: fast dividing and slow dividing [30]. The
slow-dividing are a subset of SC that have been shown
to retain stemness and long-term self-renewal ability Page 7 of 12 Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle Fig. 2 (See legend on next page.) page.) Fig. 2 (See legend on next page.) Fig. 2 (See legend on next Page 8 of 12 Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle (See figure on previous page.)
Fig. 2 Serial expansion and long-term proliferation potential of SCs using the ICT method. a Representative bright field images of SCs at day 3 of
culture in GM following ICT 1, ICT 2, and ICT 3, and differentiated cells at day 3 of culture in DM following ICT 3 (n = 10 independent
experiments). b Total number of SCs at day 3 of culture in GM, after 1 and 10 ICTs (n = 10 independent experiments). c Pax7 gene expression in
SCs at day 2 of culture in GM after ICT 1 and ICT 2 analyzed by quantitative real time PCR (n = 3 independent experiments). d Myogenin
expression in SCs at day 5 of culture in GM after ICT1 and ICT 2 analyzed by quantitative real-time PCR (n = 3 independent experiments). e
Representative images of SCs at day 2 of culture in GM, after 1–3 detachments with ICT (top panels) and trypsin (bottom panels). f Pax7 gene
expression in SCs detached with ICT or trypsin, at day 2 of culture in GM, analyzed by quantitative real-time PCR (n = 3 independent
experiments). g Myogenin gene expression in SCs detached with ICT or trypsin, at day 5 of culture in GM, analyzed by quantitative real time PCR
(n = 3 independent experiments). h Representative overlay of histogram plots of CFSE labeled ICT and trypsin detached SCs at time 0 and day 4
of culture in GM. Discussion Over the past decade, a considerable progress has been
made in the development of new methodologies for the
isolation of SCs. Nevertheless, each of the available
methods suffers from at least one disadvantage be it pur-
ity, cost, expertise, or a combination of these [11]. In this
study, we describe a novel method for the isolation of
pure mouse and human SCs, that is inexpensive, simple
to perform, and reproducibly efficient. The ICT method
takes advantage of the differing adhesive properties of
muscle cells as well as the ability of SCs to rapidly re-
spond to stress stimuli [5, 11, 14]. Thus, the combin-
ation of a mild cold-stress stimulus and cold-induced
reduction in adhesion, leads to the detachment of only
the SCs. Exposure of mammalian cells to cold stress can
slow down the progression through the cell cycle and in-
hibit protein synthesis. Moreover, depending on the in-
tensity
and
duration,
cold
stress
can
activate
the
apoptotic program, or lead to necrosis [34]. Thus, it is
conceivable
that
prolonged
exposure
to
cold
temperature could interfere with the myogenic proper-
ties of SCs. Nevertheless, we found that the brief period
of exposure to ice-cold temperature did not interfere
with the ability of SCs to proliferate and differentiate
in vitro or with their in vivo regeneration potential, sug-
gesting that ICT does not alter SC function. Indeed, SCs
isolated by ICT behaved similarly to those isolated using
the pre-plating method or the magnetic bead isolation
kit. Of note, both the magnetic bead and the FACS sort-
ing isolation methods involve far lengthier incubation
times on ice compared to our method without interfer-
ing with SC behavior and function in vitro and in vivo
[16, 17, 21, 22]. Interestingly, Marg A et al. recently
found that storing human muscle biopsies at 4 °C, in
low serum medium and no O2 for up to 35 days and
subsequent culture at 37 °C and 21% oxygen led to SCs
expansion outside the fiber fragments. Surprisingly, the
purity of the outgrowing colonies of SCs was 100% myo-
genic cells, since non-myogenic cells such as fibroblasts The ICT approach can also be used for serial passaging
and long-term expansion of SCs i Graph showing percent CFSE-low or slow-proliferating SCs after ICT or trypsin detachment, at day 4 of culture in GM (n = 3
independent experiments). Error bars represent mean ± sem, *p < 0.05, calculated by Student’s t test. SCs and SCs isolated by magnetic beads to regenerate
injured muscle (Figure S3C and D). These experiments
confirmed that SCs isolated with the ICT method suc-
cessfully engraft after transplantation and do not lose
their potential to regenerate injured muscle after expan-
sion in vitro. SCs and SCs isolated by magnetic beads to regenerate
injured muscle (Figure S3C and D). These experiments
confirmed that SCs isolated with the ICT method suc-
cessfully engraft after transplantation and do not lose
their potential to regenerate injured muscle after expan-
sion in vitro. These data show that the ICT method performs
equally well when used for the isolation of human SCs. Efficient isolation, serial expansion and long-term culture
of human satellite cells with the ICT method Representative bright field images of ICT- isolated human
SCs following 1, 3, 5, and 10 ICTs, at day 3 and 10 of culture in GM, and at day 10 of culture in DM (n = 15 independent experiments). b Number
of human SCs at 24, 48, and 72 h following ICT 1. c Total number of human SCs at day 3 of culture in GM after 1 and 10 ICTs. d Representative
immunofluorescence images of human SCs stained for Pax7 (red) and nuclei (blue). Graph shows percentage of cells positive for Pax7 at day 2 of
culture in GM. e Representative immunofluorescence images of human SCs stained for myogenin (red) and nuclei (blue). Graph shows
percentage of cells positive for myogenin at day 5 of culture in GM. f Representative immunofluorescence images of human SCs stained for
desmin (red) and nuclei (blue) after differentiation (10 days in GM + 5 days in DM). g Representative immunofluorescence images of human SCs
stained for myosin heavy chain (MHC) (red) and nuclei(blue). Graph shows percent fusion after differentiation (10 days GM + 10 days DM). (n = 3
independent experiments, 10 images analyzed per experiment) Scale bar = 100 μm. Error bars represent mean ± sem did not survive prolonged storage at hypothermic condi-
tions [33]. Therefore, it is likely that SCs tolerance for
cold stress is high compared to other cell types, and this
trait can be exploited as we did with our ICT method, to
improve the purity of SCs grown in vitro. previously been shown to retain stemness and long-term
self-renewal ability [31]. It is conceivable that being ‘true’
stem cells, the slow dividing SCs detaching in response
to cold temperature are the so-called first responders to
stress or injury in vivo [5], a hypothesis that will be the
subject of future investigation in our laboratory. Recently, Yoshioka K et al. described an improved ver-
sion of the pre-plating method, reducing the isolation
and purification procedure to 2.5 days in total, while in-
creasing the cell yield, and significantly improving the
purity of the resulting SC culture by introducing a re-
plating step [15]. The re-plating step performed at day
2.5 of culture involves the detachment of all adhered
cells including fibroblasts and SCs with trypsin, and
replating on matrigel coated dishes [15]. Efficient isolation, serial expansion and long-term culture
of human satellite cells with the ICT method While the pur-
ity of the resultant SC culture is comparable to ours, our
method involves fewer steps in total and only one over-
night pre-plating. In addition, the ICT method doubles
up as a very gentle passaging technique, allowing long-
term serial expansion of SCs ex vivo, without altering
their
proliferation
and
differentiation
properties. Whereas the FACS sorting method is and will remain
the gold standard for the study of SCs immediately after
isolation, the ICT approach will likely become the
method of choice for the in vitro expansion of SCs. After
each ICT passage, the already expanded SCs can be
cryopreserved, and stored until needed. With the ICT
method we were able to passage proliferating mouse and
human SCs for at least 10 times, expanding their num-
ber 150- and 300-fold, respectively. This represents a
clear advantage over the most commonly used passaging
reagent trypsin, which we and others have shown, typic-
ally accelerates the differentiation of passaged SCs after
only two passages [11]. Apart from being a relatively
harsh enzymatic passaging reagent, trypsin leads to the
detachment of all the cells in the dish including SCs that
are already committed to differentiate into myotubes, as
well as any contaminating cells, which might contribute
towards the loss of SC proliferative potential and accel-
erated differentiation. Indeed, even a gentler detachment
solution like Accutase leads to loss of myogenic prolifer-
ative properties and accelerates differentiation. By con-
trast, the ICT approach favors the detachment of only
the SCs that have not yet committed to differentiate,
and in particular the slow dividing population which has In a recent study, Gregory WC et al. demonstrated
that hSCs differentiate and lose their proliferative poten-
tial
when
maintained
in
high
mitogen
conditions
ex vivo. They used inhibition of p38 signalling to prevent
the differentiation of SCs and promote their expansion
[35]. Using our method, we were able to achieve the
same but with minimal manipulation, maintaining the
proliferative capacity of hSC ex vivo for an extended
period of time, to a similar degree using muscle biopsies
taken from a wide range of ages (between 50 and 90
years old). This is an important technical advance for
both basic and clinical research since it will allow re-
searchers to obtain sufficient number of cells for trans-
plantation or intervention studies, while reducing the
number of human biopsies required. Efficient isolation, serial expansion and long-term culture
of human satellite cells with the ICT method The study of human SCs (hSCs) has generally lagged be-
hind that of mouse SCs due to the difficulties associated
with obtaining muscle tissue, as well as the lack of
methods for the isolation of pure hSCs that can be ex-
panded in vitro without altering their myogenic potential
[13]. Having demonstrated the remarkable efficiency of
the ICT method in the isolation of pure mouse SCs, we
set to reproduce these findings using human muscle bi-
opsies. We obtained gluteus maximus specimens from
patients undergoing surgery, aged between 50 and 90
years. Using the same approach as described above, we
consistently and reproducibly obtained a highly pure cul-
ture of hSCs (Fig. 3a, d–g) from a heterogeneous popu-
lation of human muscle cells (Figure S4), that could be
serially expanded for more than 10 passages (Fig. 3a). As
previously reported [32], we found that hSCs proliferated
slower than mouse SCs, with a doubling time of 46 h
(Fig. 3b), reaching a peak around day 10 post isolation,
and slowing down thereafter. On average we isolated
around 20 × 103 SCs/g of muscle. Using the ICT
method, these hSCs could be expanded 300-fold over a
period of 2 months to a final total of 6 × 106/g of muscle
(Fig. 3c). Previous studies have shown that hSCs rapidly
downregulate Pax7 expression in culture [33]. In agree-
ment with others [33], we found that the expression of
Pax7 was variable and ranged between 45 and 50% at
day 2 of culture in GM (Fig. 3d). Almost 100% of the
hSCs were myogenin positive at day 5 of culture in GM,
suggesting that most of them have activated their myo-
genic program (Fig. 3e). The myogenic purity of the hSC
culture was further confirmed by desmin at day 5 (Fig. 3f) and MHC expression at day 10 after shifting to dif-
ferentiating medium (Fig. 3g). Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle Page 9 of 12 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 10 of 12 Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle (See figure on previous page.)
Fig. 3 Isolation and in vitro expansion of human satellite cells using the ICT method. A. Conclusions number of dystrophin positive fibers per TA muscle section in mdx mice
(ICT, n=5 mdx mice; ICT-expanded, n=4 mdx mice). Scale bar=100μm. Error bars represent mean ± sem. C. Representative immunofluorescence
images of dystrophin positive fibers (green) and nuclei (blue) in mdx tibi-
alis muscle at 30 days following intra-muscular injection of 15 x 103 ICT-
isolated SCs (left), or MB-freshly isolated SCs (right). B. Quantification of
the number of dystrophin positive fibers per TA section in mdx mice (ICT,
n=5 mdx mice; MB, n=5 mdx mice). Scale bar=100μm. Error bars repre-
sent mean ± sem. In the quest for new and improved SC isolation
methods, the ideal technique would permit the isolation
of pure SCs with minimal manipulation, that can be ex-
panded ex vivo without losing their stemness and regen-
erative capacity. In terms of purity of the isolated cell
population, the ICT method outperforms others such as
the pre-plating method or the magnetic beads isolation
method. Compared to other commonly used methods, it
is fast and easy to perform, and apart from the time re-
quired for enzymatic digestion (1.5 h), it involves min-
imal manipulation of the cells. Finally, using the ICT
approach, SCs can be expanded for extended periods of
time without losing their proliferation and differentiation
potential. This in turn drastically reduces the number of
mice or muscle biopsies required to obtain sufficient
number of cells. Additional file 4: Figure S4. Characterisation of the human muscle-
derived cells obtained after pre-plating and prior to ICT. A. Representative
bright field images of human muscle-derived cells at day 3 and 10 of cul-
ture in GM, and at day 10 of culture in DM. B. Representative immuno-
fluorescence images of the heterogeneous culture of human muscle-
derived cells stained for Pax7 (red) and nuclei (blue). Graph shows per-
centage of cells positive for Pax7 at day 2 of culture in GM. C. Representa-
tive immunofluorescence images of the heterogeneous human muscle
cell culture stained for myogenin (red) and nuclei (blue). Graph shows
percent of cells positive for myogenin at day 5 of culture in GM. D. Repre-
sentative immunofluorescence images of the heterogeneous culture of
human muscle-derived cells stained for MHC (red) and nuclei (blue). Graph shows percent cell fusion after differentiation (10 days in GM
followed by 10 days in DM). E. Supplementary Information
Th
l
l The online version contains supplementary material available at https://doi. org/10.1186/s13395-021-00261-w. Additional file 1: Figure S1. Myogenic properties of SCs isolated with
the ICT method. A. Percent of myogenic colony formation was calculated
as percent growing clones out of the total seeded single cells per well
(60 per 96-well plate) among ICT- and MB-isolated SCs (n=3 independent
experiments). B. Number of cells per clone in single clone-derived ICT-
and MB-isolated SCs at 48 and 72 h of culture in GM. ICT SCs, n= 37
clones analyzed per experiment. MB SCs, n= 37 clones analyzed per ex-
periment. C. Percent of ICT- and MB-isolated SCs positive for Pax7 at day
2 of culture in GM. (n=3 independent experiments). D. Percent fusion of
single clone-derived ICT and MB-isolated SCs after differentiation (4 days
in GM followed by 3 days in DM). Fusion index: number of nuclei within
myotubes divided by total number nuclei. E. Number of nuclei per myo-
tube in single clone-derived ICT and MB-isolated SCs after differentiation
(4 days in GM followed by 3 days in DM). (n=3 independent experi-
ments). Error bars represent mean ± sem. Efficient isolation, serial expansion and long-term culture
of human satellite cells with the ICT method Indeed, a major
obstacle to stem cell-based therapies has been the scar-
city of human muscle tissue specimens and the limited
number of cells that can be obtained for transplantation. Moreover, successful transplantation requires the use of
freshly isolated SCs because culturing and expanding
them in vitro greatly reduces their engraftment capacity
[13]. Here, we show that SCs isolated and expanded using
the ICT method do not lose their regenerative capacity. Another advantage of the ICT method is the im-
proved longevity of cultured myotubes. Generally, SCs
differentiate into myotubes by day 7 of culture and
shortly after, begin to detach [11, 12]. Notably, SCs
isolated and passaged with the ICT method could be
maintained in culture for up to 2 months, even once
they have differentiated into myotubes, likely due to
the lack of contaminating cells such as fibroblasts. The
purity of the isolated SCs is of paramount importance
for in vitro studies since even 97% purity is insufficient
to prevent overgrowth by non-myogenic cells, as we
demonstrated. While
important
for
cell
growth,
growth factors produced by fibroblasts, have been
linked to senescence induction in long term cultures
of mesenchymal stem cells [36]. Page 11 of 12 Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle (2021) 11:7 Conclusions Graph shows percent myogenic cells in
the human heterogenous muscle cells obtained after pre-plating or after
ICT at day 5 of culture in GM, calculated by IF staining for myogenin (n=
3 independent experiments, 10 images analysed per experiment). Scale
bar=100μm. Error bars represent mean ± sem. ****P < 0.0001 by Stu-
dent’s t-test. Overall, the cost-effectiveness, accessibility and tech-
nical simplicity of this method, as well as its remarkable
efficiency, represent major improvements over existing
methods, and will no doubt accelerate SC basic and
translational research bringing their therapeutic use
closer to the clinic. Finally, this is a proof of concept
study, and the ICT method can be further optimised,
adapted, and improved for use in different experimental
settings. Funding g
This work was supported by a grant from The Dutch Duchenne Parent
Project NL (DPP NL) to BLO; research grants from Parent Project Italy (PP,
Italy) to MB and from the University of Rome (# RP11715C7D238352,
RM118164275C7EBE, and RM11916B7E20311C to MB; and #
AR11715C7F9E158E, AR11816436905518, and AR11916B7E2A7B64 to AB). Availability of data and materials y
All data generated or analyzed during this study are included in this
published article [and its supplementary information files]. The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this
published article [and its supplementary information files]. The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. Additional file 2: Figure S2. Increased longevity in culture of ICT-
isolated SCs. A. Representative bright field images of ICT-isolated SCs at
day 3, 5, 11 and 17 of culture in DM. B. Representative bright field image
of MB isolated SCs at day 3 of culture in DM. C. Total number of days in
culture of ICT- and MB-isolated SCs. D. Representative bright field images
showing the heterogeneous muscle cell culture after pre-plating, at 3
and 5 days of culture in GM. Error bars represent mean ± sem. *P < 0.05
by Student’s t-test. Acknowledgements g
We thank Dr. Luca Madaro for critical reading of the manuscript and helpful
discussions. Authors’ contributions BLO conceived, developed, and standardized the method. BLO, AB, and MB
designed the experiments. AB performed experiments. AB and BLO analyzed
data and prepared the figures. BLO and MB supervised the study. CG, DMD,
and VC provided the human muscle biopsy specimens. BLO wrote the
manuscript. All authors critically read, edited, and approved the final
manuscript. Ethics approval and consent to participate All procedures involving mice were approved by the Italian Ministry for
Health and were conducted according to the EU regulations and the Italian
Law on Animal Research. According to the Italian law, the authors are not required to ask for approval
from an institutional review board or ethical committee for the use of
human tissue removed during surgical procedures. All patients gave their
approval to undergo intraoperative muscle biopsy and to publish the clinical
and laboratory data obtained. Additional file 3: Figure S3. In vivo functional validation of SCs
isolated using the ICT and MB method. A. Representative
immunofluorescence images of dystrophin positive fibers (green) and
nuclei (blue) in mdx tibialis muscle at 30 days following intra-muscular in-
jection of 15 x 103 SCs immediately after ICT isolation or after 3 day-
expansion in culture following ICT isolation. B. Quantification of the Additional file 3: Figure S3. In vivo functional validation of SCs
isolated using the ICT and MB method. A. Representative
immunofluorescence images of dystrophin positive fibers (green) and
nuclei (blue) in mdx tibialis muscle at 30 days following intra-muscular in-
jection of 15 x 103 SCs immediately after ICT isolation or after 3 day-
expansion in culture following ICT isolation. B. Quantification of the References Marg A, Escobar H, Gloy S, Kufeld M, Zacher J, Spuler A, et al. Human
satellite cells have regenerative capacity and are genetically manipulable. J
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1 pkcθ promotes satellite cell self-renewal. Int J Mol Sci. 2020;21:24 1Department of Anatomical, Histological, Forensic and Orthopedic Sciences,
Section of Histology and Embryology, Sapienza University of Rome, Rome,
Italy. 2Department of Anatomical, Histological, Forensic and Orthopedic
Sciences, Section of Orthopedics, Sapienza University of Rome, Rome, Italy. 3Department of Orthopaedics and Traumatology, Policlinico Umberto I,
Rome, Italy. 24. Fiore PF, Benedetti A, Sandonà M, Madaro L, de Bardi M, Saccone V, et al. Lack of PKCθ promotes regenerative ability of muscle stem cells in chronic
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The maladies of enlightenment science
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Ethics in science and environmental politics
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The maladies of enlightenment science Tim Wyatt*
Barrio A Tomada, Borreiros, Gondomar, 36378 Pontevedra, Spain Tim Wyatt* ABSTRACT: Some of the amalgam of activities which comprise traditional science (‘real’ science)
has fragmented into new modes under pressure from internal and external factors. The adoption
of the internet and the World Wide Web (which have created cyberscience and are internal fac-
tors) can be equated with the introduction of the microscope and other instruments and proce-
dures, though they have revolutionized the whole of science rather than some of its branches. External factors discussed here include post-modernism, neoliberalism, and McDonaldization. In
post-modern science, the traditional model has become burdened by social and political interests,
and concerns for practical problems in which scientific expertise can assist decision making; how-
ever scientific knowledge in itself is not a priority. Neoliberal guile has parasitized science as it has
so much else in search of profit, and has seriously damaged its host. There are also degraded or
pathological activities, ‘McScience’, in which the pursuit of knowledge has been corrupted by an
excess of bureaucratic control and over-emphasis on personal rankings. The ethos which guides
traditional science has been warped by these various outside interests, secrecy is rewarded, and
practices once considered dishonest are prospering. KEY WORDS: Enlightenment · Traditional science · Postmodernism · McDonaldization ·
Neo liberalism · Cyberscience © The author 2017. Open Access under Creative Commons by
Attribution Licence. Use, distribution and reproduction are un -
restricted. Authors and original publication must be credited.
Publisher: Inter-Research · www.int-res.com *Corresponding author: timwyatt1937@gmail.com ETHICS IN SCIENCE AND ENVIRONMENTAL POLITIC
Ethics Sci Environ Polit
Vol. 17: 51–62, 2017
https://doi.org/10.3354/esep00177 ETHICS IN SCIENCE AND ENVIRONMENTAL POLITIC
Ethics Sci Environ Polit
Vol. 17: 51–62, 2017
https://doi.org/10.3354/esep00177 ETHICS IN SCIENCE AND ENVIRONMENTAL POLITICS
Ethics Sci Environ Polit ETHICS IN SCIENCE AND ENVIRONMENTAL POLITICS
Ethics Sci Environ Polit Published October 23 Contribution to the Theme Section ‘The ethics and practice of openness in life sciences data’ © The author 2017. Open Access under Creative Commons by
Attribution Licence. Use, distribution and reproduction are un -
restricted. Authors and original publication must be credited. TRADITIONAL SCIENCE idola specus (idols of the cave), personal prejudices;
(3) the idola fori (idols of the marketplace) caused by
shared language and commerce; and (4) idola theatri
(idols of the theatre), i.e. systems of philosophy and
proof — whence came the Royal Society’s motto
‘Nullius in verba’ (which means do not take any-
body’s word for it), and the exclusion of discussions
concerning politics and religion, impediments to
clear thought, from its conduct. Science started to acquire its modern sense (as
‘natural philosophy’) during the Scientific Revolu-
tion, from Copernicus to Newton and the Age of
Enlightenment, as it gradually freed itself from the
shackles of theology and absolutism, from a thousand
years of stasis and obscurantism (Russo 1996). Under
the influence of Descartes, Leibniz, and others, faith
and dogma gave way to rationalism. ‘Gradually, the-
oreticians behind the movement that had begun as a
grand attempt to merge God and syllogisms realized
that logic did not require the link to the divine’
(Schlain 1998). When the Royal Society of London
was founded in 1660, it tried to protect itself from
intellectual fallacies, from the ‘four kinds of illusions
which block men’s minds’. These illusions, listed by
Francis Bacon in his Novum Organum Scientiarum,
were (1) the idola tribus (idols of the tribe), percep-
tual errors due to the limitations of the senses; (2) the From then until quite recently, science was almost
universally regarded as a system which formulates
laws to describe information and turn it into knowl-
edge, the systematic study of nature by methodical
processes of observation, experiment, measurement
and inference which generate that information, and
tests of the laws. These procedures are collectively
called the scientific method. ‘It is the matter-of-fact as
against the romantic, the objective as against the
subjective, the empirical, the unprejudiced, the ad
hoc as against the a priori’ (Waddington 1948, p. 61). Ethics Sci Environ Polit 17: 51–62, 2017 52 Not all branches of science fit this model. In the clas-
sical procedures of physics, single parameters are
isolated and manipulated whilst others are held con-
stant; control experiments can be set up, and replica-
tion is possible. Historical sciences such as cosmol-
ogy, geology, palaeontology, or evolutionary biology
cannot deploy these procedures. Nevertheless, we
can recognize a group of academic disciplines that
share a common identity. and many are explorers, some are artists and other
artisans. TRADITIONAL SCIENCE There are poet-scientists and philosopher-
scientists and even a few mystics’ (Medawar 1986);
but most to varying degrees subscribed to the profes-
sional ethos for which Ziman (1994) coined the
mnemonic CUDOS (communalism, universalism, dis-
interestedness, originality, skepticism), adding origi-
nality to Merton’s (1942) 4 standards of the scientific
ethos. In this ideal world, good science is produced
by ethical scientists; later, we see that the good and
the ethical have become disconnected (Nosek et al. 2012, Smaldino & McElreath 2016). Normally, hypothesis comes first, and decides what
will be observed; the dialectic between speculation
(not necessarily mere as in the pejorative tense) and
skepticism is what leads to good science (Medawar
1967). Science, then, is a search for patterns. Its laws,
or theoretical truths, when they are discovered, are
independent of any practical applications they may
subsequently be found to have — a notion unfamiliar
in some quarters. There is much in science without
any discernible economic value; do we need to know
about the Ediacaran fauna or the Permian extinction,
whales’ songs or spiders’ dances? The answer is yes
in each case, not for economic reasons, but because it
is fascinating, gripping, and above all fun. For many
scientists, understanding is more important than
technical applications, and for those who play in the
arena of pure mind, science may be a personal
search for karma. But for most, social rewards are
sought. It is true that ‘Scientific life would not be human if
it were not permeated with folly, incompetence, self-
interest, moral myopia, bureaucracy, anarchy and so
on’ (Ziman 2000, p. 5), all sources of bias and error. But the drive for objectivity in science is a feature of
the community, however partisan its individual mem-
bers might be, and has in principle been determined
by openness based on collegiality and trust. In the
well-known allegory in the Republic, Plato leads us
from the obscurity of the cave where individual opin-
ions rule (idola specus) to the outer sunshine, the
realm of episteme, knowledge, where claims must be
justified in public. Knowledge obtained by science
according to this tradition becomes part of the intel-
lectual or cultural commons, what Sprat (1667) called
the ‘publick Treasure’, freely available to all. In the kind of science just outlined, there is not nec-
essarily a direct flow from science to technology and
consumer products. NEW SCIENCES, NEW ETHEA The preceding paragraphs outlined some aspects
of the dominant tradition of how science worked for
some centuries and how much of it still works today. Ziman (2000) called this ‘real science’. However in
recent decades, this tradition has been subjected to
destructive pressures from several directions. Some
scientific problems have become much more de -
manding of capital investment and thus more vulner-
able to political and industrial tampering. In the spirit
of universal Darwinism, one might say that selection
is acting on variations in the original tradition (its
functional traits), creating new species of science. Here, the analogy refers to the social procedures of
science, and is therefore distinct from the ‘evolution-
ary epistemology’ of Kuhn (1962) or Daw kins (1976)
in which it is the content that evolves: but the proce-
dures cannot change without having an impact on
the resulting knowledge, and on its ideological
claims. Some of these new species are added to the many
pseudosciences, Medawar’s (1977) Unnatural science
(Chaucer’s unscience in Boece) which have long
accompanied traditional science, astrology, augury,
various sorts of medical quackery (Starr 1982), as
well as more recent aspirants. A short list includes
Lysenkoism (which rejected Mendelian inheritance),
psychoanalysis (Popper 1959, Gray 2014, Chapter 2),
intelligent design (Dawkins 1996, Pigliucci 2002),
homeopathy (Ernst 2010), IQ testing (Gould 1981),
parapsychology, psychokinesis; see Gardner (1952)
for more. Finally, somewhere be tween science and
pseudoscience, we can mention weird science (Hol -
lings 2008), a rag bag of topics, some pursued in secret
by government agencies, which ranged (in the 1950s
at least) from a search for ‘truth drugs’ to dianetics
and flying saucers. Science today seems to almost always be under
pressure from other constituencies, each with its dis-
tinct ethos, perhaps more so than formerly. Much of
this pressure is based on financial instruments and
bank credits, and the ambitions of John Stuart Mill’s
Homo economicus. This pressure has increased in
recent decades as neoliberal ideas have been imple-
mented. Scientific knowledge and the data which
underpins it are becoming commodified, and the tra-
ditional openness of the scientific ethos can impinge
on market values. In business, secrecy and reticence
are elementary instincts whenever something has
the smell of potential profit. ‘The tongue hath no
force when gold speaketh’ wrote Stefano Guazzo1. TRADITIONAL SCIENCE Even though much technology
today is applied science and the 2 form a conceptual
continuum, certain aspects of science can be so pure
or esoteric, even though their pursuit depends on
technology, that their value must be sought else-
where. Much technology too is independent of sci-
ence; there is no lockstep relationship between the 2. Did the invention of the sewing needle, say, or the
screwdriver, of barbed wire or Teflon, depend on sci-
ence? Practical steam engines preceded the science
of thermodynamics by nearly a century. Equally,
most discoveries depend on technology. There is no
stellar chemistry without spectroscopy, nor cell the-
ory without microscopy. In the present context, sci-
ence has been demonstrably open in some ways, at
least in times of peace and when free from bureau-
cratic meddling, while technology has not. But the
secrecy of inventors is condoned, and Galileo played
both cards. Scientific exploration of nature is in principle
rational (Popper 1959); but research is driven by pas-
sion as well as reason, and rivalry, ambition and prej-
udice are significant inputs. There are those who
crave recognition and may forego academic disci-
pline in pursuit of the rewards of fame and influence. It is against the background of this mental potpourri
that the dilemma of scientific openness and secrecy is
mediated. Thus, traditional science is embedded in a
social context, only one function of which is to strive
for objectivity. Scientists themselves are expected to
make results public so that their peers can test their
reliability. Ideas and results are shared, honed, dis-
tilled, embellished, rejected, and often ignored. All
such evaluation procedures are institutionalized to
varying degrees, most clearly in the editorial/peer re -
view system. In making their work public, re searchers
can establish priority and gain professional recogni-
tion — parts of the reward system. So the essence of
scientific communication comprises published ac -
counts of research such that, potentially, the work
can be repeated with similar results. This requires
that experimental methods and analytical tools be
described in sufficient detail for such independent
tests to be carried out. For the historical sciences and This is not to say that the scientific tradition has a
monolithic unity; far from it, it is rather an amalgam. 1Guazzo S (1581) The ciuile conuersation. George Pettie’s
translation. See Guazzo (1925) TRADITIONAL SCIENCE Scientists include ‘collectors, classifiers, and compul-
sive tidiers-up; many are detectives by temperament Wyatt: Maladies of enlightenment science 53 ics masquerading as science’ (Gray 2009), destroys
the science it wishes to emulate, just as it destroys
other social institutions (Gray 2009). for purely theoretical or speculative ad vances, com-
parable transparency is required. Good science can
withstand detailed scrutiny, and does not hide its
procedures. p
This simple ethos has long guided scientific com-
munication, although human foibles add many colors
to it. Galileo Galilei, one of the founders of modern
science, was a master of information control: his
Sidereus Nuncius (1610) in which he reported the
existence of 4 of Jupiter’s moons, was ‘a balancing
act between communication and secrecy, between
the desire to have his discoveries accepted and that
of slowing down potential replicators so that they
would not become his competitors’ (Biagioli 2006,
p. 16). Galileo withheld the technical details of his tel-
escope which ‘allowed him to develop a monopoly on
observational astronomy’ (Biagioli 2006, p. 125). Pri-
ority disputes have often been bitter, contestants’ be -
havior vitriolic. There were flagrant clashes between
Robert Hooke and Christiaan Huygens (about the
hairspring), between Evangelista Toricelli and Blaise
Pascal (mercury barometer), between Ronald Ross
and Giovanni Battista Grassi (life cycle of malarial
parasite), and especially between the unrelentingly
vindictive Isaac Newton and Gottfried Leibniz (the
calculus), a dispute which outlasted both of them. The lyrics of Tom Lehrer’s Lobachevsky spring to
mind. In more recent times, James Watson (1968), co-
discoverer of the structure of DNA, thought secrecy
should be encouraged so as to win ‘the race’ and reap
the honors. Does temporary secrecy pass as openness? POST-NORMAL SCIENCE This list is not exhaustive. Ravetz (1996, p. xiv) also
offers us shoddy science, entrepreneurial science,
reckless science, and dirty science, the ‘four horse-
men of the scientific apocalypse’. The fraud and mis-
conduct components which characterize some of
these are sometimes due to commercial pressures,
but obviously scientists themselves are often guilty,
driven by ambition and the search for laurels. We
read too of settled science, consensus science, and
other oxymorons. There are neither incontrovertible
nor inconvenient truths in science, though we do
sometimes meet convenient untruths. Some of these
new species are eroding the academic space occu-
pied by traditional science. Post-normal science is a response to the realization
that Adam Smith’s ‘invisible hand’ metaphor has
proved delusional, that unconstrained individual
greed and slash-and-burn capitalism cannot make
rational decisions about the management and con-
servation of natural resources, nor can these deci-
sions always await the results of hard science. Sci-
ence itself does not ‘possess an invisible hand
mechanism through which the naked self-interest of
individuals necessarily brings about a collectively
optimal result’ (Smaldino & McElreath 2016, p. 5). Post-normal science is a hybrid between ecology and
economics, between epistemology and governance,
stresses that evaluations cannot be reduced to one-
dimensional standards, and that the quality of policy
decisions is more important than scientific truth. It is
therefore not really science at all, but scientific infor-
mation comprises one node in complex decision
making processes. NEW SCIENCES, NEW ETHEA There is thus a conflict about how open the commu-
nication process should be, and whether scientific
data and their interpretations should be shared freely
with third parties rather than being traded as com-
modities. Neoliberalism, that is ‘faith-based econom- Some new species now discerned as distinct from
traditional science have been given names. We can
list, in no particular order, post-modern science (Po -
lanyi 1974, Feyerabend 1988), post-normal science
(Funtowicz & Ravetz 1993, Ravetz 1996), mode-2 sci-
ence (Gibbons et al. 1994), cyberscience (Wouters
1996, Nentwich 2003) and science 2.0 (Shneiderman
2008), neoliberal science (Lave et al. 2010), and junk
science (Agin 2007). Horton’s (2004a) McScience
includes some of them. Krimsky (2003) describes cor- Ethics Sci Environ Polit 17: 51–62, 2017 54 flict between constructivism and science at the oper-
ational level. The scholarly accomplishments of con-
structivism need free access to information from all
inputs to remain robust. rupted science as dishonest, lacking in integrity and
objectivity, and cut off from its ethical roots, charac-
teristics blamed partly on generous corporate fund-
ing (see below). Goldacre’s (2008) ‘bad’ science
includes McScience and other illegitimate offspring
of real science, and exposes the often uninformed
rhetoric and propaganda which accompanies it. POST-MODERN SCIENCE Proponents of post-modern science argue that all
knowledge is relative to the culture in which it is
embedded and the frailty of language, that true ob -
jectivity cannot exist. Post-modernists claim Fried -
rich Nietzsche as their own — ‘Words are but symbols
for the relations of things to one another and to us;
nowhere do they touch upon absolute truth’ (Niet-
zsche 1962, p. 83). It is in one sense an attempt to
deconstruct the ‘Enlightenment project’ (Kuntz 2012)
with its commitment to reason, thus a kind of intellec-
tual abuse or anti-science. In its extreme form, social
constructivism stands Francis Bacon on his head, and
maintains, for example, that the ‘natural world has
but a small or non-existent role in the construction of
scientific knowledge’ (Collins & Pinch 1993); this is
obviously a hostile and nihilistic view, hardly an
advance on pre-Socratic solipsism. Postmodernist
arguments of this kind were targets in the so called
‘science wars’ of the 1990s (Gross et al. 1996). Less
extreme arguments from neo-Marxists and others
maintain that scientific ideas are (and historically
have been) determined by economic and social inter-
ests; there is no doubt that study topics are influ-
enced by such interests, but this does not mean
objectivity is necessarily impugned. NEOLIBERAL SCIENCE There are powerful voices demanding that scien-
tific procedures be politicized and the products pri-
vatized and commercialized. These voices emanate
from the blinkered economic doctrine called neolib-
eralism. While constructivists pry into the accepted
rectitudes of science, a purely academic exercise,
economic pressure from the neoliberal Right is driv-
ing the practice of science into new molds. Idealism is
under pressure from ideology; this has been labeled
‘mode 2’ science by Gibbons et al. (1994), neoliberal
science by others. Much present-day scientific research activity is
now conducted in the spirit of or in apparent imita-
tion of methods to improve industrial processes (pio-
neered by Frederick Winslow Taylor). Control of
much that is done in many laboratories has been
transferred from scientists and their technical col-
leagues to line managers, and research, data analy-
sis, report writing, etc., are constrained by Gannt
charts or their modern equivalents, fashionable cha-
rades which often assume an air of spurious certainty. Even quite small collaborative projects are mission
oriented and have coordinators and associated con-
tractors and sub-contractors who prepare task
reports and technical progress reports and lists of
deliverables (= quantifiable goods or services); there
is dialogue and networking and there are milestones
and players and stakeholders. These are the sound
bites of politics and trade, not traditional science. All
this project management is coercive at the micro-
level of individuals, social engineering at the macro-
level. Informal procedures, once characteristic of
small groups of collaborators, are replaced by mana-
gerial straightjackets; a pointer moves along a scale
from open to totalitarian. It is not the quality of sci-
ence which is at stake but abstract standards derived
from outside science. Scientific issues are trans- The peer-to-peer openness and trust referred to
earlier has not been replaced (yet?), but is losing
some of its former importance as newer systems of
evaluation are imposed. These are linked to social
or political values defined by evaluators who often
have no expertise in science or any other kind of
research, and who make use of low-dimensional
numerical measures (cf. intelligence quotients or
the GNP) to quantify categorical variables. Some
evaluation protocols have little to say about the
quality of the science assessed. Thomasina Coverly
in Tom Stoppard’s play Arcadia finds ‘a truly won-
derful method whereby all the forms of nature must
give up their numerical secrets and draw them-
selves through numbers alone’. SCIENCE 2.0 Shneiderman’s (2008, 2016) science 2.0 is a re -
search manifesto and has much in common with
post-normal science. It advocates a multidisciplinary
programme of applied ‘science, engineering, and
design’ to tackle problems like disaster response sys-
tems or environmental sustainability based on col-
laboration using computer tools and the internet. Since science 2.0 forbids a purely reductionist ap -
proach, the claim that actionable problems are inac-
cessible to traditional science is sound enough: its
‘immense problems’ are then only remotely scientific
problems. To become accessible they need to be
broken into smaller pieces. To deal with such prob-
lems, Shneiderman outlines 5 strategies, one of
which is indeed classical reductionist science, useful
to tackle the smaller pieces! We can agree that solu-
tions to complex problems demand multidisciplinary
approaches. It is not obvious how rebranding the
search for such solutions as science 2.0 helps. New products and better community services may
result from science 2.0, but scientific truth is not a pri-
ority. Shneiderman is amongst the many who believe Constructivists have reactivated the idols in the
Baconian theatre, but this has not had any impact on
the way traditional science is done. There is no con- Wyatt: Maladies of enlightenment science 55 formed into political ones, and knowledge is treated
as a product, commodified, and subjected like con-
sumer goods to market criteria. we need to accelerate progress, as though innovation
is a panacea for the greed run riot of our neoliberal
times. Collaborative projects which call on expertise
from diverse disciplines have an important potential
reward, to reduce what is filtered from the percep-
tions of individual collaborators, of scientists as well
as other players. However, from the scientific per-
spective there is a downside. Should lay opinions or
the values of stakeholders play roles in scientific
decisions? The answer is no. This is simply appease-
ment and intellectual cowardice; it both sidelines the
science and dilutes evidence with opinion. Much research now begins with such proposal
writing, a minor art form in which the supplicant
might make ‘inferences about an unknown territory
from an unfaithful map’ (Eco 1994, p. 98), while
simultaneously attempting to persuade patrons that
the expected results will be innovative and with high
potential impact. It is as though results are already
known before a study has been started. SCIENCE 2.0 Supplicants
are well aware that scientific discoveries are not nor-
mally produced by logical arguments, but they must
persuade patrons otherwise; beyond the subterfuge,
evaluation can effect changes, presumably unin-
tended, in research methods and objectives, as well
as introduce surveillance. In addition, administrative
(i.e. artificial) time scales are imposed on research
activities, whatever natural rhythms they may have,
and there is no space for blue-sky research. The
implication is that research is a process of production
(per time unit), presumably measured with biblio-
metric indices, or in some cases by numbers of patents. This kind of evaluation goes far beyond the need to
provide accountability for public funding, and tends
to be coercive and normalizing so that, paradoxically,
it can inhibit the innovation which scores high points! ‘The path of creativity is strewn with the bones of
those consumed by the vultures of mediocrity, ac -
countability, and responsibility’ (Loehle 1990, p. 129). Outside evaluation also destroys the collegial spirit
that once held sway, and is undemocratic. NEOLIBERAL SCIENCE However, the quality of sci-
ence is largely determined by its exposure to refuta-
tion and counter-argument. This process becomes
much more difficult if the circulation of research find-
ings is restricted’ (Nowotny et al. 2003, p. 183). Corporate interests can be further improved by
inventing new diseases: ‘the pharmaceutical industry
is working behind the scenes to help define and
design the latest disorders and dysfunctions in order
to create and expand markets for their newest medi-
cines’ write Moynihan & Henry (2006); ‘social anxiety
disorder’, ‘pediatric bipolar disorder’ and ‘premen-
strual dysphoric disorder’ are a few examples of such
duplicity. Invention is the mother of necessity so to
speak. Authors and editors critical of the pseudo-
science and others who expose the corruption can
expect to face litigation. Full and unrestricted access
to the raw data is not encouraged, and confidentiality
agreements with sponsors are the norm. Corporate
sponsorship ensures that stock values over-ride med-
ical ethics (Horton 2004b). Ignorance and misunderstanding, like knowledge,
are social constructions, used by scientists to obtain
funding, and by commercial and political interests
(Smithson 1989). Stocking & Holstein (2009) provide
a detailed case history concerning industrial pig pro-
duction in North Carolina. Manufactured ignorance
can be purposefully blended with the genuinely
unknown and afforded a false legitimacy in debates. Even the genuinely unknown, a product of epistemic
blind spots, can be exploited. Rachel Carson’s (1962)
exposé of the impact of pesticides is classic. Proctor
(1995) details how government funding of cancer
research fostered ignorance with respect to its envi-
ronmental and industrial causes. The current debate
about the problem of colony collapse disorder in bees
is following the same pattern. Market interests often
employ expressions like ‘sound science’, ‘the science
is settled’, or alternatively, ‘there is no conclusive sci-
entific evidence’ to create ambiguities and manipu-
late public perceptions. Neoliberal pressure on scientific communication
has affected the shifting balance between coopera-
tion and competition, which moves the cursor along a
vector from openness to secrecy. Use of the internet,
development of electronic data bases, and advances
in the speed and power of computing have added
new ingredients to the desire to protect priority,
credit, and intellectual property rights (Resnik 2006). Even before neoliberalism became mainstream, mar-
ket forces distorted the open nature of traditional sci-
ence, and indeed science itself. Hobbes’ (1668, p. NEOLIBERAL SCIENCE It does not work in
the real world. Each individual scientist and evaluator has his or
her own self-interest; individuals in both groups wish
to promote their own power, the former with science
and ambition, the latter with power and ambition. Innovative thinking cannot be imposed by budgets or Ethics Sci Environ Polit 17: 51–62, 2017 56 other means (Medawar 1967). ‘Indeed, in our reckon-
ing, an aspiration of excellence seems as much amor-
phous as futile; a truly Sisyphean pursuit’ (Water-
meyer & Olssen 2016, p. 206). viewed by Lave et al. (2010) are ‘narrowing of
research agendas to focus on the needs of commer-
cial actors’, ‘fortification of intellectual property in an
attempt to commercialize knowledge, impeding the
production and dissemination of science’, meaning-
less openness, and changes in ‘the methods, organi-
zation, and content of science’. A unique feature of neoliberal biomedical science
is the way work is subcontracted. For example, clini-
cal trials can be put out to tender, and contracts
agreed with commercial research organizations. The
results of such trials are then compiled and analysed
by ghostwriters working for companies that mimic
scientific papers; with the help of a little baksheesh
from the pharmaceutical industry, academics of
standing (thought leaders) are next identified who
allow their names to be attached as honorary authors
(Horton 2004b, McHenry 2010). These services are
not cheap; a single manuscript can cost as much as
the annual salary of a scientist. A decade ago, it was
reported that more than 50% of articles about clinical
trials published in The Lancet, the New England
Journal of Medicine and the Journal of the American
Medical Association are ghostwritten (Anon 2005);
medical journals ‘have evolved into information
laundering operations for the pharmaceutical indus-
try’ (Horton 2004b). Public research funding, once disbursed in a fiduci-
ary capacity, now like corporate funding often seeks
results with market potential, and socio-economic
expectations move the cursor in the direction of
secrecy. Worse yet, we are witness to ‘the transmo-
grification of the university from free creative space
to corporate [and government] panopticon’ (Water-
meyer & Olssen 2016, p. 203). So ‘the exploitation of
intellectual property challenges the ideal of scientific
knowledge as a public good. If intellectual property
is valuable, it cannot be given away freely by open
publication in peer-reviewed journals, or at scientific
conferences open to all. McSCIENCE Horton’s McScience recalls Ritzer’s (1998, 2011)
McDonaldization (itself something of a franchise,
with 7 editions in 15 yr!), a sociological model which
focuses on 4 features of the fast food industry; pro-
duction (quantity) must aim to be efficient (minimize
the time to do things), quantifiable (quantity is more
important than quality), predictable (work tasks are
repetitive, routine), and controllable (employees are
standardized). Everything is reduced to numbers,
and the product is cheap, easy to make, fast (obvi-
ously!), but short on authenticity. The model could be
an overture to a repackaged and sanitized science, a
kindergarten science. Some science is already a
manufacturing enterprise, churning out papers
which maintain production rates, but at the cost of
quality. The fragmentation of factory work which
Frederick Taylor counseled is now met in laborato-
ries, many now staffed by a generation of highly
trained people with McJobs, amassing data for
McThinkers who in turn are calculating how thinly
the salami can be sliced, and dreaming of boosting
their McImpact factors. According to Douglas Engelbart, inventor of the
mouse, ‘the digital revolution is far more significant
than the invention of writing or even of printing’
(quoted by Bauerlein 2009). It seems probable that
Marshall McLuhan would have agreed had he lived
to witness it. Twenty years ago as the digital world
was taking shape, Jon Katz (1997) wrote that the
generation to whom it had already become second
nature ‘take no one’s word for anything’! The motto
of the Royal Society has found a home in a new con-
stituency. Katz also wrote: ‘The Internet is still a wild
frontier. The hackers and geeks who founded and
shaped it believed that there should be no obstacles
between people and information, and there are still
vibrant, almost outlaw communities that enforce this
notion: cypherpunks who act as technoanarchists,
flamers who challenge punditry, hackers who breech
the barriers constantly being thrown up by govern-
ment and business.’ Twenty years later such barriers
continue to be erected — and torn down or bypassed. There are lobbies in science as well as in politics,
and in some industries there have been (and are)
strategies to provide scientific validity for conclusions
which have been decided in advance, as well as
obfuscate or denigrate embarrassing data. Michaels
(2008) gives a detailed account of such perversions
from a variety of industries (pharmaceuticals, to -
bacco, asbestos, pesticides, dyes, …). NEOLIBERAL SCIENCE 25)
admonition ‘against the lucrative vices of men of
trade’ falls on deaf ears. The results so far as re - We see that the ethical codes and canons of tradi-
tional science have been partially abandoned, and
that this has led to a splintering or disintegration of at
least one component of the enlightenment pro-
gramme. There is no such animal as ‘settled science’. The consensus can be wrong (or fibbing!); science
deals in probabilities, not certainties. ‘Para-journal-
ism’ and ‘infotainment’ spread these dishonest mes-
sages. ‘The class which has the means of material Wyatt: Maladies of enlightenment science 57 CYBERSCIENCE production at its disposal, has control at the same
time over the means of mental production, …’ (Marx
1846). Freedom to differ from majority opinion, and
to resist pressure to conform, is essential for scientific
progress, so that legislation to silence nonconformist
views is counterproductive. History often shows that
unorthodox science can come in from the cold. Alfred
Wegener’s hypothesis of continental drift, long
mocked, is an example (Oreskes 1999); its descen-
dent, plate tectonics, is still not necessarily settled
(Storetvedt 1997). Cyberscience (also e-science) refers to science car-
ried out in cyberspace, to take advantage of net-
worked computers and state-of-the-art information
and communication applications (grid technology)
and create virtual research environments. It goes
beyond the use of individual computers as tools for
storing and processing information, provides collabo-
rators in geographically dispersed scientific projects
the ability to archive and access very large data col-
lections, and enables them to manipulate, model, and
visualize with almost unlimited computing power. Cyberspace offers new opportunities for research,
just as the telescope and the microscope have in the
past (and of course still do). The tools of cyberspace
provide new means ‘that will ease the extraction of
information from data, and of knowledge from infor-
mation’ (Hey 2006, p. vii). OPENNESS Not only have individuals succumbed to the new
norms, but large sections of Academe too. Tuchman
(2009) maintains that the traditional model for higher
education has been abandoned — decisions formerly
entrusted democratically to faculty members are now
made by administrators, and agreements formerly
guided by the common good are now replaced by
edicts of an audit culture, coercion again. Universi-
ties no longer ‘lead the minds of students to grasp
truth; to grapple with intellectual possibilities; to ap -
preciate the best in art, music and other forms of cul-
ture; and to work towards both enlightened politics
and public service’ (Tuchman 2009, p. 41). Many uni-
versities now give priority to their bottom lines. ‘Each
infringement on its [the university’s] unwritten con-
tract with society to avoid secrecy whenever possible
and maintain its independence from government or
corporate pressure weakens its integrity’ (Klein
2000). A rather natural proprietary attitude to data has
probably always accompanied the traditional ethos
of science and set a limit on full openness. The free
software movement, online mechanisms for scientific
collaboration, and the Creative Commons project,
which aims to regulate downstream use of intellec-
tual property rights, partially free culture from the
grip of corporate control and provide new models to
avoid the open-closed impasse. Mayo Fuster Morell
(2015), Lawrence Lessig (2004) and others are pro-
moting openness in the digital world. These trends
are ‘becoming the equivalent of the socialist move-
ment in the industrial age’ (Bauwens 2005). Privati-
zation and top-down control of information flows
counter such projects, and capitalism sometimes
dominates the relationship; but signs of a more bal-
anced symbiosis are emerging. The interface between transparency and secrecy
has complex topography. Openness provides infor-
mation to listeners and readers as well as peers, but
the information transmitted can be filtered and its
timing controlled to serve personal, institutional,
or corporate needs, to ensure priority, or to feed
motives related to patronage. Knowledge itself is
power (ipsa scientia potestas est), wrote Francis
Bacon in his Meditationes Sacrae (1597). As in a fit-
ness landscape, movements towards open peaks or
secret valleys can be driven by a variety of compet-
ing interests as mentioned at various points in the
preceding pages. These range from the idealistic
openness of the traditional ethos (peaks) to the
secrecy which surrounds the inventions and innova- This article began with Francis Bacon’s 4 idols. McSCIENCE Recently, collu-
sion between the sugar industry and Harvard aca-
demics in the 1960s and 1970s has come to light; this
deflected attention from the link between sucrose
consumption and coronary heart disease, and
pointed instead at fats and cholesterol (Bes-Rastrollo
et al. 2013, Kearns et al. 2016). Funding by the Sugar
Research Foundation was concealed. These practices
are pure McScience. Research areas ranging from genomics and pro-
teomics to particle physics and meteorology are now
heavily dependent on world-wide online databases
and the tools to exploit them. Cyberscience is then a
direct descendant of traditional science, undiluted by
social and political inputs, and so distinct from post-
modern science and science 2.0. The internet may in the long run have a more pro-
found impact on science than neoliberalism. The
virtues of the internet as a tool for cyberscience are
intermingled with its character as a consumer habi-
tat, inimical to sustained thought. Combined with
interruptions by e-mail, text messages and telephone
calls, the pings and beeps of the digital environment, Ethics Sci Environ Polit 17: 51–62, 2017 58 tions of individuals, corporations, or political and
military interests (valleys). Between these extremes
(on the slopes) lie the reticence of scientists who
wish to assure priority and recognition for them-
selves or their colleagues, the exposure necessary
for patent claims, the demands of society to be in -
formed. Movements through this landscape are reg-
ulated by individual ethics, by contractual agree-
ments of different kinds, by economic necessities,
and by laws, but also by plagiarism, industrial and
military espionage, by hacking, and by permuta-
tions of them. Each of these components of the land-
scape also has its time constants, and all of them
have varied in importance through history (Long
2001). There are no simple tracks between the open
peaks and the secret valleys. Like a categorical vari-
able, the landscape of openness and secrecy resists
quantification. Neither extreme can be reduced
meaningfully to a single integer. performance is disrupted, error rates increased, and
attention replaced by distraction (Jackson 2008). This
frenetic activity leaves little time for thought. The
fragmentation of the technical part of research is
matched by intellectual wounds. McSCIENCE Screen time ‘super-
powers [young adults] social impulses, but it blocks
intellectual gains’ (Bauerlein 2009), and the transi-
tion from scholarly work to infotainment is only a
mouse click away; ‘the screen becomes not a vein of
truth but a mirror of desire’ (Bauerlein 2009), and e-
literacy is replaced by illiteracy. Simultaneously, syn-
ergy between the internet and neoliberalism offers
an open road to the malignant narcissism Eric Fromm
wrote about in The Heart of Man (Fromm 1964);
there is positive feedback between the psychological
gratification of rising impact factors and the severity
of the condition. DISCUSSION Science has not only been commodified but also
infected with deceit. Scientific misconduct is fre-
quently in the news, and editors and referees detect
much of it. Misconduct by the gatekeepers of scien-
tific publications has attracted less public attention,
but recent revelations concerning the journal Food
and Chemical Toxicology (Foucart 2016) detail some
corporate lobbying of publishing. Science, like other
aspects of culture, is ruthlessly perverted in the serv-
ice of the market, sometimes with the connivance of
editors with elastic views of the truth, willing to
ignore or suppress the ethical codes and canons of
traditional science. If science provides information
which could disturb market interests, then corruption
of that science is very likely (Michaels 2008, Oreskes
& Conway 2012). Democracy is a social system based on formal
equality of rights and privileges, and in its European
mid-20th century manifestation went hand in hand
with collective rights to social protection and the
redistribution of productivity. In contrast, corpo-
ratism seeks to reserve those benefits for a particular
caste. These are the ‘sacred’ and ‘satanic’ (to twist
Carey’s Manichean world view) protagonists of mod-
ern western society, an unequal symbiosis verging
on parasitism. Control of the flow of information, an
essential ingredient of complex social systems, is a
major factor giving an advantage to corporatism. Corporate propaganda, a means to protect itself from
democratic demands (Carey 1997), is part of this con-
trol, and scientific data and scientific knowledge
generally are both used and misused in pursuit of
corporate interests, as indicated earlier. In this system, space for the motives and aspira-
tions of scientists is reduced, and inspiration is not on
the menu. The parameters of acceptable thought are
fenced in, as among the twitterati, safe-space freaks,
and others who would return to Plato’s idola specus. The ideological component has transformed tradi-
tional science, by treating it as a manufacturing pro-
cess with an end product, by rewarding raised pro-
ductivity, and by harnessing the entire enterprise to
its narrow mercantile interests. Freed from the dic-
tates of tradition, new science is forced into more
rigid institutional molds, and is explicitly goal-orien-
tated. OPENNESS Ernst Gombrich (1979) wrote of 4 more idols in the
context of the humanities, but applicable to some
patterns of practice in science. His idola quantitatis
expresses the belief that numeration is intrinsically
praiseworthy, that a mathematical treatment of a
theme is the ultimate in reductionism, or at least that
it adds an air of necessary rigour. This is methodolog-
ical naiveté. Intelligence quotients (Gould 1981)
and impact factors are examples of the disease (Ra -
manathan 2002), as are the one-dimensional stan-
dards denounced by post-normal science. Idola novi-
tatis refers to a cult of feigned originality of research,
and idola temporis to the cult of the new as the focal
point of interest. For example, ‘it appears that the Wyatt: Maladies of enlightenment science 59 The Red Queen hypothesis of co-evolutionary theory
(Van Valen 1973, Hamilton 1995) models the arms
race needed to maintain demographic stability be -
tween predator and prey or host and parasite. It
might be used to model the conflicts between mer-
chants and consumers, or between computer security
systems and hackers, and would predict constant
efforts by both sides to gain advantage. But typically,
successful parasites are eventually tamed by their
hosts and lose virulence. An analysis of trends in the
ways scientific results are communicated as well as
initiatives such as the San Francisco Declaration are
signs that the level of virulence is being reduced
(Moskovkin & Serkina 2016). innovation rate has been skyrocketing. Or claims at
innovation, at any rate. In the years between 1974
and 2014, the frequency of the words ‘innovative’,
‘groundbreaking’ and ‘novel’ in PubMed abstracts
increased by 2500% or more’ (Smaldino & McElreath
2016, p. 3). These are some of the words used to con-
struct the assertive titles mentioned below. The pro-
tective and possessive instincts, the myopia of disci-
plinary boundaries and their jealous control, belong
in the shrine of idola academica; the free flow of
information and ideas is congealed or frozen by the
devotees. DISCUSSION Many scientists assure
themselves these indices mirror their own achieve-
ments without realizing they are being persuaded to
behave correctly in conformist communities. These
ersatz scientists compete to be promoted and to win
some paltry rewards from power. The corollaries are
dismissal of the ethical norms of science, and collab-
oration in corroding the mental space once available
to an abandoned calling. Gaming these new norms
brings success to the few. Feyerabend (1988) was
persuaded that ‘anything goes’, but even he never
imagined how far anything could go. At the micro-level, this new science offers many
opportunities for gaming the system. Smoke and mir-
rors replace transparency. Statistical analyses are
used to obscure understanding rather than explore
possibilities, or in attempts to provide a professional
gloss. Subtly woven post hoc fallacies creep into
funding proposals, wittingly or otherwise, to per-
suade the unwary. The new values of this new sci-
ence are reinforced by social conformity. Lapses in
the design of protocols provide opportunities for cor-
ruption — and for the writing of more and more
baroque regulations to counteract it. One end result is a ranking or hierarchy of scien-
tists, winners and losers. Any solidarity the collective
might have had is being crushed, like that of labour
unions. It is a curious paradox that individualism
among scientists, supposedly a virtue, is being elimi-
nated by bureaucratic planning — a shadow of what
Friedrich Hayek, one of the founders of neoliberal
economic philosophy, bemoaned. This new paradigm
may be making some kinds of scientific activity more
efficient or productive, whatever those words might
mean in a creative enterprise, but it is simultaneously
changing the character of science. Pringle (2013,
p. 3281) refers to this ‘fouling of the nest’, to ‘point-
less and demeaning squabbles about priority and
authorship’ and to ‘behavior driven by lust for power,
money, and fame rather than by any desire to under-
stand nature and (perhaps) improve human well-
being in the process’. Avant-garde research is hob-
bled and objectivity is weakened (Fanelli 2012). Has
knowledge production been improved? There is no
control. g
g
g
Ethical obligations are removed and replaced by
bureaucratic fetters: ‘When people learn no tools of
judgement and merely follow their hopes, the seeds
of political manipulation are sown.’ (Gould 1987,
p. 245). DISCUSSION What we can now witness in some directions is
not the modern view of science which emerged with
Polanyi’s Personal Knowledge (Polanyi 1958) and
Kuhn’s The Structure of Scientific Revolutions (Kuhn
1962), and with the debate between Lakatos & Feyer-
abend (1999), but some eviscerated forms of tradi-
tional science. In this system, space for the motives and aspira-
tions of scientists is reduced, and inspiration is not on
the menu. The parameters of acceptable thought are
fenced in, as among the twitterati, safe-space freaks,
and others who would return to Plato’s idola specus. The currently fashionable satanic neoliberalism is
busily dismantling the Keynesian welfare state and
replacing it, impervious to social and environmental
losses. Freedom is redefined as economic freedom. Under the influence of pure market values, the for-
mer openness of science has mutated with the intro-
duction of new concepts of intellectual property, and
as resources formerly shared freely are privatized,
the production and dissemination of science is
retarded. Mirowski (2011) claims that these trends
have qualitatively degraded the resulting scientific
knowledge. Ongoing challenges to genetic engineer-
ing and gene patents, to attempts to own traditional
medicinal plants, and other conflicts between privati-
zation and the commons are testing neoliberal
extremism. As Huxley (1932) wrote in Brave New
World, ‘Primroses and landscapes … have one grave
defect: they are gratuitous’! ‘Science is dangerous; we have to keep it most
carefully chained and muzzled’ wrote Huxley (1932). At one time, Soviet ideologists looked unfavorably on
Pauling’s chemical bonds and Heisenberg’s uncer- It is questionable whether secrecy and privacy can
really be maintained long in our electronic universe. Ethics Sci Environ Polit 17: 51–62, 2017 60 burglar to distract the watchdog of the mind’
(McLuhan 1962). tainty principle, which it was argued were in conflict
with Marxism. Are present day ideologists and their
ways of managing research also eliminating the
mental space within which ideas of equivalent
importance might be conceived? The danger Huxley
sensed is partially contained. Many of its practition-
ers have been diverted; they squabble with each
other for a share of the tribute, and compete for fatu-
ous symbols of prestige like citation indices, fetishes
which elicit the drooling responses of Pavlov’s dogs. The algorithms which calculate pecking order recall
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Genome composition and GC content influence loci distribution in reduced representation genomic studies
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Abstract Background Genomic architecture is a key evolutionary trait for living organisms. Due to multiple complex adaptive
and neutral forces which impose evolutionary pressures on genomes, there is a huge variability of genomic features. However, their variability and the extent to which genomic content determines the distribution of recovered loci in
reduced representation sequencing studies is largely unexplored. Results Here, by using 80 genome assemblies, we observed that whereas plants primarily increase their genome
size by expanding their intergenic regions, animals expand both intergenic and intronic regions, although the
expansion patterns differ between deuterostomes and protostomes. Loci mapping in introns, exons, and intergenic
categories obtained by in silico digestion using 2b-enzymes are positively correlated with the percentage of these
regions in the corresponding genomes, suggesting that loci distribution mostly mirrors genomic architecture of the
selected taxon. However, exonic regions showed a significant enrichment of loci in all groups regardless of the used
enzyme. Moreover, when using selective adaptors to obtain a secondarily reduced loci dataset, the percentage and
distribution of retained loci also varied. Adaptors with G/C terminals recovered a lower percentage of selected loci,
with a further enrichment of exonic regions, while adaptors with A/T terminals retained a higher percentage of loci
and slightly selected more intronic regions than expected. Conclusions Our results highlight how genome composition, genome GC content, RAD enzyme choice and use of
base-selective adaptors influence reduced genome representation techniques. This is important to acknowledge in
population and conservation genomic studies, as it determines the abundance and distribution of loci. Keywords Protostomes, Deuterostomes, Plants, Genome categories, Restriction enzyme, Secondary selection †Xavier Turon, Carlos Carreras, and Marta Pascual jointly supervised
this work. *Correspondence:
Carles Galià-Camps
cgaliacamps@gmail.com 1Departament de Genètica, Microbiologia i Estadística, Universitat de
Barcelona, Avinguda Diagonal 643, Barcelona 08028, Spain
2Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona
(UB), Barcelona, Spain 3Department of Marine Ecology, Centre d’Estudis Avançats de Blanes
(CEAB-CSIC), Accés Cala Sant Francesc 14, Blanes 17300, Spain Carles Galià-Camps1,2,3*, Cinta Pegueroles1,2, Xavier Turon3†, Carlos Carreras1,2† and Marta Pascual1,2† arles Galià-Camps1,2,3*, Cinta Pegueroles1,2, Xavier Turon3†, Carlos Carreras1,2† and Marta Pascual1,2† BMC Genomics BMC Genomics Galià-Camps et al. BMC Genomics (2024) 25:410
https://doi.org/10.1186/s12864-024-10312-3 Open Access Backgroundh a few random nucleotides [24]. All generated fragments
can be sequenced following standard protocols with the
ligation of fully degenerated adaptors to the sticky ends
for library building. Fully degenerated 2b-RAD adaptors
are characterized by two terminal “N”s, which allow bind
ing to all fragments resulting from digestion. Nonethe
less, this enzyme family allows the use of base-selective
adaptors, which select fragments with specific nucleo
tides in their sticky ends [8, 9, 23]. This unique feature of
2b-RAD technique provides the option to perform a sec
ondary reduction of the number of loci, allowing to work
with species with large genomes at a reduced cost, and
therefore including many more individuals given a locked
budget. Compared to the first reduction, produced by
the enzymes’ recognition sites, secondary reduction via
base selection could be directed to specific regions rich
in nucleotides fixed in the base selective adaptors. How
ever, this assumption has not been formally tested, and
the number and distribution of loci retained after base
selection needs to be evaluated. g
The availability of genomes is blooming. In the last five
years, methodological advances in the sequencing of
long fragments have enhanced exponentially the quan
tity and quality of genomic resources, and several initia
tives from global to regional scope have arisen aiming to
produce genomes of all biodiversity [1, 2]. Nonetheless,
when working with species with big genome sizes or
without reference genomes, most genomic studies rely on
reduced genome sequencing techniques using restriction
site digestion enzymes (RAD) to obtain genome-wide
markers of targeted species [3–6]. In population genom
ics, RAD methods allow working with many individuals
without compromising SNP calling accuracy, since high
sequencing depth is required for reliable genotyping [7,
8]. The distribution of the sites digested by restriction
enzymes is expected to be random, and the resultant
fragments are assumed to mirror the genomic structure
of the original genome [7, 9]. Consequently, the percent
age of loci in a genomic category should be representative
of the percentage of the genome in that same category. This would imply a uniform distribution of recognition
sites, in which the nucleotide content of the genome nor
the nucleotide content of the enzyme’s target have an
effect on the distribution of digested fragments. © The Author(s) 2024, corrected publication 2024. 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. Page 2 of 12 Galià-Camps et al. BMC Genomics (2024) 25:410 Galià-Camps et al. BMC Genomics (2024) 25:410 (2024) 25:410 Galià-Camps et al. BMC Genomics Backgroundh How
ever, whether this assumption holds true across taxa,
needs to be properly evaluated, as recent simulated and
empirical studies with fish and sea urchins demonstrated
that some genomic regions are enriched according to the
GC content of the enzyme recognition site [10]. Here, we aim to analyze the genomic composition
between taxonomic groups, and to assess its effect on the
number and distribution of loci obtained with reduced
representation sequencing techniques using different
enzymes and base selective adaptors. To do so, we down
loaded 80 publicly available reference genomes from
three major taxonomic groups (plants, protostomes, and
deuterostomes) and evaluated the relative abundance
of three genomic categories (intergenic, intronic, and
exonic). We simulated the digestion of three 2b-enzymes
on the downloaded genomes to test the number and
distribution of the loci potentially being recovered in
population genomic studies, according to genome size
and taxonomic group. Finally, we simulated the effect of
two types of base selective adaptors, with different GC
content, to determine the number and distribution of
loci obtained in the three genomic categories with this
secondary reduction. Our results show how genome
architecture influences reduced genome representation
techniques in terms of number and distribution of loci,
which must be acknowledged in population and conser
vation genomics. In this context, genome availability provides an unprec
edented opportunity to dig deep into the genome com
position of living organisms [11, 12]. Recent studies
have shed light on plant genome sizes [13], abundance
of repeated elements in mammals and arthropods [14,
15], and the existence of duplicated regions in insects
and hexapods [16, 17]. Also, the genome GC content has
been explored in detail [18, 19], and the percentage of
intergenic and genic regions has been evaluated in both
plants and animals [20, 21]. Genome evolution processes
are complex and involve many heterogeneous mecha
nisms among taxa. All these processes might impact
population genomic studies, as a key element when
assessing genomic structural variants and during SNP
calling [22]. However, genomes across different taxo
nomic groups should be analyzed using the same meth
odology for a correct inference of the features that might
be relevant for population genomic studies using RAD
seq techniques. Materials and methods
Reference genome datasets We downloaded and analyzed 80 reference genome
assemblies publicly available from GeneBank (Fig. 1). They ranged from 102 Mb to 4.7Gb, and comprised both
plants and animals (Data S1). Information on the number
of contigs, L50, N50, and GC content for each genome
(Data S1) was obtained with ABySS-2.4.4 [25]. A bench
marking of universal single copy orthologs (BUSCO) was
conducted for all genome assemblies using the software
BUSCO-5.3.2 and the dataset eukaryota_odb10 [26] to Among RAD techniques, 2b-RAD allows working with
degraded DNA [23]. This is because it uses 2b-enzymes
that identify a recognition site of 6–8 bp and cleave DNA
upstream and downstream at a given length, generating
small fragments of 32–34 bp with sticky ends including Page 3 of 12 Galià-Camps et al. BMC Genomics Fig. 1 Schematic flowchart followed in the present study. Red boxes identify initial datasets obtained from public databases, blue boxes identify interme
diate datasets, green boxes identify final datasets, and gray boxes identify steps where a software was applied. Note that for “Total loci”, in light blue, only
GLMM and Post-Hoc were carried out. Single headed arrows indicate the flow of the processes, while double headed arrows indicate that correlations
were carried out between two datasets Fig. 1 Schematic flowchart followed in the present study. Red boxes identify initial datasets obtained from public databases, blue boxes identify interme
diate datasets, green boxes identify final datasets, and gray boxes identify steps where a software was applied. Note that for “Total loci”, in light blue, only
GLMM and Post-Hoc were carried out. Single headed arrows indicate the flow of the processes, while double headed arrows indicate that correlations
were carried out between two datasets Fig. 2 Phylogenetic representation of the 80 genomes used and their genomic composition. (a) Phylogenetic tree, in which monophyletic groups with
six or more species are indicated by colored branches and identified with a shape (Plants = green, Arthropods = orange, Fishes = blue, Amphibians = pink,
Mammals = brown and Birds = violet). The species names are highlighted with background color according to the three Supergroups considered
(Plants = green, Protostomes = orange, Deuterostomes = purple). Species names in bold indicate genomes with annotation information. Pie diagrams
indicate the GC content of each species (GC = Light gray, AT = Dark gray), and bars their genome sizes. Graphics and statistical analyses Dispersion plots and violin plots were drawn with the
R package “ggplot2” [31] Regression formulas and their
R2 and p-values were calculated using the “stats” pack
age from R (Fig. 1). General Linear Mixed-Effects Mod
els (GLMMs) were estimated with the R package “lme4”
[32], and “car” [33] was used to assess statistically signifi
cant effects of the explanatory factors (Fig. 1). Normaliza
tion of the data was achieved through an arcsine-square
root transformation for factors with frequency values
(i.e.: percentage of unique loci, percentage of genome in
each genomic category and percentage of unique loci in
each genomic category). Normalization of the data was
achieved through a logarithmic transformation for fac
tors with count values (i.e.: number of total loci, number
of unique loci, and genome size). The package “rsq” [34]
was used to check the coefficient of determination (R2)
for the whole model and for the fixed factors included. For statistically significant effects, multiple-level post-hoc
comparisons were carried out with the function contrast
of the R package “emmeans” [35] applying a Tukey-
adjustment of the p-value to avoid Type I errors (Fig. 1),
and plots were generated with the function emmip from
the same package. Materials and methods
Reference genome datasets (b) Percentage of intergenic, intronic and exonic
regions related to genome size for the 44 species with annotated genomes grouped by supergroup (Data S1) Fig. 2 Phylogenetic representation of the 80 genomes used and their genomic composition. (a) Phylogenetic tree, in which monophyletic groups with
six or more species are indicated by colored branches and identified with a shape (Plants = green, Arthropods = orange, Fishes = blue, Amphibians = pink,
Mammals = brown and Birds = violet). The species names are highlighted with background color according to the three Supergroups considered
(Plants = green, Protostomes = orange, Deuterostomes = purple). Species names in bold indicate genomes with annotation information. Pie diagrams
indicate the GC content of each species (GC = Light gray, AT = Dark gray), and bars their genome sizes. (b) Percentage of intergenic, intronic and exonic
regions related to genome size for the 44 species with annotated genomes grouped by supergroup (Data S1) ensure that the reference genomes are of decent qual
ity. The percentage of complete BUSCO values ranged
from 89.8 to 100% with a mean value of 97.11 ± 2.67 (Data
S1). We obtained a phylogenetic tree of the selected
taxa using Timetree web server (http://www.timetree. org/) [27]. We defined 3 different supergroup clusters,
composed of 13 plants, 18 protostomes, and 49 deu
terostomes (Data S1) (Fig. 2a). Additionally, a total of
12 different groups were defined: plants (13), molluscs
(5), nematodes (1), arthropods (12), echinoderms (1), Galià-Camps et al. BMC Genomics (2024) 25:410 (2024) 25:410 Page 4 of 12 Galià-Camps et al. BMC Genomics the selection with S- and W-adaptors compared with the
initial number of unique loci for each of the 80 species
and each of the three enzymes. tunicates (1), fishes (14), amphibians (6), mammals (12),
lepidosaurs (3), testudines (2) and birds (10). The groups
with more than six species were further evaluated sepa
rately (Fig. 2a). We retrieved the annotation files of the
same genomes in GFF format, obtaining the annotation
for 44 genomes (Fig. 1): 10 plants, 2 mollusks, 1 nema
tode, 9 arthropods, 1 tunicate, 7 fishes, 2 amphibians, 5
mammals, 1 lepidosaur, 2 testudines, and 4 birds (Fig. 2a,
Data S1). tunicates (1), fishes (14), amphibians (6), mammals (12),
lepidosaurs (3), testudines (2) and birds (10). The groups
with more than six species were further evaluated sepa
rately (Fig. 2a). Materials and methods
Reference genome datasets We retrieved the annotation files of the
same genomes in GFF format, obtaining the annotation
for 44 genomes (Fig. 1): 10 plants, 2 mollusks, 1 nema
tode, 9 arthropods, 1 tunicate, 7 fishes, 2 amphibians, 5
mammals, 1 lepidosaur, 2 testudines, and 4 birds (Fig. 2a,
Data S1). Genomic composition and functional categories For every annotated genome assembly, we calculated the
number of base pairs in exons, introns and intergenic
regions, herein designated as genomic categories, using
the genomecov function with -d -split options from BED
Tools (Fig. 1, Data S1) [28]. To do so, we first converted
the GFF files to bed12 format using the gff3_file_to_bed. pl utility from Transdecoder (Haas, BJ.https://github. com/TransDecoder/TransDecoder). In silico digestions simulating the use of base-selective
adaptors for secondary reductionf We simulated the effect of using base-selective adap
tors with the bash script select_bases_fasta_2.0.sh [23]
(Fig. 1). We performed in silico base selections of the
unique loci ending with G or C (S) sticky ends (termi
nal nucleotides are G-G, G-C, C-C, and C-G) and end
ing with A or T (W) sticky ends (terminal nucleotides are
A-A, A-T, T-T, and T-A) in order to determine the per
centage of unique loci that would be retained with these
secondary selections, since they can be easily imple
mented in empirical and in silico studies [8, 23]. Finally,
we calculated the percentage of retained sequences after Categorical profiling of 2b-RAD loci To calculate which proportion of unique loci corre
spond to intergenic, intronic, and exonic categories, we
first selected the sequences of the unique loci result
ing from the in silico digestion with the three enzymes
for the 44 annotated genomes. To identify the loca
tion of unique loci in their corresponding reference
genome we used BLAST [30] (same size, 100% of iden
tity, e-value = 1^10− 16) (Fig. 1). Afterward, we used the
in-house script classifyBlastOut.py pipeline to classify
unique hits as exonic, intronic, or intergenic (https://
github.com/EvolutionaryGenetics-UB-CEAB/classi
fyBlastOut/)(Fig. 1). The blast hits that included both
exonic and intronic regions were classified as exonic. Finally, we estimated the percentage of unique loci cor
responding to each genomic category in S-selected and
W-selected datasets for each annotated genome and
enzyme. Genomic in silico digestions We computationally digested the 80 genomes with
2b-enzymes using the program Phyper.pl [29] (Fig. 1). This program recognizes 2b-enzyme targets, cleaves
the DNA, and exports the obtained fragments. We car
ried out the analyses with three 2b-enzymes with differ
ent GC content in their recognition sites: AlfI ([10/12]
GCA[N6]TGC[12/10],
66%
GC),
CspCI
([11/13]
CAA[N5]GTGG[12/10], 57% GC) and BaeI ([10/15]
AC[N4]GTAYC[12/7], 50% GC). For every in silico diges
tion, we obtained two fasta files corresponding to the
total (all digested fragments) and unique (fragments that
were present only once in the genome) sequences [29]
(Fig. 1). Unique loci are of prime interest in population
genomic analysis, since loci found in multiple locations
are routinely removed in the filtering steps. To reduce the
effect of genome size, as larger genomes are expected to
provide more fragments than smaller genomes, we cal
culated the percentage of unique loci to standardize the
data for comparisons. Evolutionary trends on genome compositionh Protostomes
increased genome size by proportionally increasing inter
genic and intronic regions, which are found at high per
centages in their genomes. Deuterostomes also showed
a high percentage of intergenic and intronic regions, but
when increasing genome size only intergenic regions
increased significantly (Table S1). In accordance with
these observations, General Linear Mixed-Effects Models
(GLMM) using the percentage of each genomic category
as the dependent variable detected significant differ
ences in the interactions considering the three factors:
Genomic category, Supergroup, and Genome size (Table
S2). For the significant interaction between Genomic cat
egory and Supergroup, no differences in exonic regions were observed by the post-hoc tests between taxonomic
groups. However, the percentages of intronic and inter
genic regions were significantly different between plants
and animals (Table S3). Evolutionary trends on genome compositionh y
g
p
The compilation of the 80 genomes (Fig. 2a, Data S1)
highlighted different trends among supergroups (plants,
protostomes, and deuterostomes) regarding how the
three considered genomic categories (intergenic, intronic, Galià-Camps et al. BMC Genomics (2024) 25:410 (2024) 25:410 Galià-Camps et al. BMC Genomics Page 5 of 12 and exonic) change in relation to genome size (Fig. 2b). In all three taxonomic supergroups, species with small
genome sizes had a higher percentage of exonic regions,
as shown by the significant negative slopes of their
regression equations and their high coefficient of deter
mination (Fig. 2b, Table S1). The percentage of inter
genic regions in plants increased with genome size, while
both intronic and exonic regions were found at low per
centages and with significant negative regressions with
genome size (Fig. 2b, Table S1). On the other hand, ani
mal genomes increased in size by expanding both inter
genic and intronic regions (Fig. 2b). However, the two
animal supergroups differed in the increment pattern of
these categories with genome size (Fig. 2b). Protostomes
increased genome size by proportionally increasing inter
genic and intronic regions, which are found at high per
centages in their genomes. Deuterostomes also showed
a high percentage of intergenic and intronic regions, but
when increasing genome size only intergenic regions
increased significantly (Table S1). In accordance with
these observations, General Linear Mixed-Effects Models
(GLMM) using the percentage of each genomic category
as the dependent variable detected significant differ
ences in the interactions considering the three factors:
Genomic category, Supergroup, and Genome size (Table
S2). For the significant interaction between Genomic cat
egory and Supergroup, no differences in exonic regions and exonic) change in relation to genome size (Fig. 2b). In all three taxonomic supergroups, species with small
genome sizes had a higher percentage of exonic regions,
as shown by the significant negative slopes of their
regression equations and their high coefficient of deter
mination (Fig. 2b, Table S1). The percentage of inter
genic regions in plants increased with genome size, while
both intronic and exonic regions were found at low per
centages and with significant negative regressions with
genome size (Fig. 2b, Table S1). On the other hand, ani
mal genomes increased in size by expanding both inter
genic and intronic regions (Fig. 2b). However, the two
animal supergroups differed in the increment pattern of
these categories with genome size (Fig. 2b). In silico genome digestions using 2b-RAD enzymes Our results from the in silico digestion of the 80 genomes
with the three 2b-enzymes, showed that the number of
total loci (all fragments) and unique loci (those fragments
whose sequence was present only once in the genome)
were highly correlated (Figure S1). The number of total
and unique loci significantly increased with genome size
in all enzymes, regardless of taxonomic level, presenting
high coefficients of determination (R2) for most of the
regressions (Fig. 3, Table S4). Mammals were the excep
tion to the global trends (Table S4), since two genomes in
this group (platypus and red deer, the smallest and largest
genomes analyzed, respectively) had low numbers of loci
(Data S1). In the three GLMM models tested (Table S5), consid
ering all species together (Total model), split by super
group (Supergroup model) or by group considering those
with 6 or more species (Group model), the proportion
of the variance explained by the fixed factors Enzyme,
Genome size, and Supergroup or Group (depending on
the model), was high. For the Total model, significant
differences were found among enzymes (AlfI, CspCI, Fig. 3 Linear regressions of the loci yielded by each enzyme (AlfI, CspCI, BaeI) according to each species’ genome size for total and unique loci. (a-b)
Linear regressions considering all 80 genomes. (c-d) Linear regressions for each supergroup independently (plants, protostomes and deuterostomes). (e-f) Independent linear regressions for those groups with six or more species (plants, arthropods, fishes, amphibians, mammals and birds). Regression
equations can be found in Table S4 Fig. 3 Linear regressions of the loci yielded by each enzyme (AlfI, CspCI, BaeI) according to each species’ genome size for total and unique loci. (a-b)
Linear regressions considering all 80 genomes. (c-d) Linear regressions for each supergroup independently (plants, protostomes and deuterostomes). (e-f) Independent linear regressions for those groups with six or more species (plants, arthropods, fishes, amphibians, mammals and birds). Regression
equations can be found in Table S4 Page 6 of 12 Galià-Camps et al. BMC Genomics (2024) 25:410 Galià-Camps et al. BMC Genomics (2024) 25:410 Galià-Camps et al. BMC Genomics (2024) 25:410 BaeI), with genome sizes, and their interaction (Table
S5). Differences were due to the higher number of loci
obtained with AlfI, being this number intermediate for
CspCI and smallest for BaeI, and to the increase of the
number of loci with genome size, with different slopes
for each enzyme (Fig. 3, Table S4). In silico genome digestions using 2b-RAD enzymes For the Supergroup
model, the fixed factors explained 92% of the vari
ance for total loci and 90% for unique loci. Supergroup,
enzyme and genome size had significant effects, as well
as the interaction enzyme*supergroup in both total and
unique loci (Table S5). Tukey’s post-hoc pairwise com
parisons indicated major significant differences between
deuterostomes and the other two taxa for all enzymes
but BaeI (Table S6). Similar results were obtained when
considering the group model, with 93% of the variance
explained by the fixed factors (Table S5). As in the Super
group model, no differences between taxonomic groups
were found when using BaeI (Table S7). However, AlfI
presented significant differences in total and unique loci
when comparing plants and arthropods against the other
groups as assessed with Tukey’s post-hoc tests (Table S7). For CspCI, significant differences were only found when
comparing arthropods’ unique loci with other groups. with genome size when considering all species together
(Fig. 3a, Table S8). However, these percentages were
dependent on the taxa analyzed, since the variance of
the full model explained by fixed factors increased when
the species were combined in lower-level phylogenetic
groups, suggesting lineage-specific variation (Table S8). The Supergroup model showed significantly higher per
centages of unique loci on deuterostomes than on plants
and protostomes (Fig. 4a, Table S9). Finally, the Group
model showed different behaviors depending on the
groups, since mammals (0.958 ± 0.036, mean ± SE) and
birds (0.972 ± 0.027) presented a higher percentage of
unique loci (Fig. 4a). However, this effect was only signifi
cant in mammals when compared to plants, arthropods,
or fishes (Table S10). Birds did not show significantly dif
ferent values despite their high percentage of unique loci
and low dispersion values (Fig. 4a). The model in birds
presented a large 95% confidence interval on the per
centage of unique loci, which overlapped with all other
groups (Figure S2). 2b-RAD digestions slightly enrich exonic loci We BLASTed unique loci to their reference annotated
genomes and identified to which genomic category they
belonged (Data S2). For all enzymes and species being
tested, all loci BLASTed randomly over the entire ref
erence genomes indicating that loci were not aggre
gated, thus mirroring reference genomes without biases The percentage of unique loci varies across taxa Overall, the percentage of variation explained by all
regression equations was very good, as indicated by the
coefficients of determination of the full models (R2, Table
S11). Loci mapping in exonic regions were more frequent
than expected, since the values fell above the percent
age of unique loci in a given genomic category expected
under the null hypothesis of random distribution of loci
(dotted line y = 0 + 1x, Fig. 4b). On the contrary, the per
centage of loci in intergenic regions were inferior to the
expected ones as they always fell below the dotted line
(Fig. 4b). Finally, the intronic regions had a percentage of
unique loci roughly proportional to the percentage of the
corresponding category (close to the dotted line, Fig. 4b).h proportion of unique loci in the exonic category than ani
mals, and protostomes had a significantly higher propor
tion of unique loci in the intronic category than plants. All genomic categories had significantly different propor
tions of loci in each supergroup except the comparisons
between intergenic and intronic regions for plants and
deuterostomes (Table S13, Figure S3). The percentage of unique loci varies across taxa Loci mapping in exonic regions were more frequent
than expected, since the values fell above the percent
age of unique loci in a given genomic category expected
under the null hypothesis of random distribution of loci
(dotted line y = 0 + 1x, Fig. 4b). On the contrary, the per
centage of loci in intergenic regions were inferior to the
expected ones as they always fell below the dotted line
(Fig. 4b). Finally, the intronic regions had a percentage of
unique loci roughly proportional to the percentage of the
corresponding category (close to the dotted line, Fig. 4b). The GLMM using as the dependent variable the ratio
between the percentage of unique loci in a genomic
category and the percentage of the same category in
the genome, identified significant differences among
enzymes, supergroups, genomic categories, and their
pairwise interactions (Table S12). For the pairwise
interactions, there were significant differences between
genomic categories for all enzymes with the exception
of the comparison between intergenic and intronic cat
egories for AlfI and BaeI (Table S13). The percentage of
unique loci in intergenic regions did not differ between
supergroups. However, plants had a significantly higher towards any specific region. For the three supergroups,
the percentage of unique loci in a given category sig
nificantly increased with the percentage of the same
genomic category in the genome (Fig. 4b, Table S11, Data
S2). Overall, the percentage of variation explained by all
regression equations was very good, as indicated by the
coefficients of determination of the full models (R2, Table
S11). Loci mapping in exonic regions were more frequent
than expected, since the values fell above the percent
age of unique loci in a given genomic category expected
under the null hypothesis of random distribution of loci
(dotted line y = 0 + 1x, Fig. 4b). On the contrary, the per
centage of loci in intergenic regions were inferior to the
expected ones as they always fell below the dotted line
(Fig. 4b). Finally, the intronic regions had a percentage of
unique loci roughly proportional to the percentage of the
corresponding category (close to the dotted line, Fig. 4b).h towards any specific region. For the three supergroups,
the percentage of unique loci in a given category sig
nificantly increased with the percentage of the same
genomic category in the genome (Fig. 4b, Table S11, Data
S2). The percentage of unique loci varies across taxa Our GLMM showed that the percentage of recov
ered unique loci relative to the number of total loci
was not different across enzymes and did not change Fig. 4 Percentage of unique loci and genomic category distribution across taxa. (a) Violin plots of the percentage of unique loci, in relation to the total
number of loci, obtained after in silico genome digestion with 2b-enzymes. (b) Percentage of unique loci assigned to each genomic category (intergenic,
intronic and exonic) compared to the percentage of the same genomic category in the genome. Dotted lines indicate the percentage of loci in a ge
nomic category expected under the null hypothesis of random distribution of loci Fig. 4 Percentage of unique loci and genomic category distribution across taxa. (a) Violin plots of the percentage of unique loci, in relation to the total
number of loci, obtained after in silico genome digestion with 2b-enzymes. (b) Percentage of unique loci assigned to each genomic category (intergenic,
intronic and exonic) compared to the percentage of the same genomic category in the genome. Dotted lines indicate the percentage of loci in a ge
nomic category expected under the null hypothesis of random distribution of loci Fig. 4 Percentage of unique loci and genomic category distribution across taxa. (a) Violin plots of the percentage of unique loci, in relation to the total
number of loci, obtained after in silico genome digestion with 2b-enzymes. (b) Percentage of unique loci assigned to each genomic category (intergenic,
intronic and exonic) compared to the percentage of the same genomic category in the genome. Dotted lines indicate the percentage of loci in a ge
nomic category expected under the null hypothesis of random distribution of loci Page 7 of 12 Galià-Camps et al. BMC Genomics (2024) 25:410 (2024) 25:410 Galià-Camps et al. BMC Genomics (2024) 25:410 towards any specific region. For the three supergroups,
the percentage of unique loci in a given category sig
nificantly increased with the percentage of the same
genomic category in the genome (Fig. 4b, Table S11, Data
S2). Overall, the percentage of variation explained by all
regression equations was very good, as indicated by the
coefficients of determination of the full models (R2, Table
S11). Base selection enhances differential recovery of genomic
categories After base-selection, we BLASTed the selected unique
loci to their reference annotated genomes and identified
to which genomic category they belonged (Data S2). As
found for unique loci, all selected loci were randomly
distributed across their respective reference genome. The percentage of unique loci in a genomic category was
again highly correlated with the percentage of the same
category in the genome (Table S19). As found without
base selection, the percentage of unique loci was above
the expectation (dotted line, Fig. 6) for exons, below for
the intergenic regions, and the closest to the expecta
tion for introns. However, base selection also had an
effect on top of this general pattern, as the regression
lines obtained for all enzymes using the S-selection were
always above those obtained with the W-selection in
exons. This pattern was reversed for the intronic regions,
in which W-selection regression lines were above those
of the S-selection ones. The number of retained loci could be highly influ
enced by the nucleotide content of the genomes, gener
ally richer in AT than in GC (Data S1). We observed that
the percentage of unique loci retained with S-selection
within each supergroup was positively correlated with
the species genome GC content, while the percentage
of loci retained with W-selection was negatively corre
lated with the species GC content (Table S18, Fig. 5b). Thus, more loci were retained with S-selection when
the genome GC content was higher, and the reverse
occurred with W-selection (Fig. 5b). Overall, considering
the species GC content, which is on average 39.93 ± 3.9%
(Data S1), the expected mean number of loci retained
with the S-selection for the 80 analyzed species would
be 15.94% (% of GC2/100). Similarly, the W-selection
expected number of loci given an average AT content
of 60.07 ± 3.9% would be 36.08% after applying the same
equation (% of AT2/100). The observed values of percent
ages of loci retained with W-selection (32.7 ± 0.029%) and
S-selection (19.2 ± 0.025%) in the simulations with the 80 The General Linear Mixed-Effects Models (GLMM)
showed that all pairwise interactions were significant
except for the enzyme*selection interaction (Table S20). The triple interaction involving enzyme, supergroup and
genomic category was also significant (Table S20). All the Fig. 6 Percentage of base-selective unique loci assigned to each genomic category compared to the percentage of the same genomic category in
the genome. Base selection performance depends on genome GC
content BMC Genomics genomes were quite similar to those expected consider
ing only the genomes’ GC content. Fig. 4a). For the Group model, mammals showed sig
nificantly higher percentage of unique loci retained with
S-selection and lower with W-selection when compared
to all other groups but birds (Fig. 5a, Table S17). For all
groups but birds there was a significantly higher percent
age of loci retained with W-selection (Table S17, Figure
S4). Base selection performance depends on genome GC
content Our results simulating the use of base-selective adaptors
on 80 genomes showed a great reduction of the number
of loci highly dependent on whether the selection was
performed for S or W sticky ends, providing ∼ 0.19% and
∼ 0.33% of original unique loci respectively compared to
not using base selection (Fig. 5). The percentage of unique
loci retained with W-selection was significantly higher
than for S-selection (Table S14, Fig. 5a). Additionally,
significant differences were found for some of the inter
actions when simulating adaptor selection for the Total,
Supergroup and Group models (Table S14). For the Total
model, BaeI presented a significantly lower percentage
of retained unique loci for both S and W-selection when
compared to AlfI, and only for S-selection compared to
CspCI (Table S15). For the Supergroup model, a signifi
cant interaction was found between selection and super
group. Deuterostomes retained a significantly higher
percentage of unique loci with S-selection and lower with
W-selection than the other two supergroups (Table S16, The GLMM using as the dependent variable the ratio
between the percentage of unique loci in a genomic
category and the percentage of the same category in
the genome, identified significant differences among
enzymes, supergroups, genomic categories, and their
pairwise interactions (Table S12). For the pairwise
interactions, there were significant differences between
genomic categories for all enzymes with the exception
of the comparison between intergenic and intronic cat
egories for AlfI and BaeI (Table S13). The percentage of
unique loci in intergenic regions did not differ between
supergroups. However, plants had a significantly higher Fig. 5 Percentage of retained unique loci across taxa when using base selective adaptors. (a) Percentage of unique loci retrieved when S (GC) and W
(AT) base-selective adaptors are in silico simulated for each enzyme (AlfI, CspCI, BaeI). (b) Linear regressions of the percentage of selected loci retained
according to the genome’s GC content Fig. 5 Percentage of retained unique loci across taxa when using base selective adaptors. (a) Percentage of unique loci retrieved when S (GC) and W
(AT) base-selective adaptors are in silico simulated for each enzyme (AlfI, CspCI, BaeI). (b) Linear regressions of the percentage of selected loci retained
according to the genome’s GC content Page 8 of 12 Galià-Camps et al. BMC Genomics (2024) 25:410 Galià-Camps et al. BMC Genomics (2024) 25:410 Galià-Camps et al. Base selection enhances differential recovery of genomic
categories Allopolyploidy has been a main evolutionary trigger for
plants, since 87.5–99.5% of plants have been subjected
to hybridization at some point during their evolution
ary history, with a posterior rediploidization [36]. This
process involves many genomic changes, with fast gene
deletion being one of the predominant mechanisms [37]. Retained homeologs in plants enhance protein fam
ily diversity without relying on introns to create differ
ent isoforms through alternative splicing, resulting in a
higher number of genes [36, 38, 39]. Thus, allopolyploidy
may help to maintain the exonic and intronic regions
at low proportion despite increasing genome sizes, as
observed in our study. Furthermore, the high percent
age of intergenic regions in plants compared to animals
is in agreement with plants increasing their genomes by
expansions of transposable elements (TE) that can be
activated by hybridization and polyploidization altering
silencing mechanisms [40, 41]. The complexity of plant
genomes deserves further detailed studies with more
species of differing architectures. post-hoc tests for the selection and genomic category
interaction resulted in significant values with the excep
tion of the comparison between S- and W-selection in
the intergenic regions (Table S21). The selection type
directly affected the percentage of unique loci in a given
genomic category with a significantly higher percentage
of intronic loci in W-selection and a significantly higher
for exonic regions in S-selection (Table S21). Regarding
the interaction between supergroup and selection, only
deuterostomes showed significant differences between
S- and W-selection with a higher proportion of unique
loci recovered by the former (Table S21). When compar
ing the different supergroups, plants presented a higher
proportion of unique loci with W-selection than proto
stomes and deuterostomes (Table S21). The three-way
interaction showed that all supergroups presented sig
nificant differences in the dependent variable between
genomic categories in the same direction regardless of
the used enzyme, with the exception of protostomes,
which did not show significant differences between inter
genic and intronic regions with any of the three enzymes
(Table S22). Plants showed a significantly higher ratio
of unique loci in exons compared to animals with all
three enzymes (Table S22). Protostomes showed signifi
cantly lower values in introns than plants when using the
enzymes AlfI and BaeI. Finally, there were no significant
differences between enzymes with the exception of AlfI
recovering more loci in the exons of plants (Table S22). 2b-RAD sequencing mostly mirrors genome composition
Th
b
f b
h
b
d d
b 2b RAD sequencing mostly mirrors genome composition
The absence of biases in the number and distribution of
loci being retained by reduced genome representations
on population genomic studies is a prerequisite for these
studies that has been assumed without evidence. By using
three different 2b-enzymes in three major eukaryotic lin
eages, we have found that, as expected, the number of
total and unique loci increased overall with genome size
in all enzymes regardless of taxonomic level. The num
ber of unique loci is a key parameter for RADseq studies,
as they are the only ones to pass the filtering processes. Base selection enhances differential recovery of genomic
categories The dotted lines indicate the percentage of loci in a genomic category expected under the null hypothesis of random distribution of loci Fig. 6 Percentage of base-selective unique loci assigned to each genomic category compared to the percentage of the same genomic category in
the genome. The dotted lines indicate the percentage of loci in a genomic category expected under the null hypothesis of random distribution of loci Fig. 6 Percentage of base-selective unique loci assigned to each genomic category compared to the percentage of the same genomic category in
the genome. The dotted lines indicate the percentage of loci in a genomic category expected under the null hypothesis of random distribution of loci Galià-Camps et al. BMC Genomics (2024) 25:410 Galià-Camps et al. BMC Genomics (2024) 25:410 Page 9 of 12 Page 9 of 12 Page 9 of 12 Galià-Camps et al. BMC Genomics post-hoc tests for the selection and genomic category
interaction resulted in significant values with the excep
tion of the comparison between S- and W-selection in
the intergenic regions (Table S21). The selection type
directly affected the percentage of unique loci in a given
genomic category with a significantly higher percentage
of intronic loci in W-selection and a significantly higher
for exonic regions in S-selection (Table S21). Regarding
the interaction between supergroup and selection, only
deuterostomes showed significant differences between
S- and W-selection with a higher proportion of unique
loci recovered by the former (Table S21). When compar
ing the different supergroups, plants presented a higher
proportion of unique loci with W-selection than proto
stomes and deuterostomes (Table S21). The three-way
interaction showed that all supergroups presented sig
nificant differences in the dependent variable between
genomic categories in the same direction regardless of
the used enzyme, with the exception of protostomes,
which did not show significant differences between inter
genic and intronic regions with any of the three enzymes
(Table S22). Plants showed a significantly higher ratio
of unique loci in exons compared to animals with all
three enzymes (Table S22). Protostomes showed signifi
cantly lower values in introns than plants when using the
enzymes AlfI and BaeI. Finally, there were no significant
differences between enzymes with the exception of AlfI
recovering more loci in the exons of plants (Table S22). Base selection enhances differential recovery of genomic
categories f
g
Conversely, we found that intronic regions were highly
abundant in animals across genome sizes. The abun
dance of intronic regions has been proposed as a mech
anism to facilitate alternative splicing, contributing to
the diversification of gene structural variants in ani
mals [42]. Furthermore, we also found that in deutero
stomes the percentage of intergenic regions significantly
increases with genome size. In the origin of vertebrates,
two ancient rounds of whole genome duplications 450
Mya occurred [43]. These duplication events followed
by TE activity might have increased the proportion of
intergenic regions, since the effect of TE could inacti
vate former duplicated genes and thus contribute to the
expansion of intergenic regions at the expense of ancient
genes [44, 45]. Furthermore, regulatory elements modu
lating gene expression, highly abundant in vertebrates,
have been identified in intergenic regions [46, 47]. How
ever, the information on regulatory elements is limited to
a few species due to the lack of comprehensive annota
tions in most organisms, highlighting the need for cor
rect annotation for the increasing number of available
reference genomes. Discussion In agreement with previous studies, our results show that
plants and animals increase genome size by differently
expanding intergenic and intronic genomic categories
respectively [20, 21]. We confirm that the genome size
and composition, in combination with the GC content of
the restriction enzyme target site, determine the number
and distribution of loci in studies based on reduced rep
resentation sequencing techniques. Moreover, we prove
that the number of markers in the different genomic cat
egories with 2b-enzymes positively correlates with the
percentage of each region in the genome, although with
a slight enrichment of loci in exons. Finally, our simula
tions of 2b-RAD secondary reduction techniques show
how each genome category GC content influences the
number and distribution of retained loci as a function of
the base-selective adaptors used. Base-selection unravels additional GC biases Base-selection unravels additional GC biases Base-selection unravels additional GC biases RADseq population studies of species with big genome
sizes will require large sequencing effort for a cor
rect genotyping since the number of loci increases with
genome size. To reduce the number of loci, enzymes with
infrequent target sites or allowing secondary reduction
can be used. 2b-RADseq is the only technique that allows
a secondary reduction by using base-selective adaptors
to further reduce sequencing costs [23]. As expected,
we have shown that base-selective adaptors efficiently
reduce the number of recovered loci and the percentage
of retained unique loci in a genomic category is highly
correlated with the percentage of the same category in
the genome. Nevertheless, the selection performed on
the unique loci and the genome’s GC content influence
the number and genomic category of the loci retained by
each methodology. As 2b-RAD secondary reduction by using base-selec
tive adaptors is performed after the digestion process,
the recovered fragments are not a reduced representation
of the whole genome, but only of the fragments already
digested by the enzyme. Accordingly, AlfI, the enzyme
with the richest GC content in the recognition site recov
ers a significantly higher percentage of S-selected loci
and lower of W-selected loci than BaeI when consider
ing all species, likely because the first genome reduction
already selected a higher proportion of fragments with
higher GC content, which are in turn a better target for
the S-selection. However, differences are also observed
among taxonomic groups in the abundance of retained
loci with adaptor selection. Deuterostomes, and more
specifically mammals, retain a significantly higher per
centage of unique loci with S-selection and lower for
W-selection compared to the other taxonomic catego
ries. Finally, we have found that the higher the GC con
tent of the genome, the higher the percentage of loci
recovered using S-selection adaptors and the lower the
percentage recovered with W-selective adaptors. Thus,
we have shown that the number of the retained loci is
influenced by the type of selection performed, the taxo
nomic category, and the genome’s GC content. l
Moreover, we demonstrated that the markers obtained
with this technique mirror the overall genome category
composition, albeit with a slight enrichment of loci from
the exonic regions and a reduction of those from the
intergenic category. Genomic composition is shaped by multiscale evolutionary
processesf Our results indicate that the genome composition differs
among supergroups, which might determine the loci dis
tribution in reduced representation population genomic
studies. In the case of plants, we have shown that they
expand their intergenic regions with genome size. Page 10 of 12 Page 10 of 12 Page 10 of 12 Galià-Camps et al. BMC Genomics (2024) 25:410 Galià-Camps et al. BMC Genomics (2024) 25:410 Galià-Camps et al. BMC Genomics (2024) 25:410 (2024) 25:410 Galià-Camps et al. BMC Genomics Although their number positively increased with genome
size, not all taxonomic groups behaved similarly when
considering the percentage of unique loci in relation to
the total number of loci in the genome. In this scenario,
plants and protostomes showed lower percentage of
unique loci in comparison to deuterostomes. The abun
dance of recent TE expansions in protostomes (increas
ing the abundance of repetitive regions in the genome),
and recent polyploidy and whole genome duplication in
plants (due to autopolyploidy or hybridization), might
explain the lower proportion of unique loci found in
these two groups [15, 16, 38, 48, 49]. Among deutero
stomes, mammals and birds share many genomic evo
lutionary traits, such as an active expansion of TEs and
large DNA deletions [44, 50] that might determine [15,
16, 38, 48, 49] the higher percentage of unique loci found
in these two groups in the present study. However, birds
did not show significant differences compared to the
other groups despite their high percentage of unique
loci and low dispersion values. Birds are known for hav
ing compact genome sizes (ranging from 0.9 to 1.6Gb),
thought to be driven by flight constraints [44, 50]. Conse
quently, the model needs to generate estimates for a large
range of values resulting in high confidence intervals. Finally, fishes present a high dispersion in the number
of unique loci among species, mostly the result of cypri
nids having extremely low values. Cyprinids suffered two
extra genome duplications which may explain the lower
number of unique loci found in fishes [51, 52]. Overall,
we can confirm that the loci obtained from digestion
with 2b-enzymes generally reflect genome composition. Base-selection unravels additional GC biases Other RADseq population studies
have recently shown that loci in different genomic cate
gories might be enriched depending on the used enzyme,
and suggested an effect of the GC content of the recog
nition site [10]. Our results point in the same direction
since the enzymes used in our study (AlfI, CspCI and
BaeI) have a percentage of GC content over 50% in their
recognition sites. Thus, finding an overrepresentation
of loci from exonic regions is expected given that these
regions are GC enriched across eukaryotes while introns
and intergenic regions overall contain lower GC content
levels [19, 53]. Our results support the idea that the GC
content of the enzymes’ recognition site influences the
genomic categories being recovered in RADseq genomic
studies regardless of taxonomic group and genome size. Thus, the effect of different enzymes preferentially target
ing certain genomic regions, specifically exons, should be
considered in the design of population genomic studies. Moreover, although loci in exons were already enriched
by the initial digestion, the secondary reduction showed
that S-selection further enhances the recovery of loci
from exonic regions compared to W-selection. These
results are in line with the above-mentioned enrich
ment of GC in exonic regions across eukaryotes, render
ing them a better target for S-selective adaptors. On the
other hand, the percentage of loci recovered in introns
and intergenic regions, albeit overall below expecta
tion after the initial digestion, were higher when using
W-selection than S-selection, again reflecting that non-
coding regions have poorer GC content. Thus, the per
centage of GC in a genomic category can be used as a Page 11 of 12 (2024) 25:410 Galià-Camps et al. BMC Genomics (2024) 25:410 Galià-Camps et al. BMC Genomics prior to predict the performance of both S-selective and
W-selective secondary reductions. prior to predict the performance of both S-selective and
W-selective secondary reductions. Supplementary Material 23: Table S21
Supplementary Material 24: Table S22
Supplementary Material 25: Figure S1
Supplementary Material 26: Figure S2
Supplementary Material 27: Figure S3
Supplementary Material 28: Figure S4 Conclusions Our results highlight different modes of evolution of
genomes across broad taxonomic categories. Whereas
plants primarily increase their genome size by expanding
their intergenic regions, animals expand both intergenic
and intronic regions, although the patterns differ between
deuterostomes and protostomes. The results presented in
this work are also highly relevant to the design of popula
tion genomic studies. We have shown that the GC con
tent of the enzymes determines an enrichment of loci
in particular genomic regions. Further, the use of base-
selective instead of fully-degenerated adaptors provides
an efficient means to reduce the number of loci to make
the research more cost-effective, but the type of selection
reinforces bias in certain genomic categories. Overall, we
highlight that the enzyme we used for digestion, type of
secondary selection, species’ genomic composition and
GC content may determine differences in the distribu
tion of the loci in population and conservation genomic
studies. Acknowledgements
CG h
k
h
b CG thanks the members of his family for providing animal pictures for Fig. 1a,
in particular of his brother’s dog Keni, outlined under the vertebrates’ clade. Supplementary Information y
The datasets supporting the conclusions of this article, including genome
assemblies’ accession numbers and all values needed to replicate the study,
are included within the article (Data S1, Data S2). The online version contains supplementary material available at https://doi. org/10.1186/s12864-024-10312-3. Supplementary Material 1: Data S1
Supplementary Material 2: Data S2
Supplementary Material 3: Table S1
Supplementary Material 4: Table S2
Supplementary Material 5: Table S3
Supplementary Material 6: Table S4
Supplementary Material 7: Table S5
Supplementary Material 8: Table S6
Supplementary Material 9: Table S7
Supplementary Material 10: Table S8
Supplementary Material 11: Table S9
Supplementary Material 12: Table S10
Supplementary Material 13: Table S11
Supplementary Material 14: Table S12
Supplementary Material 15: Table S13
Supplementary Material 16: Table S14
Supplementary Material 17: Table S15
Supplementary Material 18: Table S16
Supplementary Material 19: Table S17
Supplementary Material 20: Table S18
Supplementary Material 21: Table S19
Supplementary Material 22: Table S20 Supplementary Material 1: Data S1
Supplementary Material 2: Data S2
Supplementary Material 3: Table S1
Supplementary Material 4: Table S2
Supplementary Material 5: Table S3
Supplementary Material 6: Table S4
Supplementary Material 7: Table S5
Supplementary Material 8: Table S6
Supplementary Material 9: Table S7
Supplementary Material 10: Table S8
Supplementary Material 11: Table S9
Supplementary Material 12: Table S10
Supplementary Material 13: Table S11
Supplementary Material 14: Table S12
Supplementary Material 15: Table S13
Supplementary Material 16: Table S14
Supplementary Material 17: Table S15
Supplementary Material 18: Table S16
Supplementary Material 19: Table S17
Supplementary Material 20: Table S18
Supplementary Material 21: Table S19
Supplementary Material 22: Table S20 Competing interests Competing interests
Authors declare they have no competing interests. Competing interests
Authors declare they have no competing interests. p
g
Authors declare they have no competing interests. Received: 8 January 2024 / Accepted: 15 April 2024 Received: 8 January 2024 / Accepted: 15 April 2024 Funding This research was funded by the Spanish Government with the project
MarGech [PID2020-118550RB, MICIU/AEI/10.13039/501100011033; https://
doi.org/10.13039/501100011033]. CG held a predoctoral contract [PRE-2018-
085227 - MICIU/AEI/10.13039/501100011033] by the Spanish Ministry of
Science, Innovation and Universities, and by ESF “invests in your future”. The
authors CG, CP, CC and MP are members of the research group [2021 SGR
01271], and XT is member of the research group [2021 SGR 00405], both
funded by the Generalitat de Catalunya (AGAUR). Author contributions All authors contributed to the conceptualization of the study, manuscript
drafting, and manuscript revision. CG retrieved genome data, conducted in
silico digestion analyses and graphic design. CG and MP curated the data, and
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Abstract argumentation and the rational man
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Abstract argumentation and the rational
man TIMOTHEUS KAMPIK, Department of Computing Science, Umeå University,
90187 Umeå, Sweden. E-mail: tkampik@cs.umu.se TIMOTHEUS KAMPIK, Department of Computing Science, Umeå University,
90187 Umeå, Sweden. E-mail: tkampik@cs.umu.se JUAN CARLOS NIEVES, Department of Computing Science, Umeå University,
90187 Umeå, Sweden. Abstract Abstract argumentation has emerged as a method for non-monotonic reasoning that has gained popularity in the symbolic
artificial intelligence community. In the literature, the different approaches to abstract argumentation that were refined over
the years are typically evaluated from a formal logics perspective; an analysis that is based on models of economically rational
decision-making does not exist. In this paper, we work towards addressing this issue by analysing abstract argumentation
from the perspective of the rational man paradigm in microeconomic theory. To assess under which conditions abstract
argumentation-based decision-making can be considered economically rational, we derive reference independence as a non-
monotonic inference property from a formal model of economic rationality and create a new argumentation principle that
ensures compliance with this property. We then compare the reference independence principle with other reasoning principles,
in particular with cautious monotony and rational monotony. We show that the argumentation semantics as proposed in
Dung’s seminal paper, as well as other semantics we evaluate, with the exception of naive semantics and the SCC-recursive
CF2 semantics, violate the reference independence principle. Consequently, we investigate how structural properties of
argumentation frameworks impact the reference independence principle and identify cyclic expansions (both even and odd
cycles) as the root of the problem. Finally, we put reference independence into the context of preference-based argumentation
and show that for this argumentation variant, which explicitly models preferences, reference independence cannot be ensured
in a straight-forward manner. Keywords: formal argumentation, economic rationality, non-monotonic reasoning Argumentation corner Argumentation corner 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 reuse, distribution, and reproduction in any medium,
provided the original work is properly cited. 1
Introduction In recent decades, a large body of research emerged on argumentation as a method to instil
intelligence, i.e. reasoning capabilities, into computing systems. In this context, a popular theory
for formal argumentation is so-called abstract argumentation, as initially developed by Dung [22],
which is frequently used as the foundation of research on formal argumentation and hence spawned
a variety of frameworks that extend the initial work [2]. A key aspect of abstract argumentation
research is the definition and evaluation of different argumentation semantics. An argumentation
semantics is a decision function that specifies how an argumentation framework (a graph of
arguments) should be resolved, i.e. which arguments can be considered valid conclusions and which
cannot. A variety of argumentation semantics, as well as a range of advanced frameworks that aim
to augment Dung’s basic definition of an argumentation framework, exist; i.e. the way how an
argumentation framework is to be resolved depends on the application scenario and the intended
meaning of arguments and attacks. Determining the arguments of an argumentation framework that can be considered valid conclu-
sions is a decision process. From the perspective of economic theory, a classical model of decision-
making is the notion of the rational man (see, e.g. Rubinstein [34]), whose decisions imply clear Vol. 31, No. 2, © The Author(s) 2021. Published by Oxford University Press. 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 reuse, distribution, and reproduction in any medium,
provided the original work is properly cited. p
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p
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Advance Access Publication on 4 February 2021
doi:10.1093/logcom/exab003 655 Abstract argumentation and the rational man and consistent preferences. While the rational man as a sufficiently precise formal model of human
decision-making has been debunked by a body of empirical research [27], it can still be considered
relevant as a cornerstone of economic theory, e.g. for prescriptive modelling of an ideal agent that
strictly optimizes according to clear preferences when determining the decision outcome. Aligning
abstract argumentation with the rational man paradigm requires, as this paper shows, the introduction
of a new argumentation principle—an axiomatic requirement to an argumentation semantics [40]—
that existing argumentation semantics typically do not satisfy. 1
Introduction In this paper, we use a novel1 formal
approach to explore the intersection of abstract argumentation as a non-monotonic reasoning method
and (bounded) economic rationality and show that abstract argumentation can lead to economically
not rational decision outcomes. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 Let us provide an example that illustrates the problem this paper focuses on. An agent A1 uses
abstract argumentation for deciding on a subset A∗of a set A = {a, b} (e.g. to determine which
actions in a set Acts ⊆A should be executed and/or which epistemic propositions Ep ⊆A
should be considered true). For this, the agent constructs the attack (a, b), i.e. a attacks b. Given
the attack relation {(a, b)}, the arguments {a, b} are typically resolved as {a}. Let us refer to the
constructed argumentation framework (AR, Attacks) = ({a, b}, {(a, b)}) as AF. According to the
rational economic man paradigm, the agent’s decision of {a} implies that the agent prefers accepting
{a} over all other possible options; i.e. the agent constructs the following (implicit) preference
relation: {a} ⪰{a, b}, {a} ⪰{b}, {a} ⪰{}. Now, let us assume that a second agent A2 consults A1 to
consider a third item c, i.e. the set of which the agent determines a subset of valid conclusions is now
{a, b, c}. A2 recommends to A1 to add the attacks (b, c) and (c, a). Note that no attacks are added
between the previously existing arguments a and b: AF′ is a normal expansion of AF [9]. Given the
attack relation {(a, b), (b, c), (c, a)}, many argumentation semantics resolve the arguments {a, b, c} as
{}. Let us refer to the constructed argumentation framework ({a, b, c}, {(a, b), (b, c), (c, a)}) as AF′. If A1 adopts the recommended attack relation, this implies, according to the rational man paradigm,
that it now prefers accepting {} over all elements in 2{a,b,c}, which in turn implies the preference
{} ⪰{a}. This is inconsistent with the preference {a} ⪰{} that A1 has given the set {a, b} (see
Figure 1). 1While Caminada introduces rationality postulates for abstract argumentation semantics [15], his work focuses on
rationality from a logics perspective, i.e. on properties of argumentation semantics with regards to specific argumentation
frameworks, without considering the expansions of any argumentation framework. Also, let us note that an application of the
rational man’s argumentation principles we establish in this work to Amgoud’s and Cayrol’s preference-based argumentation
is provided in Section 10. 1
Introduction In this case, A1 can be considered to behave economically irrationally: it has changed its
preference order on the powerset of {a, b}, although (i) no attacks have been added between a and
b and (ii) all arguments that have been added to the initial framework AF are not considered valid
conclusions and the attacks between existing arguments have not changed; i.e. the ceteris paribus
assumption (‘all else unchanged’) holds true. Figure 1 depicts the argumentation graphs of AF
and AF′. Let us note that we neither distinguish between different types of arguments (e.g. utilitarian
and epistemic arguments), nor do we assume that abstract argumentation frameworks model an
exchange of natural language arguments. In any case, the phenomenon of reference independence
can be considered potentially problematic. Given an agent that makes decisions on which utilitarian
arguments in an argumentation framework should be considered valid conclusions, the analogy to
economic theory and the decision-maker who has to choose a subset of a set of decision options is
straight-forward: in both cases, a utilitarian preference order is established. But also given an agent
that makes decisions on which epistemic arguments should be considered as true and false, it can 656
Abstract argumentation and the rational man FIGURE 1 Inconsistent preferences: given an argumentation semantics σ, such that σ(AF) = {{a}}
and σ(AF′) = {{}}, AF implies that accepting {a} is preferred over accepting {}, while AF′ implies
accepting {} is preferred over accepting {a}. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 FIGURE 1 Inconsistent preferences: given an argumentation semantics σ, such that σ(AF) = {{a}}
and σ(AF′) = {{}}, AF implies that accepting {a} is preferred over accepting {}, while AF′ implies
accepting {} is preferred over accepting {a}. be problematic if an agent changes its truth assessment of existing propositional atoms if the new
atoms it considers are all disregarded2 and the relations between existing atoms remain unchanged. For example, given an agent has a knowledge base with the propositional atoms a and b, and—based
on a binary relation between a and b—considers b as true and a as false, it does not make sense that
the addition of a new atom c that the agent considers false changes the assessment of b to false or of
a to true, as long as the relation between a and b remains unchanged. 2Note that in this context, we assume the agent wants to ‘commit’ to a set of arguments it considers a valid conclusion.
This is aligned with the clear preference principle of economic decision-making (see Rubinstein [34,p. 7 et sqq.]). 1
Introduction In economics, the phenomenon that the presence of additional decision options changes existing
preferences—in this case {a} ⪰{} to {} ⪰{a}—is called reference dependence. In this paper, we use
the notion of preferences analogously but with a broader intended meaning. An agent’s preference
over two sets A ⪰B means that the agent considers the acceptance of the set B preferable over the
acceptance of the set A; this can imply the following: •
The agent prefers accepting a set of epistemic arguments B as true over accepting a set of
epistemic arguments A as true. •
The agent prefers accepting a set of epistemic arguments B as true over accepting a set of
epistemic arguments A as true. •
The agent prefers executing a set of actions B over executing a set of actions A. •
The agent prefers executing a set of actions B′ ⊆B and accepting a set of epistemic arguments
B′′ = B \ B′ as true over executing a set of actions A′ ⊆A and considering a set of epistemic
arguments A′′ = A \ A′ as true. (A and B are sets of propositional atoms, and the agent
interprets each propositional atom either as an action or as an epistemic argument.) We show in this paper that changes in preferences can, given most argumentation semantics, be
achieved without adding direct attacks between the arguments in an initial argumentation framework
AF, i.e. by normally expanding AF. We show in this paper that changes in preferences can, given most argumentation semantics, be
achieved without adding direct attacks between the arguments in an initial argumentation framework
AF, i.e. by normally expanding AF. This means that in our scenario, A2 can attempt to deceive A1 into changing its preferences
without noticing. From the perspective of economic rationality, the existence of the ‘invalid’
argument c should not change the preferences A1 has already established given the arguments {a, b}. Indeed, such a change of preferences can be problematic in practice. For example, when an intelligent
system needs to determine a set of actions, deciding {a} are valid conclusions given {a, b}, while
deciding no arguments are valid conclusions ({}) given {a, b, c} does typically not make sense, given
all other things remain the same (the so-called ceteris paribus assumption in economic theory). 1
Introduction 657 Abstract argumentation and the rational man Note that we do not distinguish between arguments that are ‘out’ and ‘undecided’ (in contrast
to many formal argumentation approaches, see, e.g. Wu and Caminada for three-valued labelling
[43]). A binary distinction of arguments that are either element of an extension or not is better
aligned with the clear preferences property that is inherent in the rational economic man paradigm
(see Definition 3.2) and that allows us to infer a clear preference relation from an argumentation
framework, given a specific extension. While we concede that a variant of boundedly economically
rational argumentation that considers the ‘undecided’ status of arguments can be relevant future
work, we consider this out of scope of this paper, analogously to formal argumentation approaches
that qualify or quantify the uncertainty of arguments in other ways. Let us highlight that with this
approach, we are well aligned with the problem in microeconomic theory to have a model that
‘include[s] a description of the resolution of all uncertainties that influence [a] decision maker’s
interests’ [34, p. 42],3 as well as with the purely extension-based approach many other argumentation
principles take (see Van der Torre and Vesic [40] for a survey). Let us also note that we do not
claim that the consistency of preference relations given an argumentation framework and its normal
expansion is a principle that must be enforced given any application scenario. Similarly to the general
notion of economically rational decision-making, the practical usefulness of the principle may be
use case dependent. In this regard, this paper can be considered a first bridge between abstract
argumentation and economically rational decision-making that can serve as a point of departure
for more research, e.g. on argumentation-based models that can be of relevance to the economics
community. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 To analyse the problem of reference dependence in abstract argumentation, this paper provides the
following contributions: 1. It characterizes the concepts of reference independence, cautious monotony and rational
monotony in the context of argumentation semantics and argumentation framework expan-
sions, introducing strong and weak notions of each of the three concepts. 1. It characterizes the concepts of reference independence, cautious monotony and rational
monotony in the context of argumentation semantics and argumentation framework expan-
sions, introducing strong and weak notions of each of the three concepts. 2. 3Emphasis added by the authors 658
Abstract argumentation and the rational man 658
Abstract argumentation and the rational man 5. It puts reference independence and rational man’s expansions in the context of preference-
based argumentation, an argumentation approach that explicitly models agent preferences. This
shows that reference dependence is also a problem for preference-based argumentation, which
can, however, in this case not be solved with the rational man’s expansion that this paper
introduces. 6. It introduces potential application examples for the presented theoretical results. An imple-
mentation of a dialogue reasoning engine that makes use of the theoretical results is presented
in a short paper [28]. 6. It introduces potential application examples for the presented theoretical results. An imple-
mentation of a dialogue reasoning engine that makes use of the theoretical results is presented
in a short paper [28]. The rest of this paper is organized as follows: Section 2 grounds this paper in the context of research
traditions at the intersection of economics and artificial intelligence, whereas Section 3 provides
an overview of abstract argumentation frameworks, semantics and principles, as well as the relevant
definitions of formal models of economic rationality. Then, Section 4 defines a set of non-monotonic
reasoning properties in the context of abstract argumentation, before Section 5 presents argumen-
tation frameworks in the context of decision functions. Section 6 establishes the argumentation
principle the rational man paradigm requires. Hereby, the new rational man’s reference independence
principle is defined, based on which Section 7 shows that typically, with the notable exception of
CF2 semantics (naive semantics are another exception), argumentation semantics do not guarantee
reference-independent decision-making. To still allow for abstract argumentation in accordance
with the rational man paradigm, a new argumentation framework expansion that provides a naive
approach for guaranteeing economic rationality by (severely) limiting the allowed expansions to
normal, non-cyclic expansions that also do not ‘add’ new attacks that originate from cycles is
defined in Section 8. Subsequently, Section 9 provides examples of how the established concepts
can be applied in different scenarios. Finally, Section 10 shows that preference-based argumentation
cannot guarantee reference-independent decision-making, either, before Section 11 concludes the
paper by highlighting relevant future work, in particular the importance of developing ‘loop-busting’
approaches to allow for reference-independent argumentation. 1
Introduction It
analyses
the
relation
between
reference
independence
and
monotony,
cautious
monotony, as well as rational monotony. It proves that for semantics whose extensions
are maximal conflict-free sets or maximal admissible sets (w.r.t. set inclusion), reference
independence does not imply monotony but monotony implies reference independence. Reference independence does not imply cautious and rational monotony, and strong rational
monotony does not imply strong reference independence, but weak rational monotony implies
weak reference independence. However, the non-naive semantics that are evaluated in this
paper all violate weak rational monotony, which indicates that weak rational monotony may
be too strict to be useful as an argumentation principle. The paper also analyses the relation
between weak reference independence and directionality and SCC-recursiveness and shows
that weak reference independence neither implies directionality nor SCC-recursiveness (and
vice versa). 3. It proves that most well-established argumentation semantics cannot guarantee reference
independence for normal argumentation framework expansions. A notably exception are CF2
semantics, which are weakly reference independent. 3. It proves that most well-established argumentation semantics cannot guarantee reference
independence for normal argumentation framework expansions. A notably exception are CF2
semantics, which are weakly reference independent. 4. It proves that argumentation that is based on normal, non-cyclic expansions that also do not
‘add/change’ attack sequences that originate from cycles—which we refer to as rational man’s
expansions—guarantees reference independence for many argumentation semantics. 3Emphasis added by the authors 2
Economic rationality and artificial intelligence Artificial intelligence research frequently draws from concepts that have first been established in
economic theory. For example, the notions of utility functions and preferences that are central to
many models and algorithms of autonomous agents and multi-agent systems stem from ideas of
the philosophers and economists Jeremy Bentham and John Stuart Mill [37]. Von Neumann’s and
Morgenstern’s ground-breaking game theoretical work Theory of Games and Economic Behavior
[42] has influenced generations of both economics and artificial intelligence researchers. More
recently, the works of Daniel Kahneman and Richard Thaler, who both have received the Nobel
Memorial Prize in Economic Sciences for their research on behavioural economics, have inspired
new work on and discussions about ethics and responsibility in artificial intelligence [19]. A key concept at the intersection of economics and artificial intelligence is the notion of economic
rationality. In the same way that a rational economic decision-maker (traditionally a human agent)
is ideally expected to act according to clear and consistent preferences [34, p. 7 et sqq.], artificially
intelligent agents should ‘maximize [their] performance measure’ as stated in the definition of a
rational agent by Russel and Norvig [35, p. 37]. The concept of economical rationality provides
a formal foundation for many ground-breaking works in economics, such as the Nash equilibrium
[31], and Tversky’s and Kahneman’s empirical work on the limits of human agents to act rationally
in the economic sense [27]. 659 Abstract argumentation and the rational man An important property in the context of economic rationality is reference independence: a
decision-maker’s preference order on a set of items S should not be affected by the presence,
or absence, of additional items T. Reference dependence, the negation of reference independence
as established as a theory by Tversky et al. [39] and empirically validated as a phenomenon in
human decision-making by, e.g. Bateman et al. [6], stipulates that an agent might change their initial
preference a ⪰b out of the set of possible decision outcomes A, with a, b ∈A to b ⪰a, depending
on reference points that are either added to the set of decision options itself or provided as additional
context but that do not impact the value or quality of either a or b. As a real-world example, let us
summarize a study by Doyle et al. [21]. In a grocery store, two brands of baked beans x and y are
sold. 2
Economic rationality and artificial intelligence x and y are sold in cans of the same size. Although x is cheaper than y, only 19% of bean sales
are of brand x. By adding a new option x′—a smaller can of beans by brand x that is sold at the
same prize as the original can x—the share of x increases to 33% of total sales (while sales of x′ are
negligible). From the body of research we summarize above, it is clear that human decision-making is often
not reference independent. However, reference independence can potentially be considered a desired
property of the decision process of artificially intelligent agents, as we have demonstrated above,
in the example that is visualized by Figure 1. Yet, when evaluating artificially intelligence systems,
economic rationality in general, and reference dependence in particular, is typically not considered. Instead, research results are commonly evaluated based on traditional performance criteria like
accuracy and computational complexity. In this context, a group of well-established artificial
intelligence researchers advocates for a paradigm shift in the evaluation of ‘machine behaviour’
[33] that can be considered a continuation of the cross-disciplinary work of Herbert Simon, who
initially coined the term bounded rationality [38]. In the spirit of Simon, but also of Kahneman, who
uses empirical methods to systematically specify the boundaries of human rationality, this paper
presents an application of the study of economic rationality to the area of formal argumentation. As
we study—in contrast to Kahneman—formally specified frameworks and not humans, we are able to
use formal methods in our evaluations. Let us highlight that the interface between formal models of
economic rationality and formal argumentation that this paper establishes can potentially also help
to apply formal argumentation as a method to solve problems in microeconomic theory. 3
Theoretical background This section introduces the theoretical foundations upon which this paper builds: the rational man
paradigm in economic theory and abstract argumentation. 3.1 The rational economic man paradigm 3.1 The rational economic man paradigm 4In this paper, we often use the shortened term rational man.
5However, in contrast to the model used by Rubinstein, we use a decision model that allows for choosing any subset
A∗⊆A of the set of decision options A instead of exactly one option (x∗∈A). 3.1 The rational economic man paradigm As a prerequisite, we introduce a definition of a partially ordered set [18]. As a prerequisite, we introduce a definition of a partially ordered set [18]. DEFINITION 3.1
Let Q be a set. An order (or partial order) on Q is a binary relation ⪰on Q such that, for all
x, y, z ∈Q: DEFINITION 3.1
Let Q be a set. An order (or partial order) on Q is a binary relation ⪰on Q such that, for all
x, y, z ∈Q: 1. x ⪰x (reflexivity); 2. x ⪰y and y ⪰x imply x = y (antisymmetry); 3. x ⪰y and y ⪰z imply x ⪰z (transitivity). 660
Abstract argumentation and the rational man FIGURE 2 Example: Hasse diagram of a set, in which the following preference relation of the
rational man holds: ∀A′ ∈{{coffee}, {snack}, {}}, {coffee, snack} ⪰A′. FIGURE 2 Example: Hasse diagram of a set, in which the following preference relation of the
rational man holds: ∀A′ ∈{{coffee}, {snack}, {}}, {coffee, snack} ⪰A′. We refer to a set Q that is equipped with an order relation ⪰as an ordered set (or partially ordered
set). We refer to a set Q that is equipped with an order relation ⪰as an ordered set (or partially ordered
set). In economic theory, the model of a rational decision maker—the rational economic man
paradigm4 —can be described as follows (based on a definition by Rubinstein [34,p. 7 et sqq.])5. 4In this paper, we often use the shortened term rational man.
5 In this paper, we often use the shortened term rational man.
5However, in contrast to the model used by Rubinstein, we use a decision model that allows for choosing any subset
A∗⊆A of the set of decision options A instead of exactly one option (x∗∈A). 3.2 Abstract argumentation To allow for a concise overview of the relevant foundations of abstract argumentation, we introduce a
formal definition of the basic structure of an argumentation framework (AF), provide the definitions
of well-established argumentation semantics and explain the notions of argumentation expansions
and principles. EXAMPLE 3.4 If a decision-maker, given the set {tea, cookie} chooses {tea} and given the set {tea, cookie, coffee}
chooses {tea, cookie} the decision is not rational; the presence of the ‘new’ irrelevant alternatives
{coffee}, {tea, coffee}, {cookie, coffee} and {tea, cookie, coffee} causes the preference {tea}
⪰
{tea, cookie} to change to {tea, cookie} ⪰{tea}. The reference independence property implies that the expansion of a set A to A′ ⊃A does not add
new knowledge about the elements in A that affects the decision-maker’s preferences over any two
elements in 2A. Yet, in reality, human decision-makers can use the addition of options to a set as a way
to infer new information about the quality of the original options, as e.g. shown by Doyle et al. [21]. Still, in the context of computational argumentation-based decision-making, it can be useful to be
able to distinguish between changes in options and outcomes that satisfy the reference independence
property and those that do not. For example, in a consultation scenario, a consulted agent may want
to check if the consulting agent is providing a proposal that is seemingly barely adding new options
but covertly also altering the preference order on existing options. Such a scenario is presented in
greater detail in Section 9. DEFINITION 3.2 DEFINITION 3.2 DEFINITION 3.2 (Rational Economic Man). Given a set of decision options A, the rational economic man’s decision A∗⊆A implies the
preference order (partial order) ⪰, such that ∀A′ ∈2A, A∗⪰A′; i.e. A∗is the rational man’s preferred
option when compared to all possible alternatives. For example, given the set {coffee, snack}, the rational man’s preferred option could be
{coffee, snack}, given he chooses this element from the set 2{coffee,snack}. The decision implies
the preference relation ∀A′ ∈{{coffee}, {snack}, {}}, {coffee, snack} ⪰A′, as depicted in Figure 2
(note that technically also {coffee, snack} ⪰{coffee, snack}). In the context of this paper, an important property of the rational man’s decision process is
reference independence. DEFINITION 3.3
(Reference independence). Given two sets of options A and A′, such that A ⊆A′ and the rational man’s decisions A∗⊆A and
A′∗⊆A′, if A′∗⊆A, then A′∗= A∗. Colloquially expressed, the rational man’s decision outcome is not affected if options that the agent
does not prefer over the ‘previously’ existing options are added to the set of potential options. 661 Abstract argumentation and the rational man Abstract argumentation and the rational man 662
Abstract argumentation and the rational man 662
Abstract argumentation and the rational man •
A conflict-free set S ⊆AR is admissible iff each argument in S is acceptable with regards to
S. •
A conflict-free set S ⊆AR is admissible iff each argument in S is acceptable with regards to
S. •
A set S ⊆AR is strongly admissible iff ∀c ∈S, there exists a set Ssub ⊆S \ {c}, such that c is
acceptable with regards to Ssub and Ssub is strongly admissible. •
A set S ⊆AR is strongly admissible iff ∀c ∈S, there exists a set Ssub ⊆S \ {c}, such that c is
acceptable with regards to Ssub and Ssub is strongly admissible. Based on these concepts, Dung and others define different semantics for resolving argumentation
frameworks. DEFINITION 3.7 (Argumentation semantics [22]). (
g
[
])
An argumentation semantics σ is a function that takes an argumentation framework AF
=
(AR, Attacks) and returns a set of sets of arguments denoted by σ(AF). σ : AF →22AR, σ : AF →22AR, with AF denoting the set of all possible argumentation frameworks. Each set of σ(AF) is called a
σ-extension. with AF denoting the set of all possible argumentation frameworks. Each set of σ(AF) is called a
σ-extension. Some argumentation semantics make use of the notion of range. rgumentation semantics make use of the notion of range. DEFINITION 3.8 (Range [41]). (
g
)
Let AF = (AR, Attacks) be an argumentation framework and let S ⊆AR. We define the range of S
as S ∪S+, where S+ = {b|b ∈AR, a ∈S, (a, b) ∈Attacks}. Below, we provide an overview of some of the semantics that are commonly applied and analysed
by the formal argumentation community. Below, we provide an overview of some of the semantics that are commonly applied and analysed
by the formal argumentation community. DEFINITION 3.9 DEFINITION 3.9 (Argumentation extensions and semantics [2, 14, 16, 22, 23, 41]). (Argumentation extensions and semantics [2, 14, 16, 22, 23, 41]). (Argumentation extensions and semantics [2, 14, 16, 22, 23, 41]). Given an argumentation framework AF = (AR, Attacks), an admissible set S ⊆AR is a (
g
[ ,
,
,
,
,
])
Given an argumentation framework AF = (AR, Attacks), an admissible set S ⊆AR is a •
Stable extension of AF iff S attacks each argument that does not belong to S. (Argumentation framework [22]). (Argumentation framework [22]). (
g
[
])
An argumentation framework is a pair AF = (AR, Attacks), where AR is a finite set of arguments
and Attacks is a binary relation on AR, i.e. Attacks ⊆AR × AR. An argumentation framework is a pair AF = (AR, Attacks), where AR is a finite set of arguments
and Attacks is a binary relation on AR, i.e. Attacks ⊆AR × AR. Note that we diverge from the original definition by Dung in that we constrain an argumentation
framework to a finite set of arguments. In the context of an argumentation framework AF =
(AR, Attacks), a attacks b means that for a, b ∈AR it holds true that (a, b) ∈Attacks. Similarly,
any set S ⊆AR attacks b if b is attacked by an argument in S. We say that an argument c ∈AR is
unattacked iff ∄d ∈AR, such that d attacks c and we say that an argument g ∈AR is self-attacking
iff g attacks g. To select coherent sets of arguments from an argumentation framework, Dung introduces the notion
of conflict-free and admissible sets; based on the notion of admissible sets, Baroni et al. define
strongly admissible sets. DEFINITION 3.6
(Conflict-free, acceptable and (strongly) admissible arguments [4, 22]). Let AF = (AR, Attacks) be an argumentation framework. DEFINITION 3.6
(Conflict-free, acceptable and (strongly) admissible arguments [4, 22]). Let AF = (AR, Attacks) be an argumentation framework. Let AF = (AR, Attacks) be an argumentation framework. •
A set S ⊆AR is conflict-free iff there are no arguments a, b in S such that a attacks •
An argument a ∈AR is acceptable w.r.t. an argument set S ⊆
it holds true that if b attacks a, then b is attacked by S. •
An argument a ∈AR is acceptable w.r.t. an argument set S ⊆AR iff for each argument b ∈AR
it holds true that if b attacks a, then b is attacked by S. 662
Abstract argumentation and the rational man Stable semantics
σstable(AF) returns all stable extensions of AF. •
Preferred extension of AF iff S is maximal (w.r.t. set inclusion) among admissible sets. Preferred semantics σpreferred(AF) returns all preferred extensions of AF. •
Complete extension of AF iff each argument that is acceptable w.r.t. S belongs to S. Complete
semantics σcomplete(AF) returns all complete extensions of AF. p
•
Grounded extension of AF iff S is the minimal (w.r.t. set inclusion) complete extension. Grounded semantics σgrounded(AF) denotes the set {S}, such that S is the grounded extension
of AF. •
Ideal extension of AF iff S is the maximal (w.r.t. set inclusion) ideal set, where a set S′ ⊆AR
is ideal iff S′ is a subset of every preferred extension of AF. Ideal semantics σideal(AF) denotes
the set {S}, such that S is the ideal extension of AF. •
Semi-stable extension of AF iff S is a complete extension where S ∪S+ is maximal (w.r.t. set inclusion) among complete extensions. Semi-stable semantics σsemi−stable(AF) returns all
semi-stable extensions of AF. Abstract argumentation and the rational man 663 •
Eager extension of AF iff S is the maximal (w.r.t. set inclusion) admissible set which is
included in every semi-stable extension. Eager semantics σeager(AF) denotes the set {S}, such
that S is the eager extension of AF. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 Given an argumentation framework AF = (AR, Attacks), a set S ⊆AR is a •
Naive extension iff S is a maximal (w.r.t. set inclusion) conflict-free set. Naive •
Naive extension iff S is a maximal (w.r.t. set inclusion) conflict-free set. Naive semantics
σnaive(AF) returns all naive extensions of AF. + •
Stage extension of AF, iff S is conflict-free and S∪S+ is maximal (w.r.t. set inclusion) among
all conflict-free sets, i.e. there is no conflict-free set S′ ⊆AR, such that (S′ ∪S′+) ⊃(S∪S+). Stage semantics σstage(AF) returns all stage extensions of AF. To introduce the definitions of stage2 and CF2 semantics, let us first define the notion of attack
sequences and strongly connected components. To introduce the definitions of stage2 and CF2 semantics, let us first define the notion of attack
sequences and strongly connected components. DEFINITION 3.11 (Strongly connected components). Let AF = (AR, Attacks) be an argumentation framework. S ⊆AR is a strongly connected component
of AF, iff ∀a, b ∈S, a is reachable from b and b is reachable from a and ∄c ∈AR \ S, such that a is
reachable from c and c is reachable from a. Let us denote the strongly connected components of AF
as SCCSAF. For example, the graph of the argumentation framework ({a, b, c}, {(a, b), (b, a), (b, c)}) has the
SCCS {a, b} and {c}. Let us also define the concept of a restriction of an argumentation framework to a subset of its
arguments. DEFINITION 3.10 (Attack sequences in argumentation frameworks). (Attack sequences in argumentation frameworks). An attack sequence Va1,an in an argumentation framework AF = (AR, Attacks) is an argument
sequence ⟨a1, ..., an⟩such that (ai, ai+1) ∈Attacks, ai ∈AR, ai ̸= aj, 1 ≤j ≤i, 1 ≤i ≤n−1. V(AF)
denotes all attack sequences of AF. Given an attack sequence Va1,an, ARV
a1,an denotes all arguments
that occur in V. An argument b is reachable from an argument a iff there exists an attack sequence
Va1,an ∈V(AF), such that a1 = a and an = b. An attack sequence Va1,an in an argumentation framework AF = (AR, Attacks) is an argument
sequence ⟨a1, ..., an⟩such that (ai, ai+1) ∈Attacks, ai ∈AR, ai ̸= aj, 1 ≤j ≤i, 1 ≤i ≤n−1. V(AF)
denotes all attack sequences of AF. Given an attack sequence Va1,an, ARV
a1,an denotes all arguments
that occur in V. An argument b is reachable from an argument a iff there exists an attack sequence
Va1,an ∈V(AF), such that a1 = a and an = b. Strongly connected components (SCCS) ‘provide a unique partition of a directed graph into
disjoint parts where all nodes are mutually reachable’ [2]. (
g
[ ])
Let AF = (AR, Attacks) be an argumentation framework and let S ⊆AR be a set of arguments. The
restriction of AF to S, denoted by AF ↓S, is the argumentation framework (AR∩S, Attacks∩(S×S)).
Let us introduce some additional preliminaries DEFINITION 3.15 DEFINITION 3.15
(Stage2 extensions [24]). Let AF = (AR, Attacks) be an argumentation framework. A set S ⊆AR is an extensions of stage2
semantics, iff Let AF = (AR, Attacks) be an argumentation framework. A set S ⊆AR is an extensions of stage2
semantics, iff •
S is a stage extension of AF, if |SCCSAF| = 1; •
∀Args ∈SCSSAF, (S ∩Args) ∈σstage2(AF ↓UPAF(Args,S)), otherwise. •
∀Args ∈SCSSAF, (S ∩Args) ∈σstage2(AF ↓UPAF(Args,S)), otherwis
Stage2 semantics σ
(AF) returns all stage2 extensions of AF •
∀Args ∈SCSSAF, (S ∩Args) ∈σstage2(AF ↓UPAF(Args,S)), otherwise. Stage2 semantics σstage2(AF) returns all stage2 extensions of AF. •
∀Args ∈SCSSAF, (S ∩Args) ∈σstage2(AF ↓UPAF(Args,S)), oth
Stage2 semantics σstage2(AF) returns all stage2 extensions of AF. tage2 semantics σstage2(AF) returns all stage2 extensions of AF. Stage2 semantics σstage2(AF) returns all stage2 extensions of AF. In words, CF2 and stage2 semantics decompose an argumentation framework into SCCS and
recursively resolve the framework component by component, starting with the SCCS that are not
attacked by any other SCC. Note that a detailed explanation of CF2 and stage2 semantics is beyond
the scope of the paper. Finally, we provide the established definitions for credulous and skeptical acceptance of arguments
[20].6 6From a decision-making perspective, credulous and skeptical acceptance are often referred to as objective and subjective
acceptance [12]; however, we use the terms credulous and skeptical because they are arguably more prevalent in the
community and because our work is relevant no matter the type of reasoning (making a decision about an action to take
or deciding which beliefs in a set should be considered as true). DEFINITION 3.12 A set of arguments S ⊆AR is an extension
of CF2 semantics iff: (
[ ])
Let AF = (AR, Attacks) be an argumentation framework. A set of arguments S ⊆AR is an extension
of CF2 semantics iff: •
S is a naive extension of AF, if |SCCSAF| = 1; •
S is a naive extension of AF, if |SCCSAF| = 1; •
∀Args ∈SCSSAF, (S ∩Args) ∈σCF2(AF ↓UPAF(Args,S)), otherwise. •
∀Args ∈SCSSAF, (S ∩Args) ∈σCF2(AF ↓UPAF(Args,S)), otherwise. CF2 semantics σCF2(AF) returns all CF2 extensions of AF. •
∀Args ∈SCSSAF, (S ∩Args) ∈σCF2(AF ↓UPAF(Args,S)), otherwise. CF2 semantics σCF2(AF) returns all CF2 extensions of AF. •
∀Args ∈SCSSAF, (S ∩Args) ∈σCF2(AF ↓UPAF(Args,S)), otherwise. CF2 semantics
(AF) ret rns all CF2 e tensions of AF •
∀Args ∈SCSSAF, (S ∩Args) ∈σCF2(AF ↓UPAF(Args,S)), otherwise. CF2 semantics σCF2(AF) returns all CF2 extensions of AF. •
∀Args ∈SCSSAF, (S ∩Args) ∈σCF2(AF ↓UPAF(Args,S)), otherwise. CF2 semantics σCF2(AF) returns all CF2 extensions of AF. •
∀Args ∈SCSSAF, (S ∩Args) ∈σCF2(AF ↓UPAF(Args,S)
CF2 semantics σCF2(AF) returns all CF2 extensions of AF. CF2 semantics σCF2(AF) returns all CF2 extensions of AF. DEFINITION 3.12 (Argumentation framework restriction [2]). Let AF = (AR, Attacks) be an argumentation framework and let S ⊆AR be a set of arguments. The
restriction of AF to S, denoted by AF ↓S, is the argumentation framework (AR∩S, Attacks∩(S×S)). Let AF = (AR, Attacks) be an argumentation framework and let S ⊆AR be a set of arguments. The
restriction of AF to S, denoted by AF ↓S, is the argumentation framework (AR∩S, Attacks∩(S×S)). Let us introduce some additional preliminaries. DEFINITION 3.13
(D, P, U and UP [5]). Let AF = (AR, Attacks) be an argumentation framework, let E ⊆AR and let S be a strongly Abstract argumentation and the rational man 664 connected component of AF (S ∈SCCS(AF)). Let us define S−
out = {a|a ∈AR, a ̸∈S and a
attacks S}. Given an argument a ∈AR, let us define a−= {b|b ∈AR and a attacks b}. We define connected component of AF (S ∈SCCS(AF)). Let us define S−
out = {a|a ∈AR, a ̸∈S and a
attacks S}. Given an argument a ∈AR, let us define a−= {b|b ∈AR and a attacks b}. We define •
DAF(S, E) = {a|a ∈S, (E ∩S−
out) attacks a}; •
DAF(S, E) = {a|a ∈S, (E ∩S−
out) attacks a}; •
DAF(S, E)
{a|a ∈S, (E ∩Sout) attacks a};
•
PAF(S, E) = {a|a ∈S, (E ∩S−
out) does not attack a and ∃b ∈(S−
out ∩a−) such that E does not
attack b}; out
•
PAF(S, E) = {a|a ∈S, (E ∩S−
out) does not attack a and ∃b ∈(S−
out ∩a−) such that E does not
attack b}; };
•
UAF(S, E) = S \ (DAF(S, E) ∪PAF(S, E)); \
•
UPAF(S, E) = UAF(S, E) ∪PAF(S, E). Now, we have the necessary concepts established that allow us to define SCC-recursive extensions. Now, we have the necessary concepts established that allow us to define SCC-recursive extensions. DEFINITION 3.14
(CF2 extensions [5]). Let AF = (AR, Attacks) be an argumentation framework. A set of arguments S ⊆AR is an extension
of CF2 semantics iff: DEFINITION 3.14
(CF2 extensions [5]). Let AF = (AR, Attacks) be an argumentation framework. A set of arguments S ⊆AR is an extension
of CF2 semantics iff: DEFINITION 3.14
(CF2 extensions [5]). Let AF = (AR, Attacks) be an argumentation framework. DEFINITION 3.17 (Argumentation framework expansion [9]). (Argumentation framework expansion [9]). An argumentation framework AF′ = (AR′, Attacks′) is an expansion of another argumentation
framework AF = (AR, Attacks) (denoted by AF ⪯E AF′) iff AR ⊆AR′ and Attacks ⊆Attacks′. An argumentation framework AF′ = (AR′, Attacks′) is an expansion of another argumentation
framework AF = (AR, Attacks) (denoted by AF ⪯E AF′) iff AR ⊆AR′ and Attacks ⊆Attacks′. An argumentation framework AF′ = (AR′, Attacks′) is an expansion of another argumentation
framework AF = (AR, Attacks) (denoted by AF ⪯E AF′) iff AR ⊆AR′ and Attacks ⊆Attacks′. Several expansion types have been defined in the literature. In the context of this paper, the notion
of a normal expansion is of relevance. DEFINITION 3.18
(Normal expansion [9]). An argumentation framework AF′ = (AR′, Attacks′) is a normal expansion of an argumentation
framework AF = (AR, Attacks) (denoted by AF ⪯N AF′) iff AF ⪯E AF′ and ∄(a, b) ∈Attacks′ \
Attacks, such that a ∈AR ∧b ∈AR. DEFINITION 3.18 (Normal expansion [9]). (Normal expansion [9]). An argumentation framework AF′ = (AR′, Attacks′) is a normal expansion of an argumentation
framework AF = (AR, Attacks) (denoted by AF ⪯N AF′) iff AF ⪯E AF′ and ∄(a, b) ∈Attacks′ \
Attacks, such that a ∈AR ∧b ∈AR. In words, a normal expansion of an argumentation framework adds additional arguments to the
framework that can attack and be attacked by any other argument but neither removes existing
arguments nor changes attacks (neither adds nor removes) between existing arguments. DEFINITION 3.16 (Credulous and skeptical reasoning modes) (Credulous and skeptical reasoning modes). Given an argumentation framework AF = (AR, Attacks) and an argumentation semantics σ, we say
that Given an argumentation framework AF = (AR, Attacks) and an argumentation semantics σ, we say
that •
an argument a ∈AR is credulously accepted w.r.t. σ iff it is contained in at least one of the
extensions of σ(AF); •
an argument a ∈AR is credulously accepted w.r.t. σ iff it is contained in at least one of the
extensions of σ(AF); 6From a decision-making perspective, credulous and skeptical acceptance are often referred to as objective and subjective
acceptance [12]; however, we use the terms credulous and skeptical because they are arguably more prevalent in the
community and because our work is relevant no matter the type of reasoning (making a decision about an action to take
or deciding which beliefs in a set should be considered as true). 665 Abstract argumentation and the rational man •
an argument a ∈AR is skeptically accepted w.r.t. σ iff it is contained in all of the extensions of
σ(AF). The skeptical extension of an argumentation framework AF w.r.t. σ is the intersection
of all extensions of σ(AF) (σ ∩(AF) = {
E∈σ(AF) E}). •
an argument a ∈AR is skeptically accepted w.r.t. σ iff it is contained in all of the extensions of
σ(AF). The skeptical extension of an argumentation framework AF w.r.t. σ is the intersection
of all extensions of σ(AF) (σ ∩(AF) = {
E∈σ(AF) E}). Note that to be consistent with the general function signature of argumentation semantics, we
provide the aggregation as the intersection of sets as made by a semantics’ skeptical reasoning mode
wrapped into an additional set. Given the variety of argumentation semantics that have been established throughout the years, it
can be challenging to assess which semantics are suitable for a specific application. Consequently,
argumentation principles have been defined; evaluating the satisfaction of one or multiple principles
can guide the assessment of argumentation semantics in the context of a specific use case. For
example, a principle can specify that an argumentation semantics should determine exactly one
extension for every argumentation framework. An overview of argumentation principles is provided
by Van der Torre and Vesic [40]. 3.3 Argumentation expansions To describe a specific type of relation between argumentation frameworks, the notion of an
argumentation framework expansion has been introduced by Baumann and Brewka [9]. A concise
introduction and overview of expansions are presented by Baumann and Woltran [11]. The general
concept of an argumentation framework expansion can be defined as follows. 4
Properties of non-monotonic inference in argumentation Previous research exists that explores the relation of non-monotonic inference properties and formal
argumentation methods. In particular, ˇCyras and Toni provide a series of proofs that show which
semantics (among grounded, ideal, skeptically preferred, stable, preferred and complete semantics) Abstract argumentation and the rational man 666 satisfy the cautions monotony properties7 for assumption-based argumentation [17]. However,
ˇCyras’ and Toni’s definition cannot be applied to abstract argumentation as it relies on specific
characteristics of assumption-based argumentation. We use the same notions of strong and weak
monotony properties as introduced by ˇCyras and Toni to characterize monotony in the context of
abstract argumentation. Let us note that we define the properties in the context of normal expansions
because changes to attacks between existing arguments will obviously cause problems for most
semantics w.r.t. compliance with the properties. As a preliminary, let us introduce the definition
of universally defined semantics, in our case given finite argumentation semantics (see Baumann
[8]). Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 DEFINITION 4.1 DEFINITION 4.1
(Universally defined semantics). (Universally defined semantics). An argumentation semantics σ is universally defined iff for every argumentation framework AF =
(AR, Attacks) it holds true that |σ(AF)| ≥1. Let us now define monotony as an argumentation principle. DEFINITION 4.2 (Monotony in argumentation semantics). Let σ be an argumentation semantics. σ satisfies •
Strong monotony iff σ is universally defined and for every two argumentation frameworks
AF = (AR, Attacks) and AF′ = (AR′, Attacks′), such that AF ⪯N AF′, ∀E ∈σ(AF), ∀E′ ∈
σ(AF′) it holds true that E ⊆E′. •
Strong monotony iff σ is universally defined and for every two argumentation fr •
Weak monotony iff for every two argumentation frameworks AF = (AR, Attacks) and AF′ =
(AR′, Attacks′), such that AF ⪯N AF′, ∀E ∈σ(AF), it holds true that ∃E′ ∈σ(AF′), such that
E ⊆E′. •
Weak monotony iff for every two argumentation frameworks AF = (AR, Attacks) and AF′ =
(AR′, Attacks′), such that AF ⪯N AF′, ∀E ∈σ(AF), it holds true that ∃E′ ∈σ(AF′), such that
E ⊆E′. Now, let us define cautious monotony, based on a definition provided (e.g.) by Schröder et al. [36], i.e. if (a ∼b) and (a ∼c) then (a ∧b ∼c). In words, the property can be described as if
we infer c from a, and we infer b from a, then we infer c from ‘a and b’. In the context of abstract
argumentation, we can say that an argumentation semantics ‘infers sets of arguments as parts of
extensions from argumentation frameworks’. Using an argumentation semantics to infer the sets of
arguments c and b from an argumentation framework a implies that b is a subset of the arguments
in argumentation framework a; i.e. a straight-forward adaptation of the cautious monotony principle
is not useful in the case of abstract argumentation (this problem does not exist in assumption-based
argumentation, see ˇCyras and Toni [17]). Instead, we state that •
Given a semantics σ, if we infer a set of arguments c from an argumentation framework a,
and •
Given a semantics σ, if we infer a set of arguments c from an argumentation framework a,
and •
If normally expanding a by the arguments and attacks b′ to an argumentation framework b
and arguments in b′ do not attack c,
h
i f
f
b •
Then we can infer c from b. We can consequently define cautious monotony for argumentation semantics as follows 7Cautions monotony is based on a property initially introduced by Gabbay as restricted monotony [26]. Abstract argumentation and the rational man 667 DEFINITION 4.3 (Cautious monotony in argumentation semantics). (Cautious monotony in argumentation semantics). Let σ be an argumentation semantics. σ satisfies Let σ be an argumentation semantics. σ satisfies Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 •
Strong cautious monotony iff σ is universally defined and for every two argumentation
frameworks AF
= (AR, Attacks) and AF′ = (AR′, Attacks′), such that AF
⪯N
AF′,
∀E ∈σ(AF) and ∀E′ ∈σ(AF′) it holds true that if {(a, b) | (a, b) ∈Attacks′, a ∈AR′\AR, b ∈
E} = ∅then E ⊆E′; •
Weak cautious monotony: iff for every two argumentation frameworks AF = (AR, Attacks)
and AF′ = (AR′, Attacks′), such that AF ⪯N AF′ and ∀E ∈σ(AF), it holds true that if
{(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅then ∃E′ ∈σ(AF′) such that E ⊆E′. A property that has so far not been used to analyse abstract argumentation approaches is rational
monotony, as e.g. provided by Benferhat et al. as if ¬(a ∼¬b) and (a ∼c) then (a∧b ∼c) [13]. In
words, the property can be described as if we infer c from a, and we do not infer ‘¬b’ from a, then we
infer c from ‘a and b’. Analogously to cautious monotony, which requires an adaptation to be useful
in the context of abstract argumentation, we need to ‘tweak’ the ¬(a ∼¬b) condition and we state
that •
Given a semantics σ, if we infer a set of arguments c from an argumentation framework a,
and •
If normally expanding a by the arguments and attacks b′ to an argumentation framework b
and arguments in b′ that are in any σ-extension of b do not attack c, •
Then we can infer c from b. •
Then we can infer c from b. DEFINITION 4.4 This follows from the fact that the only difference between the rational monotony and
cautious monotony argumentation principles is that the constraints of rational monotony, given
an argumentation semantics σ and two argumentation frameworks AF = (AR, Attacks), AF′ =
(AF′, Attacks′), such that AF ⪯N AF′, are weaker •
Rational monotony:
{(a, b) | (a, b) ∈Attacks′, a ∈
E′∈σ(AF′)(E′ \ AR), b ∈E} = ∅. •
Cautious monotony:
{(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅. •
Rational monotony:
{(a, b) | (a, b) ∈Attacks′, a ∈
E′∈σ(AF′)(E′ \ AR), b ∈E} = ∅. •
Cautious monotony:
{(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅. •
Rational monotony: •
Cautious monotony: Considering the paper’s primary focus on the intersection of economic rationality and abstract
argumentation, a detailed analysis of cautious and rational monotony can be considered out of scope. The purpose of the establishment of the cautious monotony and rational monotony argumentation
principles is to show the difference between these variants of ‘relaxed’ monotony and the reference
independence argumentation principle we derive from a formal model of economic rationality. Considering the paper’s primary focus on the intersection of economic rationality
argumentation, a detailed analysis of cautious and rational monotony can be considered 5
Rational argumentation-based decision functions In this section, we define the concept of economically rational argumentation-based decision
functions. DEFINITION 4.4 (Rational monotony in argumentation semantics). (Rational monotony in argumentation semantics). Let σ be an argumentation semantics. σ satisfies Let σ be an argumentation semantics. σ satisfies •
Strong rational monotony: iff σ is universally defined and for every two argumentation
frameworks AF = (AR, Attacks) and AF′ = (AR′, Attacks′), such that AF ⪯N AF′, ∀E ∈
σ(AF) and ∀E′ ∈σ(AF′) it holds true that if {(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} =
∅then E ⊆E′, where UE′
new =
E′∈σ(AF′)(E′ \ AR); ∈σ(
)
•
Weak rational monotony: iff for every two argumentation frameworks AF = (AR, Attacks)
and AF′ = (AR′, Attacks′), such that AF ⪯N AF′ and ∀E ∈σ(AF), it holds true that if
{(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅then ∃E′ ∈σ(AF′) such that E ⊆E′, where
UE′
new =
E′∈σ(AF′)(E′ \ AR). Let us observe that when considering the two principles of non-monotonic reasoning, rational
monotony implies cautious monotony: •
Rational monotony: if ¬(a ∼¬b) and (a ∼c) then (a ∧b ∼c). •
Rational monotony: if ¬(a ∼¬b) and (a ∼c) then (a ∧b ∼c). •
Cautious monotony: if (a ∼
b) and (a ∼c) then (a ∧b ∼c). We can see that the if-condition of rational monotony is weaker, i.e. (a ∼b) implies ¬(a ∼¬b), but
not vice versa, from which it follows that the principle of rational monotony is stricter. Analogously,
the rational monotony argumentation principle implies the cautious monotony argumentation
principle, assuming that an argumentation semantics, given any argumentation framework AF =
(AR, Attacks), and E ∈σ(AF), ∀AF′ = (AR′, Attacks′), such that AR′ = AR and Attacks ⊆Attacks′, Abstract argumentation and the rational man 668 Attacks′ ⊇(Attacks \ {(a, b)|(a, b) ∈AR′, b ∈E}), it holds true that ∃E′ ∈σ(AF′), such that
E ⊆E′. Colloquially speaking, we assume that ‘removing’ attacks from arguments in AR to an
extension E does not cause any argument in E to be no longer considered part of a valid conclusion
(extension). 5.1 Rational argumentation-based decision-making To build the foundation for exploring the intersection of abstract argumentation semantics and
economic rationality, we introduce the notion of an argumentation-based decision function. DEFINITION 5.1 (Argumentation-based decision function). (Argumentation-based decision function). The argumentation-based decision function g ◦σ of an agent is the function composition between
a function g and an argumentation semantics σ that takes an argumentation framework AF =
(AR, Attacks) as its input and returns a set of decision outcomes AR∗⊆AR: g ◦σ : AF →2AR. Because we assume an economically rational decision function, given an argumentation frame-
work AF = (AR, Attacks), A∗= g ◦σ(AF) implies ∀A ∈2AR, A∗⪰A (i.e. the agent establishes the
preference relation A∗⪰A for all A ∈2AR). To determine the function’s input, an economically rational agent needs to construct an argumen-
tation framework AF that consists of the propositional atoms AR and an attack relation Attacks ⊆
AR × AR. The framework is resolved by an argumentation semantics σ; different argumentation
semantics can be used. In the context of rational economic decision-making, we want to determine
exactly one set of arguments that can be considered valid conclusions. To achieve this, the decision
function can be defined, for instance, as g∪◦σ(AF) =
E∈σ(AF) E; i.e. if a semantics σ returns
more than one extension, the decision function g∪◦σ(AF) =
E∈σ(AF) E returns the union of the 669 Abstract argumentation and the rational man set of extensions returned by σ. We call such a function a lenient decision function. For example,
given a particular argumentation semantics, we can define argumentation-based decision functions
as follows: Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 1. g∩◦σstage(AF) corresponds to the skeptical reasoning mode of stage semantics. ∩
∪ 1. g∩◦σstage(AF) corresponds to the skeptical reasoning mode of stage semantics. ∩
∪ 2. g∩◦σgrounded(AF) and g∪◦σgrounded(AF) return the grounded extension of AF. There is
always exactly one grounded extension, from which it follows that g∩◦σgrounded(AF) = g∪◦
σgrounded(AF). The same applies to ideal and eager semantics. 2. g∩◦σgrounded(AF) and g∪◦σgrounded(AF) return the grounded extension of AF. There is
always exactly one grounded extension, from which it follows that g∩◦σgrounded(AF) = g∪◦
σgrounded(AF). The same applies to ideal and eager semantics. Other approaches to use g as an aggregate function for ‘selecting’ an extension from the set of
extensions a semantics returns are possible, of course. Other approaches to use g as an aggregate function for ‘selecting’ an extension from the set of
extensions a semantics returns are possible, of course. EXAMPLE 5.2 As an example, let us assume we have a consultant who has to suggest whether or not to launch a
product p. Initially, she does not find any arguments against launching the product, i.e. she establishes
the argumentation framework AF = ({lp}, {}), where lp stands for ‘launch product’. When asked
by a decision-maker about whether to launch the product or not, our consultant can give a clear
recommendation given any of the common argumentation semantics, let us assume, e.g., stage
semantics σstage or preferred semantics σpreferred: σstage(AF) = σpreferred(AF) = {{lp}}. From the
perspective of microeconomic decision theory, we can infer that this result implies the consultant
has established the preference order that {lp} is preferred over all other items in 2{lp} = {{}, {lp}}, i.e. ∀A ∈{{}, {lp}}, {lp} ⪰A. However, let us now assume that the decision-maker asks the consultant to
collect feedback from different management stakeholders on whether or not the product should be
launched. After doing so, the consultant constructs an expansion of AF that reflects the different
arguments for and against launching the product (directly or indirectly). She ends up with the
argumentation framework AF′ = ({lp, a, b, c}, {(a, lp), (a, b), (b, c), (c, a)}). Let us assume she again
uses either preferred or stage semantics to determine the framework’s extensions: •
Preferred semantics. σpreferred(AF) = {{}}. This means that the addition of the arguments a, b,
and c changes the status of lp although a, b and c are all not considered valid conclusions and
although the relationships between the arguments in AF have not changed; i.e. the consultant
reverses a preference from {lp} ⪯{} to {} ⪯{lp} and is economically not rational. Of course,
the consultant could search for new knowledge until she can make an economically rational
decision (see work on ‘loop-busting’ in abstract argumentation as presented by Gabbay [25]),
but preferred semantics do not allow her to commit to an economically rational decision at
the moment. •
Stage semantics. σstage(AF) = {{lp, b}, {lp, c}, {a}}. This result means that the consultant can
‘pick’ an extension (either {lp, b} or {lp, c} or {a}) that implies preferences that are consistent
with the previous recommendation based on AF. (Argumentation-based decision function). It is important to note that argumentation-based decision-making does not necessarily imply
choice from a set of goods or indeed any type of scenario that is typical for economic decision-
making examples but can cover any decision process, e.g. the selection of epistemic arguments from
an argumentation framework that an agent will consider as valid. EXAMPLE 5.2 This can be considered a useful property in
this scenario, where we can assume that the decision-maker expects a clear recommendation
on whether the product should be launched or not that is consistent with regards to the previous
interactions between decision-maker and consultant, and indecisiveness is most likely not an
option. Figure 3 depicts the example’s argumentation graphs. 670
Abstract argumentation and the rational man FIGURE 3
Inconsistent preferences, given preferred semantics: σpreferred(AF) = {{lp}} implies
accepting {lp} is preferred over accepting {}, while σpreferred(AF′) = {{}} implies accepting {} is
preferred over accepting {lp}. FIGURE 3
Inconsistent preferences, given preferred semantics: σpreferred(AF) = {{lp}} implies
accepting {lp} is preferred over accepting {}, while σpreferred(AF′) = {{}} implies accepting {} is
preferred over accepting {lp}. Let us now formally put argumentation-based decision functions in the context of economic ratio-
nality. By considering Definition 3.2, we define the clear preferences principle for argumentation-
based decision-making. DEFINITION 5.3
(Clear preferences for argumentation-based decision-making). Let g ◦σ be an agent’s argumentation-based decision function. Given any argumentation framework
AF = (AR, Attacks), it follows from A∗= g ◦σ(AF) that the agent has established the following
preference order: ∀A ∈2AR, A∗⪰A. Let g ◦σ be an agent’s argumentation-based decision function. Given any argumentation framework
AF = (AR, Attacks), it follows from A∗= g ◦σ(AF) that the agent has established the following
preference order: ∀A ∈2AR, A∗⪰A. In words, the agent’s decision A∗from 2AR means that the agent prefers A∗over any of the other
sets in 2AR. The clear preferences principle is obvious when evaluating one-off argumentation-based decision-
making. However, when considering an argumentation process or dialogue, during which arguments
and attacks are added to an argumentation framework over time, it is clear that we need to consider
the reference independence property as introduced in Definition 3.3. We prove that, analogous to
the economic principles, reference independence is implied by the clear preferences property in the
context of argumentation-based decision-making. ION 5.4 PROPOSITION 5.4
(Reference independence for argumentation-based decision-making). Let AF = (AR, Attacks) and AF′ = (AR′, Attacks′) be two argumentation frameworks for which it
holds true that AR ⊆AR′ and let g◦σ be an argumentation-based decision function. An economically
rational argumentation-based decision A∗= g ◦σ(AF) implies that if g ◦σ(AF′) ⊆AR, then
g ◦σ(AF′) = g ◦σ(AF). nce independence for argumentation-based decision-making). (Reference independence for argumentation-based decision-making). EXAMPLE 5.2 Let AF = (AR, Attacks) and AF′ = (AR′, Attacks′) be two argumentation frameworks for which it
holds true that AR ⊆AR′ and let g◦σ be an argumentation-based decision function. An economically
rational argumentation-based decision A∗= g ◦σ(AF) implies that if g ◦σ(AF′) ⊆AR, then
g ◦σ(AF′) = g ◦σ(AF). Let AF = (AR, Attacks) and AF′ = (AR′, Attacks′) be two argumentation frameworks for which it
holds true that AR ⊆AR′ and let g◦σ be an argumentation-based decision function. An economically
rational argumentation-based decision A∗= g ◦σ(AF) implies that if g ◦σ(AF′) ⊆AR, then
g ◦σ(AF′) = g ◦σ(AF). PROOF. We provide a proof by contradiction. Let us suppose that g ◦σ(AF) ̸= g ◦σ(AF′). It follows
that g ◦σ(AF) ̸⊆g ◦σ(AF′) ∨g ◦σ(AF′) ̸⊆g ◦σ(AF). 1. If g ◦σ(AF) ̸⊆g ◦σ(AF′), then ∃a ∈g ◦σ(AF), such that a ̸∈g ◦σ(AF′). It follows that
i) By definition of g ◦σ(AF), ∃A∗∈2AR, such that ∀A ∈2AR, A∗⪰A ∧a ∈A∗,
ii) By definition of g ◦σ(AF′), ∃A′∗∈2AR such that ∀A ∈2AR, A′∗⪰A ∧a ̸∈A′∗. ⇒Because A′∗̸= A∗, (i) contradicts (ii). 1. If g ◦σ(AF) ̸⊆g ◦σ(AF′), then ∃a ∈g ◦σ(AF), such that a ̸∈g ◦σ(AF′). It follows that
AR
AR i) By definition of g ◦σ(AF), ∃A∗∈2AR, such that ∀A ∈2AR, A∗⪰A ∧a ∈A∗, ii) By definition of g ◦σ(AF′), ∃A′∗∈2AR such that ∀A ∈2AR, A′∗⪰A ∧a ̸∈A′∗. ⇒Because A′∗̸= A∗, (i) contradicts (ii). Abstract argumentation and the rational man 671 2. If g ◦σ(AF′) ̸⊆g ◦σ(AF), then ∃a ∈g ◦σ(AF′) such that a ̸∈g ◦σ(AF). It follows that:
i) By definition of g ◦σ(AF), ∃A∗∈2AR, such that ∀A ∈2AR, A∗⪰A ∧a ̸∈A∗,
ii) By definition of g ◦σ(AF′), ∃A′∗∈2AR, such that ∀A ∈2AR, A′∗⪰A ∧a ∈A′∗. ⇒Because A′∗̸= A∗, (i) contradicts (ii). 2. If g ◦σ(AF′) ̸⊆g ◦σ(AF), then ∃a ∈g ◦σ(AF′) such that a ̸∈g ◦σ(AF). It follows that:
i) By definition of g ◦σ(AF), ∃A∗∈2AR, such that ∀A ∈2AR, A∗⪰A ∧a ̸∈A∗,
ii) By definition of g ◦σ(AF′), ∃A′∗∈2AR, such that ∀A ∈2AR, A′∗⪰A ∧a ∈A′∗. ⇒Because A′∗̸= A∗, (i) contradicts (ii). □ In words, given a decision A∗from 2AR that implies ∀A ∈2AR, A∗⪰A, no decision A′ from
2AR′ with AR′ ⊇AR should change the preferences implied by A∗. EXAMPLE 5.2 Colloquially speaking, the
proof is the ‘select a subset of a set’-equivalent to the economics proof that clear preferences imply
consistent preferences for ‘select an item from a set’-scenarios, as e.g. presented by Rubinstein [34,
p. 11]. Note that in the proof, attack relations are irrelevant. However, it can be assumed that given
two argumentation frameworks AF = (AR, Attacks) and AF′ = (AR′, Attacks′), with AF′ being
an expansion, but not a normal expansion of AF, the reference independence property does not
need to hold, because the knowledge that is modelled in the ‘original’ argumentation framework
AF has changed. In contrast, if AF ⪯N AF′, the relationships between arguments in AR have not
changed, which means that a rational decision-maker should maintain the previously established
preference order on the items in the powerset of AR given that no ‘new’ arguments are considered
valid conclusions. EXAMPLE 6.2 EXAMPLE 6.2
We have the following argumentation frameworks: 1. AF = (AR, Attacks) = ({a, b}, {(a, b)}) Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 2. AF′ = (AR′, Attacks′) = ({a, b, c}, {(a, b), (b, c), (c, a)})
Note that AF ⪯N AF′. Now, we apply complete semantics σcomplete to both framew 2. AF′ = (AR′, Attacks′) = ({a, b, c}, {(a, b), (b, c), (c, a)}) Note that AF ⪯N AF′. Now, we apply complete semantics σcomplete to both framework Note that AF ⪯N AF′. Now, we apply complete semantics σco 1. g∩◦σcomplete(AF′) = {a}, which implies ∀Args ∈2{a,b}, {a} ⪰Args. { b } 2. g∩◦σcomplete(AF′) = {}, which implies ∀Args ∈2{a,b,c}, {} ⪰Args. 2. g∩◦σcomplete(AF′) = {}, which implies ∀Args ∈2{a,b,c}, {} ⪰Args. g∩◦σcomplete(AF′) ⊂AR and g∩◦σcomplete(AF′) ̸= g∩◦σcomplete(AF); i.e. the decisions of {a}
given {a, b} and {} given {a, b, c} are economically not rational, because the preference orders they
imply are inconsistent: g∩◦σcomplete(AF) implies {a} ⪰{}, whereas g∩◦σcomplete(AF′) implies {} ⪰
{a}. Hence, it is clear that the argumentation semantics do not satisfy the reference independence
principle. 2. g
◦σcomplete(AF ) = {}, which implies ∀Args ∈2{ , , }, {} ⪰Args. g∩◦σcomplete(AF′) ⊂AR and g∩◦σcomplete(AF′) ̸= g∩◦σcomplete(AF); i.e. the decisions of {a}
given {a, b} and {} given {a, b, c} are economically not rational, because the preference orders they
imply are inconsistent: g∩◦σcomplete(AF) implies {a} ⪰{}, whereas g∩◦σcomplete(AF′) implies {} ⪰
{a}. Hence, it is clear that the argumentation semantics do not satisfy the reference independence
principle. Let us again highlight that the principle applies by definition only to normal expansions of a
framework (AF ⪯N AF′). We introduced this restriction because any expansion AF′ of AF =
(AR, Attacks) with AF
̸⪯N
AF′ that adds attacks between arguments of AF is revising the
assumptions about AF, i.e. the ceteris paribus assumption of economic rationality does not hold
true. Let us now introduce the reference independence principle for argumentation seman 6
An argumentation principle for the rational man From the rational man properties that we have established in the context of argumentation-based
decision functions, we derive a principle that an argumentation semantics σ needs to satisfy to
guarantee rational argumentation-based decision-making, given a decision function g ◦σ. Ideally,
the argumentation semantics σ of an argumentation-based choice function g ◦σ is uniquely defined
(|σ(AF)| = 1 for any argumentation framework AF) and hence satisfies the clear preferences
principle of the rational economic man, and the aggregation function g merely needs to flatten
the set σ returns. An overview of semantics that fulfil this principle is provided in Appendix
C, based on an analysis by Baumann [8]. Let us note that the clear preferences principle only
implies clear preferences in one-off decision-making i.e. it is self-evident that (i) argumentation
semantics that always return exactly one extension do not necessarily return extensions that imply
consistent preference relations when resolving a sequence of normally expanding argumentation
frameworks and (ii) argumentation semantics that may return multiple extensions may allow to
‘pick’ an extension that implies consistent preference relations when resolving a normally expanding
sequence. The focus of this paper is on exactly this problem, which we analyse using the reference
independence principle. DEFINITION 6.1
(Reference independence in argumentation-based decision functions). An argumentation-based decision function g ◦σ satisfies the reference independence principle iff
for every two argumentation frameworks AF = (AR, Attacks) and AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, the following implication holds true, given an argumentation-based decision function
g ◦σ: (Reference independence in argumentation-based decision functions). An argumentation-based decision function g ◦σ satisfies the reference independence principle iff
for every two argumentation frameworks AF = (AR, Attacks) and AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, the following implication holds true, given an argumentation-based decision function If
g ◦σ(AF′) ⊆AR
then
g ◦σ(AF′) = g ◦σ(AF). To illustrate the principle, let us introduce an example. 672
Abstract argumentation and the rational man 672 EXAMPLE 6.2
We have the following argumentation frameworks: DEFINITION 6.3 (Reference independence principle for argumentation semantics). (Reference independence principle for argumentation semantics). Let σ be an argumentation semantics. σ satisfies: Let σ be an argumentation semantics. σ satisfies: •
Strong reference independence: iff σ is universally defined and for every two argumentation
frameworks AF = (AR, Attacks) and AF′ = (AR′, Attacks′), such that AF ⪯N AF′, ∀E ∈
σ(AF), ∀E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E. •
Weak reference independence: iff for every two argumentation frameworks AF
=
(AR, Attacks) and AF′ = (AR′, Attacks′), such that AF ⪯N AF′, ∀E ∈σ(AF), it holds
true that ∃E′ ∈σ(AF′), such that E′ ̸⊆AR ∨E′ = E. It is clear that strong reference independence cannot be guaranteed by a ‘reasonable’ abstract
argumentation semantics. To illustrate this, let us claim that σ(({a}, {})) = {{a}}, σ(({b}, {})) =
{{b}} are ‘reasonable’ assumptions about the behaviour of an argumentation semantics. Let us now
consider the frameworks AF = (AR, Attacks), such that AF = ({a, b}, {(a, b), (b, a)}) and AF′ =
(AR′, Attacks′), such that AF′ = ({a, b, c}, {(a, b), (b, a), (b, c), (c, b), (c, a), (a, c)}) (note that AF ⪯N AF′). One can see that σ(AF) needs to return {{a}, {b}}
(if it returned the empty set, we could not achieve weak reference independence when expanding
from an argumentation framework containing only a (({a}, {})) or ({b}, {})), whereas σ(AF′) needs to
return {{a}, {b}, {c}} to be weakly reference independent. However, σ(AF′) is not strongly reference
independent because ∀E ∈σ(AF) it does not hold true that ∀E′ ∈σ(AF′), (E′ ̸⊆AR ∨E′ = E): •
Let E be {b} and let E′ be {a}. Note that {b} ∈σ(AF) and {a} ∈σ(AF′). {a} ⊆AR∧{a} ̸= {b}. •
Let E be {a} and let E′ be {b}. Note that {a} ∈σ(AF) and {b} ∈σ(AF′). {b} ⊆AR∧{b} ̸= {a}. In contrast, we show further below that weak reference independence can be satisfied by an argu-
mentation semantics; hence, it is the more relevant property in the context of abstract argumentation Abstract argumentation and the rational man 673 and we may refer to it as reference independence without any further qualifier. DEFINITION 6.3 It is important to
highlight that the strong reference independence principle is still a crucial building block for this
paper, as it can be derived in a more obvious manner from the reference independence property of
economic rationality; showing that relaxing this principle is necessary to motivate the weak reference
independence principle. Let us colloquially summarize how we have transformed a formal model of
an economically rational decision-maker (‘rational economic man’) into the abstract argumentation
principle of weak reference independence. 1. Rational economic man selects a subset of a set of items, which are, in the case of abstract
argumentation, arguments in an argumentation framework. 1. Rational economic man selects a subset of a set of items, which are, in the case of abstract
argumentation, arguments in an argumentation framework. 2. The selection implies a preference order (partial order) on the powerset of items/arguments:
the selected set is preferred over all other items in the powerset. 2. The selection implies a preference order (partial order) on the powerset of items/arguments:
the selected set is preferred over all other items in the powerset. 3. When adding items to the set, the preference order on the powerset of items remains consistent
as long as the properties of the initial items do not change. Consequently, in an argumentation
scenario, when expanding an argumentation framework, the preference order on the powerset
of arguments remains consistent as long as the relations among the initial arguments remain
unchanged, i.e. as long as the expansion is normal. Let us highlight that attacks from ‘new’
to ‘initial’ arguments must not affect the preference order as long as no ‘new’ argument is
considered a valid conclusion and if a ‘new’ argument is considered a valid conclusion (is part
of the extension) the new preference order is by definition consistent with the initial preference
order. FIGURE 4 Monotony implies reference independence, rational monotony and cautious monotony
(given any semantics that satisfies maximal conflict-freeness or maximal admissibility). Rational
monotony typically implies cautious monotony (see Section 4). Weak rational monotony implies
weak reference independence; however, rational monotony is violated by all non-naive argumenta-
tion semantics that are evaluated in this paper. We conclude that only monotony and (in the weak case) rational monotony, which are too strict
for common-sense reasoning approaches, imply reference independence. Hence, we can conclude
that reference independence is novel and useful as a property for analysing abstract argumentation
approaches from the perspective of economic rationality. Let us motivate our decision to require semantics that are based on maximal conflict-free sets
or maximal admissible sets in our proofs instead of merely requiring conflict-freeness as the only
principle that needs to be satisfied. Let AF
=
({a}, {}) and AF′
=
({a, b}, {}) be argumentation frameworks and let σ be an
argumentation semantics such that σ(AF) = {{}} and σ(AF′) = {{a}}. While σ returns conflict-
free (and also admissible) sets for both argumentation frameworks, the behaviour of σ is clearly
flawed from a common-sense perspective: the structure of the argumentation framework provides
no reason to consider a but not b part of a valid conclusion. For semantics with such behaviour, the
implication relationship between monotony and reference independence that we are demonstrating
below does not hold true. Introducing a new property that is weaker than maximal conflict-freeness and maximal admissi-
bility, yet does not allow for semantics with ‘unreasonable’ behaviour as shown in the example can
be considered relevant future work but is not in the scope of this paper. The proofs of the observations
regarding weak monotony properties and reference independence follow below. For the sake of
conciseness, the corresponding proofs regarding strong monotony and reference independence,
which are analogous to the proofs regarding the corresponding weak properties, are available in
Appendix A. In Appendix B, we show that it holds true for maximal admissible set-based semantics
that strong monotony implies strong reference independence and weak monotony implies weak
reference independence. Let us first prove that weak reference independence does not imply weak monotony. 6.1 Reference independence and monotony Considering that abstract argumentation is a method for non-monotonic reasoning, and given that
the reference independence property may seem—at first glance—to imply monotony, let us provide
an intuition for distinguishing between reference independence and the different forms of monotony
as defined in Section 4 in the context of abstract argumentation. In particular, we provide proofs that
show the following, given any argumentation semantics whose extensions are maximal conflict-free
sets, and in case an implication relationship holds, also given any argumentation semantics whose
extensions are maximal admissible sets: •
Strong monotony implies strong reference independence and weak monotony implies weak
reference independence but not vice versa. •
Strong monotony implies strong reference independence and weak monotony implies weak
reference independence but not vice versa. •
Strong rational monotony does not imply strong reference independence and vice versa. Weak
rational monotony implies weak reference independence but not vice versa. We observe that
in contrast to weak reference independence, weak rational monotony is not satisfied by any
non-naive semantics that is evaluated in this paper; i.e. weak rational monotony is too strict to
be useful in the context of abstract argumentation. •
Strong/weak cautious monotony does not imply strong/weak reference independence and vice
versa. •
Strong/weak cautious monotony does not imply strong/weak reference independence and vice
versa. By definition, strong monotony implies strong rational monotony and strong cautious monotony,
and weak monotony implies weak rational monotony and weak cautious monotony. We have
observed that typically, strong/weak rational monotony implies strong/weak cautious monotony
(see Section 4). Figure 4 visualizes the relations between monotony, cautious monotony, rational
monotony and reference independence. 674
Abstract argumentation and the rational man PROOF. Let us provide a proof by counter-example. s provide a proof by counter-example. 1. Let AF = (AR, Attacks) = ({a}, {}), AF′ = ({a, b}, {(b, a)}) and let us take CF2 semantics,
denoted by σcf 2, as an example of a semantics whose extensions are maximal conflict-free
sets. Note that AF ⪯N AF′. 1. Let AF = (AR, Attacks) = ({a}, {}), AF′ = ({a, b}, {(b, a)}) and let us take CF2 semantics,
denoted by σcf 2, as an example of a semantics whose extensions are maximal conflict-free
sets. Note that AF ⪯N AF′. ⪯N
2. σcf 2(AF) = {{a}} and σcf 2(AF′) = {{b}}. Let E = {a}, E ∈σcf 2(AF). ∀E′ ∈σ(AF′) it holds
true that E ̸⊆E′. The condition for weak monotony does not hold. 2. σcf 2(AF) = {{a}} and σcf 2(AF′) = {{b}}. Let E = {a}, E ∈σcf 2(AF). ∀E′ ∈σ(AF′) it holds
true that E ̸⊆E′. The condition for weak monotony does not hold. 3. ∃E′ ∈σ(AF′) such that E′ ̸⊆AR. The condition for weak reference independence holds. We
have proven the proposition. □ 3. ∃E′ ∈σ(AF′) such that E′ ̸⊆AR. The condition for weak reference independence holds. We
have proven the proposition. □ The proof below shows that weak monotony implies weak reference independence. The intuition
is that given any maximal conflict-free set S of an argumentation framework AF = (AR, Attacks),
adding arguments (and attacks) to the argumentation framework (without changing attacks between
existing arguments) will never cause any argument in AR \ S to be conflict-free w.r.t. S. This means
it is not possible to ‘add’ arguments within AR to a maximal conflict-free set by normally expanding
AF, which in turn means that to violate reference independence we must in our expansion of AF
successfully attack arguments that are accepted in AF, which then violates monotony. PROPOSITION 6.6 (∀E ∈σ(AF), ∃E′ ∈σ(AF′),
such that
E′ ̸⊆AR ∨E′ = E) (∀E ∈σ(AF), ∃E′ ∈σ(AF′),
such that
E′ ̸⊆AR ∨E′ = E) PROPOSITION 6.5 (Weak reference independence does not imply weak monotony). (Weak reference independence does not imply weak monotony). There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that 675 Abstract argumentation and the rational man AF ⪯N AF′, such that the following statement does not hold true: If
(∀E ∈σ(AF), ∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR ∨E′ = E))
then
(∀E ∈σ(AF), ∃E′ ∈σ(AF′),
such that
E ⊆E′). PROOF. Let us provide a proof by counter-example. 2. (i) E = E′ or PROPOSITION 6.6 (Weak monotony implies weak reference independence). For every argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set
inclusion) and every two argumentation frameworks AF = (AR, Attacks), AF′ = (AR′,
Attacks′), such that AF ⪯N AF′, the following statement holds true: For every argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set
inclusion) and every two argumentation frameworks AF = (AR, Attacks), AF′ = (AR′,
Attacks′), such that AF ⪯N AF′, the following statement holds true: If
(∀E ∈σ(AF), ∃E′ ∈σ(AF′),
such that
E ⊆E′)
then
(∀E ∈σ(AF), ∃E′ ∈σ(AF′),
such that
E′ ̸⊆AR ∨E′ = PROOF. PROOF. 1. Given σ such that each all σ-extensions are maximal conflict-free sets (w.r.t. set inclusion),
∀E ∈σ(AF), E′ ∈σ(AF′), E and E′ are maximal conflict-free (w.r.t. set inclusion). It follows
that, if E ⊆E′, we have the following cases: 2. (i) E = E′ or 3. (ii) E ⊂E′, which implies that ∃a ∈E′, such that a ̸∈E, a ∈AR′ \ AR, from which it follows
that E′ ̸⊆AR. 4. Consequently, by (i) and (ii) the following statement holds true: ∀E ∈σ(AF), ∀E′ ∈σ(AF′),
if
E ⊆E′
then
E′ ̸⊆AR ∨E′ = E. Hence, the proposition (which is implied by the proven statement) holds true. Hence, the proposition (which is implied by the proven statement) holds true. □ We can prove that weak cautious monotony does not imply weak reference independence. We can prove that weak cautious monotony does not imply weak reference independence. Abstract argumentation and the rational man 676 PROPOSITION 6.7 However, it does not hold true that ∀E ∈σ(AF), ∃E′ ∈σ(AF′), such that (E′ ̸⊆AR ∨ 4. However, it does not hold true that ∀E ∈σ(AF), ∃E′ ∈σ(AF′), such that (E′ ̸⊆AR ∨E′ = E). We have proven the proposition. □ In a similar manner, we can prove that weak reference independence does not imply weak cautious
monotony. In a similar manner, we can prove that weak reference independence does not imply weak cautious
monotony. PROPOSITION 6.8 PROPOSITION 6.7 (Weak cautious monotony does not imply weak reference independence). There exists an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′ and the following statement does not hold true: Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 ∀E ∈σ(AF),
If
({(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅
implies
∃E′ ∈σ(AF′)
such that
E ⊆E′)
then
(∃E′ ∈σ(AF′)
such that
E′ ̸⊆AR ∨E′ = E)). ∀E ∈σ(AF),
If
({(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅
implies
∃E′ ∈σ(AF′)
such that
E ⊆E′)
then
(∃E′ ∈σ(AF′)
such that
E′ ̸⊆AR ∨E′ = E)). PROOF. Let us provide a proof by counter-example. 1. Let AF = (AR, Attacks) = ({c, d}, {(c, d), (d, c)}), AF′ = ({c, d, e}, {(c, d), (d, c), (d, e),
(e, c), (e, e)}) and let us take stage semantics, denoted by σstage, as an example of a semantics
whose extensions are maximal conflict-free sets. Note that AF ⪯N AF′. 1. Let AF = (AR, Attacks) = ({c, d}, {(c, d), (d, c)}), AF = ({c, d, e}, {(c, d), (d, c), (d, e),
(e, c), (e, e)}) and let us take stage semantics, denoted by σstage, as an example of a semantics
whose extensions are maximal conflict-free sets. Note that AF ⪯N AF′. 2. σstage(AF) = {{c}, {d}} and σstage(AF′) = {{d}}. stage(
)
{{ }, { }}
stage(
)
{{ }}
3. Consequently, given E = {c}, {(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅is false and
it holds true that ∀E ∈σ(AF), ({(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅implies
∃E′ ∈σ(AF′)
such that
E ⊆E′). g
g
3. Consequently, given E = {c}, {(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅is false and
it holds true that ∀E ∈σ(AF), ({(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅implies
∃E′ ∈σ(AF′)
such that
E ⊆E′). (
)
⊆
)
4. However, it does not hold true that ∀E ∈σ(AF), ∃E′ ∈σ(AF′), such that (E′ ̸⊆AR ∨E′ = E). We have proven the proposition. □ 4. 2. σstage(AF) = {{c}, {d}, {e}} and σstage(AF′) = {{e, f }}. PROPOSITION 6.8 Let us now prove that weak rational monotony implies weak reference independence. PROPOSITION 6.8 (Weak reference independence does not imply weak cautious monotony). There exists an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′) such that
AF ⪯N AF′ and the following statement does not hold true: ∀E ∈σ(AF),
(if
(∃E′ ∈σ(AF′),
such that
E′ ̸⊆AR ∨E′ = E)
then
({(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅
implies
∃E′ ∈σ(AF′),
such that
E ⊆E′). PROOF. We provide the following proof by counter-example. PROOF. We provide the following proof by counter-example. 1. Let AF = (AR, Attacks) = ({c, d, e}, {(c, d), (d, e), (e, c)}),
AF′ = ({c, d, e, f }, {(c, d), (d, e), (e, c), (f , d)}) and let us take stage semantics, denoted by
σstage, as an example of a semantics whose extensions are maximal conflict-free sets. Note that AF ⪯N AF′. 2. σstage(AF) = {{c}, {d}, {e}} and σstage(AF′) = {{e, f }}. 2. σstage(AF) = {{c}, {d}, {e}} and σstage(AF′) = {{e, f }}. Abstract argumentation and the rational man 677 FIGURE 5 Example: weak reference independence does not imply weak cautious monotony. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 FIGURE 5 Example: weak reference independence does not imply weak cautious monotony. 3. Given the extension E ∈σstage(AF), E = {c}, it holds true that {(a, b) | (a, b) ∈Attacks′, a ∈
AR′ \ AR, b ∈E} = ∅. 4. Given E′ = {e, f } as the only extension in σ(AF′) it holds true that E′ ̸⊆AR; the result does
not violate the weak reference independence property. 5. However, given E′ = {e, f } as the only extension in σ(AF′) and given E = {c}, E ∈σstage(AF),
we have E ̸⊆E′, which violates weak cautious monotony. Hence, we have proven the
proposition. □ 5. However, given E′ = {e, f } as the only extension in σ(AF′) and given E = {c}, E ∈σstage(AF),
we have E ̸⊆E′, which violates weak cautious monotony. Hence, we have proven the
proposition. □ Figure 5 depicts the frameworks used in the proof of Proposition 6.8. Figure 5 depicts the frameworks used in the proof of Proposition 6.8. Let us now prove that weak rational monotony implies weak reference independence. PROPOSITION 6.9 (Weak rational monotony implies weak reference independence). (Weak rational monotony implies weak reference independence). For every argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set
inclusion) and every two argumentation frameworks AF = (AR, Attacks), AF′ = (AR′,
Attacks′), such that AF ⪯N AF′, the following statement holds true: h that AF ⪯N AF′, the following statement holds true: Attacks′), such that AF ⪯N AF′, the following statement holds true: ∀E ∈σ(AF), ∀E ∈σ(AF), ∀E ∈σ(AF), (If
({(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅
implies
∃E′ ∈σ(AF′),
such that
E ⊆E′)
then (∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR ∨E′ = E))), (∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR ∨E′ = E))), (∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR ∨E′ = E))), where UE′
new =
E′∈σ(AF′)(E′ \ AR). PROOF. Given any E ∈σ(AF), we have two cases for which the weak rational monotony condition
is satisfied. Case 1:
{(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} ̸= ∅: If {(a, b) | (a, b) ∈Attacks′, a ∈
UE′
new, b ∈E} ̸= ∅, then ∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR). Consequently, weak
reference independence is satisfied. Case 1:
{(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} ̸= ∅: If {(a, b) | (a, b) ∈Attacks′, a ∈
UE′
new, b ∈E} ̸= ∅, then ∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR). Consequently, weak
reference independence is satisfied. PROPOSITION 6.10 (Weak reference independence does not imply weak rational monotony). There exists an argumentation semantics σ whose extensions are maximal conflict-free sets and
argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that AF ⪯N AF′, such
that the following statement does not hold true: ∀E ∈σ(AF),
(if
(∃E′ ∈σ(AF′), such that
E′ ̸⊆AR ∨E′ = E))
then
({(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅
implies
∃E′ ∈σ(AF′),
such that
E ⊆E′)), ({(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅
implies
∃E′ ∈σ(AF′),
such that
E ⊆E′)), where UE′
new =
E′∈σ(AF′)(E′ \ AR). where UE′
new =
E′∈σ(AF′)(E′ \ AR). PROOF. Let us provide a proof by counter-example. PROOF. Let us provide a proof by counter-example. 1. Let AF = (AR, Attacks) = ({c, d, e}, {(c, d), (d, e), (e, c)}),
AF′
= ({c, d, e, f }, {(c, d), (d, e), (e, c), (f , d)}) and let us take stage semantics, denoted
by σstage, as an example of a semantics whose extensions are maximal conflict-free sets. Note that AF ⪯N AF′. 2. σstage(AF) = {{c}, {d}, {e}} and σstage(AF′) = {{e, f }}. ′
′ 3. Given E′ = {e, f } as the only extension in σ(AF′) it holds true that E′ ̸⊆AR; the result does
not violate the weak reference independence property. p
p
p
y
4. However, given E′ = {e, f } as the only extension in σ(AF′) and given E = {c}, E ∈σstage(AF),
it holds true that {(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅and E ̸⊆E′, which violates
weak rational monotony. Hence, we have proven the proposition. □ □ 8This example can also be applied to the weakly admissible set-based semantics as introduced by Baumann et al. [10] to
observe that these semantics violate weak rational monotony. Case 2: Case 2:
{(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅and ∃E′ ∈σ(AF′),
such that
E ⊆
E′: Because it holds true that ∃E′ ∈σ(AF′),
such that
E ⊆E′ and because all
σ-extensions are maximal conflict-free sets (w.r.t. set inclusion), from the proof of
Proposition 6.6 it follows that weak reference independence is satisfied. We have proven
the proposition. □ 678 Abstract argumentation and the rational man The proof that weak rational monotony implies weak reference independence in the case of
maximal admissible set-based semantics is provided in the Appendix. However, let us informally
observe that weak rational monotony is violated by all semantics that are analysed in this paper, with
the exception of naive semantics. For this, we merely need to consider the following examples: Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 •
Complete, grounded, preferred, stable, ideal, semi-stable, eager: AF1 = ({a}, {}), AF′
1 =
({a b c} {(a b) (b c) (c a)}) AF1 ⪯N AF′ 8 ; •
Complete, grounded, preferred, stable, ideal, s
({a, b, c}, {(a, b), (b, c), (c, a)}), AF1 ⪯N AF′
1
8 ; ({a, b, c}, {(a, b), (b, c), (c, a)}), AF1 ⪯N AF′
1
8 ; 1
•
Stage, CF2, Stage2: AF2 = ({a, b, c}, {(a, b), •
Stage, CF2, Stage2: AF2 = ({a, b, c}, {(a, b),
(b, c), (c, a)}), AF′
2 = ({a, b, c, d}, {(a, b), (b, c), (c, a), (d, c)}), AF2 ⪯N AF′
2. •
Stage, CF2, Stage2: AF2
({a, b, c}, {(a, b),
(b, c), (c, a)}), AF′
2 = ({a, b, c, d}, {(a, b), (b, c), (c, a), (d, c)}), AF2 ⪯N AF′
2. Now, let us prove that weak reference independence does not imply weak rational m PROPOSITION 6.10 DEFINITION 6.13 (SCC-recursiveness [5]). •
if |SCSS(AF)| = 1, E ∈BF(AR, C); •
if |SCSS(AF)| = 1, E ∈BF(AR, C); •
otherwise, ∀S ∈SCSS(AF), (E ∩S) ∈GF(AF ↓UPAF(S,E), UAF(S, E) ∩C), where BF(AF, C) is a function that, given an argumentation framework AF = (AR, Attacks), such
that |SCSS(AF)| = 1 and a set C ⊆AR, returns a subset of 2A. To show that weak reference independence does not imply directionality and SCC-recursiveness,
let us prove that naive semantics, which does not satisfy directionality and SCC-recursiveness [40],
satisfies the weak reference independence principle. PROPOSITION 6.14 Let σnaive be naive argumentation semantics. For every AF = (AR, Attacks), AF′ = (AR′, Attacks′),
such that AF ⪯N AF′, ∀E ∈σnaive(AF), the following statement holds true: 6.2 Weak reference independence, directionality and SCC-recursiveness Two argumentation principles that have received much attention because, colloquially speaking, they
reflect the intuitive idea to traverse an argumentation graph in a ‘top-down’ manner are directionality
and SCC-recursiveness [4]. Let us highlight (and show by formal analysis) that weak reference 8This example can also be applied to the weakly admissible set-based semantics as introduced by Baumann et al. [10] to
observe that these semantics violate weak rational monotony. Abstract argumentation and the rational man
679 Abstract argumentation and the rational man
679 independence does not imply directionality and SCC-recursiveness (and vice versa). Let us first
provide the definition of unattacked sets as a preliminary. independence does not imply directionality and SCC-recursiveness (and vice versa). Let us first
provide the definition of unattacked sets as a preliminary. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 DEFINITION 6.11
(Unattacked sets [4]). Let AF = (AR, Attacks) be an argumentation framework. A set S ⊆AR is unattacked iff ∄a ∈AR\S
such that a attacks S. US(AF) denotes all unattacked sets in AF. DEFINITION 6.11
(Unattacked sets [4]). Let AF = (AR, Attacks) be an argumentation framework. A set S ⊆AR is unattacked iff ∄a ∈AR\S
such that a attacks S. US(AF) denotes all unattacked sets in AF. s provide the definition of the directionality principle. Now, let us provide the definition of the directionality principle. Now, let us provide the definition of the directionality principle. DEFINITION 6.12
(Directionality [4]). An argumentation semantics σ is directional iff for every argumentation framework AF
=
(AR, Attacks), for every unattacked set of arguments U ⊆AR it holds true that σ(AF ↓U) =
{E ∩U|E ∈σ(AF)}. Let us now introduce the SCC-recursiveness principle, on which semantics like CF2 and stage2
are based. Let us now introduce the SCC-recursiveness principle, on which semantics like CF2 and stage2
are based. DEFINITION 6.13
(SCC-recursiveness [5]). Let σ be an argumentation semantics. σ is SCC-recursive iff σ(AF) = GF(AF, Attacks), where for
every AF = (AR, Attacks), C ⊆AF, GF(AF, C) ⊆2AR is defined as follows. For every E ⊆AR, E ∈
GF(AF, C) iff DEFINITION 6.13
(SCC-recursiveness [5]). Let σ be an argumentation semantics. σ is SCC-recursive iff σ(AF) = GF(AF, Attacks), where for
every AF = (AR, Attacks), C ⊆AF, GF(AF, C) ⊆2AR is defined as follows. For every E ⊆AR, E ∈
GF(AF, C) iff DEFINITION 6.13
(SCC-recursiveness [5]). 6.2 Weak reference independence, directionality and SCC-recursiveness Let σ be an argumentation semantics. σ is SCC-recursive iff σ(AF) = GF(AF, Attacks), where for
every AF = (AR, Attacks), C ⊆AF, GF(AF, C) ⊆2AR is defined as follows. For every E ⊆AR, E ∈
GF(AF, C) iff We have proven the proposition. □ From our analysis in the next section, it follows that directionality and SCC-recursiveness
do not imply weak reference independence: the directional and SCC-recursive stage2 semantics
do not satisfy weak reference independence. However, the directional and SCC-recursive CF2
semantics satisfy weak reference independence, i.e. SCC-recursiveness and directionality do not
imply violation of weak directionality. PROPOSITION 7.1
y Let σ y
x be an argumentation semantics, such that x ∈{complete, stable, grounded, preferred,
ideal, semi-stable, eager} and y ∈{∩, i}. There exist argumentation frameworks AF = (AR, Attacks)
and AF′ = (AR′, Attacks′), such that AF ⪯N AF′, for which the following statement does not hold Let σ y
x be an argumentation semantics, such that x ∈{complete, stable, grounded, preferred,
ideal semi-stable eager} and y ∈{∩i} There exist argumentation frameworks AF = (AR Attack Let σ y
x be an argumentation semantics, such that x ∈{complete, stable, grounded, prefe ideal, semi-stable, eager} and y ∈{∩, i}. There exist argumentation frameworks AF = (AR, Attacks)
and AF′ = (AR′, Attacks′), such that AF ⪯N AF′, for which the following statement does not hold
true: 7
Reference dependence in abstract argumentation Let us show violation of the weak reference independence principle of complete, stable preferred,
semi-stable, as well as grounded, ideal and eager semantics (credulous and skeptical). These
observations are formalized by the following proposition. Note that for the sake of providing more
concise proposition and proofs, we denote credulous semantics (typically denoted by σ) by σ i, with
i being the identity function, i.e. σ i(AF) = σ(AF) for every argumentation framework AF. ∃E′ ∈σnaive(AF′),
such that
E′ ̸⊆AR ∨E′ = E. ∃E′ ∈σnaive(AF′),
such that
E′ ̸⊆AR ∨E′ = E. PROOF. ∀E ∈σnaive(AF), E is a maximal conflict-free set w.r.t. set inclusion. σnaive(AF′) contains
all S ⊆AR′, such that S is a maximal conflict-free set w.r.t. set inclusion. It follows that because
AF ⪯AF′, we have two cases ∀E ∈σnaive(AF): PROOF. ∀E ∈σnaive(AF), E is a maximal conflict-free set w.r.t. set inclusion. σnaive(AF′) contains
all S ⊆AR′, such that S is a maximal conflict-free set w.r.t. set inclusion. It follows that because
AF ⪯AF′, we have two cases ∀E ∈σnaive(AF): 1. ∃a ∈AR′ \ AR, such that a does not attack E, E does not attack a, and a does not attack a. It
follows that E ∪{a} is a conflict-free set and hence it holds true that ∃E′ ∈σnaive(AF), such
that E′ ̸⊆AR. 1. ∃a ∈AR′ \ AR, such that a does not attack E, E does not attack a, and a does not attack a. It
follows that E ∪{a} is a conflict-free set and hence it holds true that ∃E′ ∈σnaive(AF), such
that E′ ̸⊆AR. 680 680
Abstract argumentation and the rational man 2. ∄a ∈AR′ \ AR, such that a does not attack E, E does not attack a and a does not attack
a. It follows that E is a maximal conflict-free (w.r.t. set inclusion) subset of AR′ and hence
∃E′ ∈σnaive(AF), such that E′ = E. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 ∃E′ ∈σnaive(AF), such that E′ = E. We have proven the proposition. ∀E ∈σ y
x (AF), (∃E′ ∈σ y
x (AF′),
such that
(E′ ̸⊆AR ∨E′ = E)). ∀E ∈σ y
x (AF), (∃E′ ∈σ y
x (AF′),
such that
(E′ ̸⊆AR ∨E′ = E)). PROOF. We provide a proof by counter-example. Let us introduce the following argumentation
frameworks: •
AF = (AR, Attacks) = ({a, b}, {(a, b)});
•
AF′ = (AR′, Attacks′) = ({a, b, c}, {(a, b), (b, c), (c, a)}). •
AF = (AR, Attacks) = ({a, b}, {(a, b)}); •
AF
(AR, Attacks)
({a, b}, {(a, b)});
•
AF′ = (AR′, Attacks′) = ({a, b, c}, {(a, b), (b, c), (c, a)}). We can see that AF ⪯N AF′. The argumentation frameworks are resolved as follows:
•
σ y(AF) = {{a}} We can see that AF ⪯N AF′. The argumentation frameworks are resolved as follows:
•
σ y
x (AF) = {{a}}. •
σ y
x (AF) = {{a}}. y •
σ y
x (AF) = {{a}}. y •
If x = stable, σ y
x (AF′) = {}; otherwise, σ y
x (AF′) = {{}}. Note that {} ⊆AR ∧{} ̸= {a}. We
have proven the proposition. □ The proof that skeptical stage, stage2 and CF2 semantics do not satisfy the weak reference
independence principle can be provided in the same way. Note that {} ⊆AR ∧{} ̸= {a}. We have proven the proposition. Note that the counter-example in the proof of Proposition 7.2 is analogous to the counter-example
in the proof of Proposition 7.1. In words, given AF, {a} is preferred over {} and given AF′, {} is
preferred over {a}. Hence, the semantics violate the reference independence principle; adding an
element c to the set of elements {a, b} can affect the preference relation an agent has on elements
in 2{a,b}. For credulous stage and stage2 semantics, another example proves violation of the weak
reference independence principle. PROPOSITION 7.2 We have proven the proposition. PROPOSITION 7.3 Let σ i
x be an argumentation semantics, such that x ∈{stage, stage2}. There exist argumentation
frameworks AF = (AR, Attacks) and AF′ = (AR′, Attacks′), such that AF ⪯N AF′, for which the
following statement does not hold true: PROPOSITION 7.2 PROPOSITION 7.2
Let σ ∩
x be an argumentation semantics, such that x ∈{stage, stage2, CF2}. There exist argumentation
frameworks AF = (AR, Attacks) and AF′ = (AR′, Attacks′), such that AF ⪯N AF′, for which the
following statement does not hold true: Let σ ∩
x be an argumentation semantics, such that x ∈{stage, stage2, CF2}. There exist argumentation
frameworks AF = (AR, Attacks) and AF′ = (AR′, Attacks′), such that AF ⪯N AF′, for which the
following statement does not hold true: ∀E ∈σ ∩
x (AF), (∃E′ ∈σ ∩
x (AF′), such that
(E′ ̸⊆AR ∨E′ = E)) ∀E ∈σ ∩
x (AF), (∃E′ ∈σ ∩
x (AF′), such that
(E′ ̸⊆AR ∨E′ = E)) ∀E ∈σ ∩
x (AF), (∃E′ ∈σ ∩
x (AF′), such that
(E′ ̸⊆AR ∨E′ = E)) PROOF. To provide a proof by counter-example, let us again introduce the following argumentation
frameworks: •
AF = (AR, Attacks) = ({a, b}, {(a, b)}); Abstract argumentation and the rational man 681 FIGURE 6 Inconsistent preferences: given the extensions σstage(AF) = {{a}, {b}}, an agent can
‘pick’ the extension {a}, which then implies the preference {a} ⪯{b}, while σstage(AF′) implies the
preference {b} ⪯{a}. FIGURE 6 Inconsistent preferences: given the extensions σstage(AF) = {{a}, {b}}, an agent can
‘pick’ the extension {a}, which then implies the preference {a} ⪯{b}, while σstage(AF′) implies the
preference {b} ⪯{a}. FIGURE 6 Inconsistent preferences: given the extensions σstage(AF) = {{a}, {b}}, an agent can
‘pick’ the extension {a}, which then implies the preference {a} ⪯{b}, while σstage(AF′) implies the
preference {b} ⪯{a}. FIGURE 6 Inconsistent preferences: given the extensions σstage(AF) = {{a}, {b}}, an agent can
‘pick’ the extension {a}, which then implies the preference {a} ⪯{b}, while σstage(AF′) implies the
preference {b} ⪯{a}. •
AF′ = (AR′, Attacks′) = ({a, b, c}, {(a, b), (b, c), (c, a)}). We can see that AF ⪯N AF′. The argumentation frameworks are resolved as follows: •
AF′ = (AR′, Attacks′) = ({a, b, c}, {(a, b), (b, c), (c, a)}). We can see that AF ⪯N AF′. The argumentation frameworks are resolved as follows: •
σ ∩
x (AF) = {{a}}. x
•
σ ∩
x (AF′) = {{}}. □ Note that {} ⊆AR ∧{} ̸= {a}. We have proven the proposition. Note that {} ⊆AR ∧{} ̸= {a}. We have proven the proposition. Note that {} ⊆AR ∧{} ̸= {a}. ∀E ∈σ i
x(AF), (∃E′ ∈σ i
x(AF′), such that
(E′ ̸⊆AR ∨E′ = E)). ∀E ∈σ i
x(AF), (∃E′ ∈σ i
x(AF′), such that
(E′ ̸⊆AR ∨E′ = E)). PROOF. To provide a proof by counter-example, let us again introduce the following argumentation
frameworks: PROOF. To provide a proof by counter-example, let us again introduce the following argumentation
frameworks: •
AF = (AR, Attacks) = ({a, b}, {(a, b), (b, a)}); AF = (AR, Attacks) = ({a, b}, {(a, b), (b, a)} (
,
)
({ , }, {( , ), ( , )});
•
AF′ = (AR′, Attacks′) = ({a, b, c}, {(a, b), (b, a), (b, c), (c, b), (c, c)}). •
AF′ = (AR′, Attacks′) = ({a, b, c}, {(a, b), (b, a), (b, c), (c, b), (c, c)}). We can see that AF ⪯N AF′. The argumentation frameworks are resolved as follows We can see that AF ⪯N AF′. The argumentation frameworks are resolved as follows: •
σ i
x(AF) = {{a}, {b}}. x
•
σ i
x(AF′) = {{b}}. □ Note that {b} ⊆AR ∧{b} ̸= {a}. We have proven the proposition. Note that {b} ⊆AR ∧{b} ̸= {a}. We have proven the proposition. □ Note that {b} ⊆AR ∧{b} ̸= {a}. We have proven the proposition. 6 shows the frameworks that the proof uses. Figure 6 shows the frameworks that the proof uses. Finally, let us prove that CF2 semantics is weakly reference independent. Note that we use the
notion of attack sequences (see Definition 3.10) in the proof. Finally, let us prove that CF2 semantics is weakly reference independent. Note that we use the
notion of attack sequences (see Definition 3.10) in the proof. 682
Abstract argumentation and the rational man 682 PROPOSITION 7.4
Let σCF2 be CF2 semantics. For every two argumentation frameworks AF = (AR, Attacks) and
AF′ = (AR′, Attacks′), such that AF ⪯N AF′, the following statement holds true: PROPOSITION 7.4
Let σCF2 be CF2 semantics. For every two argumentation frameworks AF = (AR, Attacks) and
AF′ = (AR′, Attacks′), such that AF ⪯N AF′, the following statement holds true: ∀E ∈σCF2(AF), (∃E′ ∈σCF2(AF′), such that
(E′ ̸⊆AR ∨E′ = E)). ∀E ∈σCF2(AF), (∃E′ ∈σCF2(AF′), such that
(E′ ̸⊆AR ∨E′ = E)). PROOF. 1. In the proof of Proposition 6.14, we have shown that naive semantics satisfies weak reference
independence. We have proven the proposition. We have proven the proposition. □ then
∃Va,b ∈V(AF′), such that
a ∈AR′ \ AR, a ∈E′, b ∈(E ∩SCC), Vb,a ̸∈V(AF′). 2. Now, let us observe that ∀E′ ∈σCF2(AF′), (if (∃Vc,d ∈V(AF′), Vc,d ̸∈V(AF), d ∈AR′, c ∈
AR′ \ AR, c ∈E′) then E′ ̸⊆AR). Consequently, from 1 it follows that ∀E ∈σCF2(AF), ∃E′ ∈
σCF2(AF′) such that the following statement holds true: ∨E′ ̸⊆AR. 3. By definition of CF2 semantics as an SCC-recursive, naive-based semantics, given E ∈
σCF2(AF), if (∃E′ ∈σCF2(AF′) such that ∀SCC ∈SCCSAF, (E ∩SCC) = (E′ ∩SCC)) then
E = E′ or E′ ̸⊆AR. Consequently, it follows from 2 that ∀E ∈σCF2(AF), ∃E′ ∈σCF2(AF′)
such that the following statement holds true: E = E′ ∨E′ ̸⊆AR. DEFINITION 8.1
(Attack cycles in argumentation frameworks).
An attack cycle C in an argumentation framework AF = (AR, Attacks) is an argument sequence
⟨a1, a2, ..., an−1, an⟩such that (ai, ai+1) ∈Attacks, ai ∈AR, 1 ≤i ≤n −1, a1 = an and |ARV
a1,an| = ∀E ∈σ i
x(AF), (∃E′ ∈σ i
x(AF′), such that
(E′ ̸⊆AR ∨E′ = E)). It follows that by definition of CF2 semantics as an SCC-recursive and naive-
based semantics, ∀E ∈σCF2(AF), ∃E′ ∈σCF2(AF′), such that the following statement holds
true: if
∃SCC ∈SCCSAF,
such that
(E ∩SCC) ̸= (E′ ∩SCC), DEFINITION 8.3 (Rational man’s expansion). A rational man’s argumentation expansion of two argumentation frameworks AF = (AR, Attacks)
and AF′ = (AR′, Attacks′) (denoted by AF ⪯RM AF′) is an expansion AF ⪯E AF′, for which the
following conditions hold true: 1. AF ⪯N AF′; 1. AF ⪯N AF′; 2. AF ⪯NC AF′; 2. AF ⪯NC AF′; NC
;
3. ∀a ∈AR, b ∈AR′ \ AR, such that b is reachable from a, it holds true that ∀C ∈C(AF′), a ̸∈
AR
′C. 3. ∀a ∈AR, b ∈AR′ \ AR, such that b is reachable from a, it holds true that ∀C ∈C(AF′), a ̸∈
AR
′C. Colloquially speaking, a rational man’s expansion is a normal expansion in which (i) no additional
cycles are added to the initial argumentation framework and no additional arguments are added to
existing cycles and (ii) no newly added arguments are reachable from cycles. We prove that the rational man’s expansion guarantees weak reference independence, given
complete, preferred, stage2, CF2, grounded, ideal and eager argumentation semantics. The proof
relies on the observation that given any argumentation framework, each argument that is in a strongly
admissible set is by definition always also in at least one extension of any of the aforementioned
argumentation semantics. PROPOSITION 8.4 Let σx be an argumentation semantics, such that x ∈{complete, preferred, semi −stable, stage,
stage2, CF2, grounded, ideal, eager}. For every two argumentation frameworks AF = (AR,
Attacks) and AF′ = (AR′, Attacks′), such that AF ⪯RM AF′, the following statement holds true: ∀E ∈σx(AF), ∃E′ ∈σx(AF′)
such that
E′ ̸⊆AR ∨E′ = E. ∀E ∈σx(AF), ∃E′ ∈σx(AF′)
such that
E′ ̸⊆AR ∨E′ = E. PROOF. We provide a proof by contradiction. 8
Cycles and reference independence In the previous section, we have shown that only one of the evaluated argumentation semantics—CF2
semantics—satisfies the weak reference independence principle. In order to guarantee economic
rationality, and in particular reference independence, it is relevant to look beyond argumentation
semantics. At first glance, it is striking that the example expansions in the proofs of Propositions 7.1,
7.2 and 7.3 add new cycles to the argumentation graphs. Consequently, we examine if an argumenta-
tion framework expansion can be defined that can guarantee compliance with the rational man’s
argumentation principle by further restricting the relationship of two argumentation frameworks
AF ⪯N AF′. For this, we first introduce a definition of cycles in the context of argumentation
frameworks. DEFINITION 8.1
(Attack cycles in argumentation frameworks). An attack cycle C in an argumentation framework AF = (AR, Attacks) is an argument sequence
⟨a1, a2, ..., an−1, an⟩such that (ai, ai+1) ∈Attacks, ai ∈AR, 1 ≤i ≤n −1, a1 = an and |ARV
a1,an| = Abstract argumentation and the rational man 683 n −1. C(AF) denotes all attack cycles of AF and ARC denotes the arguments that occur in an attack
cycle C. n −1. C(AF) denotes all attack cycles of AF and ARC denotes the arguments that occur in an attack
cycle C. e define the concept of a non-cyclic expansion. Now, we define the concept of a non-cyclic expansion. DEFINITION 8.2
(Non-cyclic expansion). A non-cyclic expansion of two argumentation frameworks AF
= (AR, Attacks) and AF′
=
(AR′, Attacks′) (denoted by AF ⪯NC AF′) is an expansion AF ⪯E AF′, for which it holds true
that C(AF′) = C(AF). DEFINITION 8.2
(Non-cyclic expansion). A non-cyclic expansion of two argumentation frameworks AF
= (AR, Attacks) and AF′
=
(AR′, Attacks′) (denoted by AF ⪯NC AF′) is an expansion AF ⪯E AF′, for which it holds true
that C(AF′) = C(AF). DEFINITION 8.2
(Non-cyclic expansion). A non-cyclic expansion of two argumentation frameworks AF
= (AR, Attacks) and AF′
=
(AR′, Attacks′) (denoted by AF ⪯NC AF′) is an expansion AF ⪯E AF′, for which it holds true
that C(AF′) = C(AF). This allows us to define the rational man’s expansion. b ∈S ⊆AR′,
such that
S
is strongly admissible and ∀E′ ∈σ(AF′), b ̸∈E′. b ∈S ⊆AR′,
such that
S
is strongly admissible and ∀E′ ∈σ(AF′), b ̸∈E′. 3. By definition of σx, iff ∃c ∈AR′, such that c ∈S ⊆AR′ and S is strongly admissible, then
∃E′ ∈σ(AF′), c ∈E′. Hence, ∃E′ ∈σx(AF′), such that b ∈E′. Note that b ∈AR′ \ AR. Contradiction. □ 3. By definition of σx, iff ∃c ∈AR′, such that c ∈S ⊆AR′ and S is strongly admissible, then
∃E′ ∈σ(AF′), c ∈E′. Hence, ∃E′ ∈σx(AF′), such that b ∈E′. Note that b ∈AR′ \ AR. Contradiction. □ Let us note that allowing for expansions that add even cycles to an argumentation framework
cannot guarantee reference independence in the case of many argumentation semantics. For example,
given AF = ({a, b}, {(a, b)}) and its expansion AF′ = ({a, b, c, d}, {(a, b), (b, c), cannot guarantee reference independence in the case of many argumentation semantics. For example,
given AF = ({a, b}, {(a, b)}) and its expansion AF′ = ({a, b, c, d}, {(a, b), (b, c),
(c, d), (d, a)}, grounded, ideal and eager semantics, as well as the skeptical reasoning modes of
complete, preferred, semi-stable, stage, stage2 and CF2 semantics return {{a}} for AF and {{}} for
AF′, as depicted in Figure 7. (c, d), (d, a)}, grounded, ideal and eager semantics, as well as the skeptical reasoning modes of
complete, preferred, semi-stable, stage, stage2 and CF2 semantics return {{a}} for AF and {{}} for
AF′, as depicted in Figure 7. p
g
Also, it is not sufficient that only cycles that include at least one argument arg
∈
AR
and at least one argument arg′ ∈AR′ \ AR are not allowed in an expansion AF ⪯NC AF′. This can be shown by introducing the following example. Given AF = ({a}, {}) and AF′ =
({a, b, c, d}, {(b, a), (c, b), (d, c), (b, d)}), grounded, ideal and eager semantics, as well as skeptical
complete, preferred, semi-stable, stage, stage2 and CF2 semantics return {{a}} for AF and {{}} for
AF′, as depicted in Figure 8. Given the presented findings, it is obvious that if expansion and deletion (the removal of
arguments) of an argumentation framework are allowed in any argumentation scenario, cycles should
be avoided altogether. For example, assuming preferred semantics, the argumentation framework
AF = ({a, b, c}, {(a, b), (b, c), (c, a)}) implies (among others) the preference {} ⪰{a}. PROOF. We provide a proof by contradiction. 1. Let us suppose the following statement holds true: 1. Let us suppose the following statement holds true: ∃E ∈σx(AF),
such that
∀E′ ∈σx(AF′)
it holds true that
E′ ⊆AR ∧E′ ̸= E. ∃E ∈σx(AF),
such that
∀E′ ∈σx(AF′)
it holds true that
E′ ⊆AR ∧E′ ̸= E. 2. Let us observe that by definition of σx and because AF ⪯RM AF′ implies ∀a ∈AR′ \ AR,
∀C ∈C(AF′), a ̸∈C and a is not reachable from any c ∈AR′
C, it follows from 1 that the 684
Abstract argumentation and the rational man 684
Abstract argumentation and the rational man
FIGURE 7 Importance of absence of even-length cycles, given, e.g. any of the surveyed semantics
and skeptical acceptance. AF ̸⪯RM AF′. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 FIGURE 7 Importance of absence of even-length cycles, given, e.g. any of the surveyed semantics
and skeptical acceptance. AF ̸⪯RM AF′. FIGURE 7 Importance of absence of even-length cycles, given, e.g. any of the surveyed semantics
and skeptical acceptance. AF ̸⪯RM AF′. FIGURE 7 Importance of absence of even-length cycles, given, e.g. any of the surveyed semantics
and skeptical acceptance. AF ̸⪯RM AF′. FIGURE 7 Importance of absence of even-length cycles, given, e.g. any of the surveyed semantics
and skeptical acceptance. AF ̸⪯RM AF′. following statement holds true: following statement holds true: ∃b ∈AR′ \ AR
such that
b
attacks
AR
and ∃b ∈AR′ \ AR
such that
b
attacks
AR
and b ∈S ⊆AR′,
such that
S
is strongly admissible and ∀E′ ∈σ(AF′), b ̸∈E′. Removing the
argument c from AF gives us AF′ = ({a, b}, {(a, b)}), with AF′ ⪯N AF. As AF′ implies (among
others) {a} ⪰{}, the preference relation implied by AF is inconsistent with the preference relation
implied by AF′. While it is clear that it is, in many scenarios, impractical to avoid cycles (or cyclic
expansions) altogether, the findings highlight the importance of further research on ‘loop-busting’
methods to ensure reference independence. Abstract argumentation and the rational man 685 FIGURE 8
Importance of absence of cycles among arguments Args ⊆(AR′ \ AR), given, e.g. grounded semantics. AF ̸⪯RM AF′. FIGURE 8
Importance of absence of cycles among arguments Args ⊆(AR′ \ AR), given, e.g. grounded semantics. AF ̸⪯RM AF′. FIGURE 8
Importance of absence of cycles among arguments Args ⊆(AR′ \ AR), given, e.g. grounded semantics. AF ̸⪯RM AF′. FIGURE 8
Importance of absence of cycles among arguments Args ⊆(AR′ \ AR), given, e.g. grounded semantics. AF ̸⪯RM AF′. 9
Rational man’s argumentation, belief revision and dialogues To highlight the relevance of the presented research, this section provides two examples that illustrate
how the newly established principles and expansions can be applied. EXAMPLE 9.1 (Argumentation dialogues). In a multi-agent scenario, let us assume we have a decision-maker agent A1 that receives advise from
a consultant agent A2. In this context, A1 presents its argumentation framework AF = (AR, Attacks)
to A2, who then proposes changes by providing AF′ = (AR′, Attacks′), with AF′ being a normal
expansion of AF (AF ⪯N AF′). Subsequently, A1 can accept or reject the changes. A2 can propose
two types of changes: Set-expanding changes. A2 only shows A1 that additional options to choose from exist and
how these options should be integrated into the argumentation framework. how these options should be integrated into the argumentation framework. Belief-revising changes. A2 advises A1 to change its beliefs about the decision options
contained in AF and may in addition propose set-expanding changes. Belief-revising changes. A2 advises A1 to change its beliefs about the decision options
contained in AF and may in addition propose set-expanding changes. A2might want to deceive A1 by proposing changes that A2 labels as set-expanding but that are also
belief-revising. For example, A1 presents the following argumentation framework to A2: AF = ({a, b, c}, {(b, a), (c, a)})). Then, A2 proposes the following: AF′ = ({a, b, c, d, e, f }, {(b, a), (c, a), (d, c), (e, d)(f , e)(d, f )})). AF′ = ({a, b, c, d, e, f }, {(b, a), (c, a), (d, c), (e, d)(f , e)(d, f )})). If A2 labels this proposal as set-expanding, A2 is deceiving A1 to think that A1 is barely considering
new options and not revising the assessment of the existing options. However, given the work
presented above, A1 can first detect that AF RM AF′ (AF NC AF′) and then check if its
preferences over the initial arguments in AF are still consistent, given the decision that is made
based on AF′. As this is not the case, A1 can, e.g., decide to stick to the initial decision that it made
based on AF. Figure 9 depicts the argumentation graphs of AF and AF′. 686
Abstract argumentation and the rational man Figure 9 AF ⪯N AF′, but AF NC AF′. Figure 9 AF ⪯N AF′, but AF NC AF′. Figure 9 AF ⪯N AF′, but AF NC AF′. Figure 9 AF ⪯N AF′, but AF NC AF′. Figure 10 Labelling reference-dependent argumentation graphs to improve argument mining. Figure 10 Labelling reference-dependent argumentation graphs to improve argument mining. EXAMPLE 9.2 EXAMPLE 9.2
(Argument mining). (Argument mining). (
g
g)
The ability to assess whether argumentation-based inference is reference independent can potentially
be useful in argument mining scenarios, in which argumentation graphs are generated from natural
language text or other data sources [30]. Let us introduce a scenario where an argument miner uses
machine learning techniques for natural language processing to generate argumentation frameworks
from text—e.g. from legal documents—and then hands them over to an agent that resolves the
argumentation frameworks to inform its decision-making. However, the argumentation agent is not
accepting the frameworks under any condition; instead, it is assessing the frameworks and their
relation with each other to determine if reference independence is violated. The argumentation agent
then provides the results of these assessments to the argument miner, who can use the information in
different ways. If reference independence is violated, it can either re-assess the corresponding text
and suggest an alternative, reference-independent interpretation, or consider the text as not useful Abstract argumentation and the rational man 687 FIGURE 11 AF0 ⪯N AF1, but AF0 RM AF1 (AF0 NC AF1). Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 FIGURE 11 AF0 ⪯N AF1, but AF0 RM AF1 (AF0 NC AF1). and label it accordingly to increase its ability to focus on more useful texts in the future. Figure 10
depicts the architecture of the proposed system. AF′ = ({a, b, c, d, e, f }, {(b, a), (c, a), (d, c), (e, d)(f , e)(d, f )})). As an example, let us assume the argument miner creates argumentation frameworks based
on an evolving online discussion on whether a policy should be implemented or not (denoted
by argument p). At time t0, the argument miner detects an argument a that attacks the policy
implementation proposal: AF0 = ({p, a}, {(a, p)}). The argumentation agent—using, e.g. skeptical
complete semantics σ ∩
complete—resolves AF to {a}, i.e. it decides the policy should not be imple-
mented. At time t1, the argument miner detects the additional arguments b and c, as well as the
additional attacks (a, b), (b, c) and (c, a). The argumentation agent resolves the framework AF1 =
({p, a, b, c}, {(a, p), (a, b), (b, c), (c, a)}) as {}. Now, it is clear that AF0 ⪯N AF1 but AF0 ̸⪯RM AF1. It is also clear that the preference relations implied by g ◦σ ∩
complete(AF0) and g ◦σ ∩
complete(AF0) are
inconsistent, i.e. g∩◦σ ∩
complete(AF0) implies {a} ⪰{} and g∩◦σ ∩
complete(AF1) implies {} ⪰{a}. Hence, the argumentation agent can label AF1 as faulty or not useful and provide this information to
the argument miner, who can then attempt to find alternative formal interpretations of the discussion,
or move on to a different discussion. Figure 11 depicts the argumentation graphs of AF0 and AF1. 10
Related work: preference-based argumentation and rational man’s
expansions In our work, we derive implicit preference orders on sets of arguments from abstract argumentation
frameworks. Hence, it makes sense to put our work in the context of argumentation approaches
that explicitly define preferences. Amgoud’s and Cayrol’s preference-based argumentation [1] can
be considered the most foundational work that advances this research direction. Hence, we relate
our work to preference-based argumentation and confirm the intuition that the explicit definition
of preferences does not guarantee reference independence by formal proof. Let us first introduce a
definition of a preference-based argumentation framework. DEFINITION 10.2 (Preference-based argumentation framework [1]). Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 Given a preference-based argumentation framework AFp = (AR, Attacks, Prefs), the set of accept-
able arguments Argsacc ⊆AR is determined as follows by the preference-based argumentation
function τpreferred: τpreferred(AF) = {a|a ∈AR,
such that
∀b ∈AR,
if
(b, a) ∈Attacks
then
a ⪰b}. τpreferred(AF) = {a|a ∈AR,
such that
∀b ∈AR,
if
(b, a) ∈Attacks
then
a ⪰ Note that in this paper, we only consider the acceptability status of arguments; i.e. in contrast to
Amgoud and Cayrol, we do not distinguish between rejected arguments and arguments in abeyance. This simplification is motivated by the rational decision-maker’s required ability to make clear,
unambiguous decisions.9 To analyse preference-based argumentation in the context of reference independence, let us first
define the concept of a normal expansion of a preference-based argumentation framework. DEFINITION 10.3
An expansion AF′
p = (AR′, Attacks′, Prefs′) of a preference-based argumentation framework AFp =
(AR, Attacks, Prefs) is normal (AFp ⪯NP AF′
p) iff DEFINITION 10.3
An expansion AF′
p = (AR′, Attacks′, Prefs′) of a preference-based argumentation framework AFp =
(AR, Attacks, Prefs) is normal (AFp ⪯NP AF′
p) iff An expansion AF′
p = (AR′, Attacks′, Prefs′) of a preference-based argumentation framework AFp =
(AR, Attacks, Prefs) is normal (AFp ⪯NP AF′
p) iff p
•
Attacks ⊆Attacks′ and
•
∀(a, b) ∈Attacks′ \ Attacks, a ∈AR′ \ AR ∨b ∈AR′ \ AR and
•
Prefs ⊆Prefs′ and
•
∀(a ⪰b) ∈Prefs′ \ Prefs, a ∈AR′ \ AR ∨b ∈AR′ \ AR. •
Attacks ⊆Attacks′ and
•
∀(a, b) ∈Attacks′ \ Attacks, a ∈AR′ \ AR ∨b ∈AR′ \ AR and
•
Prefs ⊆Prefs′ and
•
∀(a ⪰b) ∈Prefs′ \ Prefs, a ∈AR′ \ AR ∨b ∈AR′ \ AR. In words, considering the addition of preferences Prefs to abstract argumentation frameworks, we
assume that a normal expansion AF′
p of AFp neither changes existing preferences defined in Prefs nor
adds additional preferences between any two arguments that exist in AFp. As non-cyclic expansions
do not require a definition that is specific to preference-based argumentation, let us directly define
the rational man’s expansion in the context of preference-based argumentation. DEFINITION 10.1 (Preference-based argumentation framework [1]). A preference-based argumentation framework is a triplet (AR, Attacks, Prefs), whereby AR and
Attacks are arguments and attacks, defined according to Definition 3.5 and Prefs define a partial
or total ordering on AR × AR. In a preference-based argumentation framework, acceptability is determined as follows Abstract argumentation and the rational man 688 9Analogously, we also do not distinguish between rejected and undecided arguments in abstract argumentation. PROPOSITION 10.5 PROPOSITION 10.5
Let g∩◦τpreferred be an argumentation-based decision function for preference-based argumentation
frameworks. For every two preference-based argumentation frameworks AFp and AF′
p, such that
AFp = (AR, Attacks, Prefs), AF′
p = (AR′, Attacks′, Prefs′) and AFp ⪯NP AF′
p, the following
statement holds true: Let g∩◦τpreferred be an argumentation-based decision function for preference-based argumentation
frameworks. For every two preference-based argumentation frameworks AFp and AF′
p, such that
AFp = (AR, Attacks, Prefs), AF′
p = (AR′, Attacks′, Prefs′) and AFp ⪯NP AF′
p, the following
statement holds true: g∩◦τpreferred(AF′
p) ⊆AR
does not imply
g∩◦τpreferred(AF′
p) = g∩◦τpreferred(AFp). PROOF. The proposition can be proven by counter-example. We introduce the following preference-
based argumentation frameworks: PROOF. The proposition can be proven by counter-example. We introduce the following preference-
based argumentation frameworks: •
AFp = ({a, b, c}, {(a, b)}, (a ⪰c, b ⪰c)}); •
AFp = ({a, b, c}, {(a, b)}, (a ⪰c, b ⪰c)}); •
AFp = ({a, b, c}, {(a, b)}, (a ⪰c, b ⪰c)}); p
•
AF′
p = ({a, b, c, d}, {(a, b), (b, d), (d, a)}, (a ⪰c, b ⪰c, d ⪰c))}. We can see that AFp ⪯NP AF′
p. g∩◦τpreferred resolves the frameworks as follows: 1. g∩◦τpreferred(AFp) = {a, c}. ∩
′ 1. g∩◦τpreferred(AFp) = {a, c}. ∩ 1. g∩◦τpreferred(AFp) = {a, c}. 2
∩
(A
′ )
{ } 1. g
◦τpreferred(AFp)
{a, c}. 2. g∩◦τpreferred(AF′
p) = {c}. From g∩◦τpreferred(AF′
p) ⊆AR and g∩◦τpreferred(AF′
p) ̸= g∩◦τpreferred(AFp), it follows that
g∩◦τpreferred(AF′
p) ⊆AR
does not imply
g∩◦τpreferred(AF′
p) = g∩◦τpreferred(AFp). □ 2. g∩◦τpreferred(AF′
p) = {c}. p
From g∩◦τpreferred(AF′
p) ⊆AR and g∩◦τpreferred(AF′
p) ̸= g∩◦τpreferred(AFp), it follows that
g∩◦τpreferred(AF′
p) ⊆AR
does not imply
g∩◦τpreferred(AF′
p) = g∩◦τpreferred(AFp). □ Figure 12 depicts the argumentation frameworks used in the proof. Figure 12 depicts the argumentation frameworks used in the proof. Also, it can be proven that given two preference-based argumentation frameworks AFp and AF′
p
even when AF′
p is a rational man’s expansion of AFp (AFp ⪯RMP AF′
p), reference dependence is not
guaranteed. DEFINITION 10.2 DEFINITION 10.4
An expansion AF′
p = (AR′, Attacks′, Prefs′) of a preference-based argumentation framework AFp =
(AR, Attacks, Prefs) is a rational man’s expansion (AFp ⪯RMP AF′
p) iff: •
AFp ⪯NP AF′
p •
AFp ⪯NP AF′
p p
•
AF ⪯NC AF′ •
∀a ∈ARa, b ∈AR′
a \ ARa such that b is reachable from a, it holds true that ∀C ∈C(AF′), a ̸∈
AR
′C
a ,
where AF = (ARa, Attacksa) and AF′ = (AR′
a, Attacks′
a) are abstract argumentation frameworks,
such that ARa = AR, Attacksa = Attacks, AR′
a = AR′, Attacks′ = Attacks. Note that in the definition above, we ‘map’ preference-based argumentation frameworks to
abstract argumentation frameworks, in order to be able to use definitions we have introduced for
abstract argumentation frameworks. 9Analogously, we also do not distinguish between rejected and undecided arguments in abstract argumentation. Abstract argumentation and the rational man 689 FIGURE 12 Normal, but cyclic expansion: AFp ⪯NP AF′
p, but AFp NC AF′
p. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 FIGURE 12 Normal, but cyclic expansion: AFp ⪯NP AF′
p, but AFp NC AF′
p. Now, it can be easily shown that preference-based argumentation does not guarantee reference
independence. For this, we introduce the following proposition. statement holds true: statement holds true: g∩◦τpreferred(AF′
p) ⊆AR
does not imply
g∩◦τpreferred(AF′
p) = g∩◦τpreferred(AFp g∩◦τpreferred(AF′
p) ⊆AR
does not imply
g∩◦τpreferred(AF′
p) = g∩◦τpreferred(AFp). PROOF. Again, the proposition can be proven by counter-example. We introduce the following
preference-based argumentation frameworks: PROOF. Again, the proposition can be proven by counter-example. We introduce the following
preference-based argumentation frameworks: •
AFp = ({a, b, c}, {(a, b)}, (a ⪰c, b ⪰c)}); p
•
AF′
p = ({a, b, c, d}, {(a, b), (c, d), (d, a)}, (a ⪰c, b ⪰c, d ⪰c))}. •
AF′
p = ({a, b, c, d}, {(a, b), (c, d), (d, a)}, (a ⪰c, b ⪰c, d ⪰c))}. We can see that AFp ⪯RMP AF′
p. g∩◦τpreferred resolves the frameworks as follows: We can see that AFp ⪯RMP AF′
p. g∩◦τpreferred resolves the frameworks as follows: We can see that AFp ⪯RMP AF′
p. g∩◦τpreferred resolves the frameworks as follows: 1. g∩◦τpreferred(AFp) = {a, c}. ∩ 2. g∩◦τpreferred(AF′
p) = {c}. 2. g∩◦τpreferred(AF′
p) = {c}. From g∩◦τpreferred(AF′
p) ⊆AR and g∩◦τpreferred(AF′
p) ̸= g∩◦τpreferred(AFp), it follows that
g∩◦τpreferred(AF′
p) ⊆AR
̸⇒g∩◦τpreferred(AF′
p) = g∩◦τpreferred(AFp). □ From g∩◦τpreferred(AF′
p) ⊆AR and g∩◦τpreferred(AF′
p) ̸= g∩◦τpreferred(AFp), it follows that
g∩◦τpreferred(AF′
p) ⊆AR
̸⇒g∩◦τpreferred(AF′
p) = g∩◦τpreferred(AFp). □ The argumentation frameworks used in the proof are depicted by Figure 13. As preference-based argumentation is a special case of value-based argumentation as introduced
by Bench-Capon [12], it is also clear that normal, non-cyclic expansions cannot guarantee rationality
for value-based argumentation. Let us highlight that in contrast to these works, which introduce
explicit preference relations on arguments, our work analyses argumentation in the context of the
implicit preference relations on sets of arguments that are implied by argumentation semantics. PROPOSITION 10.6 Let g∩◦τpreferred be any argumentation-based decision function for preference-based argumentation
frameworks. For every two preference-based argumentation frameworks AFp and AF′
p, such that
AFp = (AR, Attacks, Prefs), AF′
p = (AR′, Attacks′, Prefs′), and AFp ⪯RMP AF′
p, the following Let g∩◦τpreferred be any argumentation-based decision function for preference-based argumentation
frameworks. For every two preference-based argumentation frameworks AFp and AF′
p, such that frameworks. For every two preference based argumentation frameworks AFp and AFp, such that
AFp = (AR, Attacks, Prefs), AF′
p = (AR′, Attacks′, Prefs′), and AFp ⪯RMP AF′
p, the following 690
Abstract argumentation and the rational man Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 FIGURE 13 Violation of reference independence in preference-based argumentation in absence of
cycles. FIGURE 13 Violation of reference independence in preference-based argumentation in absence of
cycles. FIGURE 13 Violation of reference independence in preference-based argumentation in absence of
cycles. 11
Conclusion and future work In this work, we observe that abstract argumentation semantics imply preferences orders on sets
of arguments. Hence, we explore these preference orders from the perspective of microeconomic
theory, and in particular the rational man paradigm. This paper shows that abstract argumentation
semantics typically do not guarantee reference-independent decision-making according to the
rational man paradigm and consequently uncovers a gap between abstract argumentation semantics
and economic rationality. Thereby, our research focuses on foundational work at the intersection
of abstract argumentation and (bounded) economic rationality. Our work sheds new light on the
question to what extent and under which circumstances the application of abstract argumentation Abstract argumentation and the rational man 691 returns ‘reasonable’ results from the perspective of economic rationality. Taking into account the
rich body of formal works on both argumentation and (boundedly) rational decision-making, plenty
of opportunities to extend our work exist. In particular, we consider the following research directions
as promising future work: •
‘Loop-busting’ to ensure economic rationality in temporal argumentation. In this paper,
we have shown that abstract argumentation approaches are typically economically not rational
when considering the normal expansion of argumentation frameworks and that economic
rationality—i.e. reference independence—can be achieved by avoiding the addition of cycles
in normal expansions. However, it can be assumed that many scenarios require well-
defined approaches to handling argumentation cycles in an economically rational (reference-
independent) manner. To devise such approaches, it should be possible to build on a
fundament of works on the resolution of cycles in argumentation graphs (also called ‘loop-
busting’) [3], as well as on methods that enforce that particular arguments are in an extension
[7]. •
Economic rationality and advanced argumentation frameworks. In addition to preference-
based and value-based argumentation, a range of other works extend Dung’s notion of an
argumentation framework, e.g. by assigning weights or intervals to attacks (e.g. probabilistic
[29] and possibilistic [32] argumentation). Given that the rational man’s expansion as
established in this paper does not guarantee reference independence in the case of preference-
/value-based argumentation, the exploration of the intersection of these approaches and
economic rationality can be considered promising future research. •
Bounded rationality and abstract argumentation. As mentioned in the introduction, from
the perspective of formal argumentation, it can be interesting to relax the clear preferences
property of the economically rational decision-maker, e.g. 11
Conclusion and future work to consider three-valued labelling
and ‘undecided’ status of arguments, or to support quantitative notions of uncertainty. Such
work can potentially be evaluated from an economics perspective, as a systematic relaxation
of rationality constraints that can create new formal models of bounded rationality. In this
context, the recently introduced family of weak admissible set-based semantics can potentially
be useful [10]. (However, let us informally observe that the semantics introduced in [10] do not
satisfy weak reference independence by considering the example argumentation frameworks
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3, 12–29, 2010. Appendix A. Strong monotony properties and reference independence This appendix provides the following proofs, given maximal conflict-free semantics and nor
expansions: •
Strong monotony implies strong reference independence but not vice versa. •
Strong rational monotony does not imply strong reference independence and vice versa. •
Strong rational monotony does not imply strong reference independence and vi •
Strong cautious monotony does not imply strong reference independence and v Let us first prove that strong reference independence does not imply strong monotony. Let us first prove that strong reference independence does not imply strong monotony. PROPOSITION A.1
(Strong reference independence does not imply strong monotony). (Strong reference independence does not imply strong monotony). There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, such that the following statement does not hold true: There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, such that the following statement does not hold true: If
∀E ∈σ(AF), ∀E′ ∈σ(AF′), (E′ ̸⊆AR ∨E′ = E)
then
(∀E ∈σ(AF), ∀E′ ∈σ(AF′), E ⊆E′). If
∀E ∈σ(AF), ∀E′ ∈σ(AF′), (E′ ̸⊆AR ∨E′ = E)
then
(∀E ∈σ(AF), ∀E′ ∈σ(AF′), E ⊆E′). PROOF. Let us provide a proof by counter-example. 1. Let AF = (AR, Attacks) = ({a}, {}), AF′ = ({a, b}, {(b, a)}) and let us take CF2 semantics,
denoted by σcf 2, as an example of a semantics whose extensions are maximal conflict-free
sets. Note that AF ⪯N AF′. 1. Let AF = (AR, Attacks) = ({a}, {}), AF′ = ({a, b}, {(b, a)}) and let us take CF2 semantics,
denoted by σcf 2, as an example of a semantics whose extensions are maximal conflict-free
sets. Note that AF ⪯N AF′. ⪯N
2. σcf 2(AF) = {{a}} and σcf 2(AF′) = {{b}}. Let E = {a}, E ∈σcf 2(AF). ∃E′ ∈σ(AF′) such that
{a} ̸⊆E′. The condition for strong monotony does not hold. ⪯N
2. σcf 2(AF) = {{a}} and σcf 2(AF′) = {{b}}. Let E = {a}, E ∈σcf 2(AF). ∃E′ ∈σ(AF′) such that
{a} ̸⊆E′. The condition for strong monotony does not hold. 3. ∀E′ ∈σ(AF′) it holds true that E′ ̸⊆AR. The condition for strong reference independence
holds. We have proven the proposition. □ 3. Appendix A. Strong monotony properties and reference independence ∀E′ ∈σ(AF′) it holds true that E′ ̸⊆AR. The condition for strong reference independence
holds. We have proven the proposition. □ Strong monotony implies strong reference independence. Strong monotony implies strong reference independence. 694
Abstract argumentation and the rational man PROPOSITION A.2 (Strong monotony implies strong reference independence). For all argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set
inclusion) and every two argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), Abstract argumentation and the rational man 695 TABLE 1
Overview: compliance with the uniqueness principle. Credulous (σ(AF))
Skeptica TABLE 1
Overview: compliance with the uniqueness principle. TABLE 1
Overview: compliance with the uniqueness principle. Credulous (σ(AF))
Skeptical (σ ∩(AF))
Complete
No
Yes
Grounded
Yes
Yes
Preferred
No
Yes
Stable
No
No
Ideal
Yes
Yes
Semi-stable
No
Yes
Eager
Yes
Yes
Stage
No
Yes
CF2
No
Yes
Stage2
No
Yes such that AF ⪯N AF′, the following statement holds true:
If
(∀E ∈σ(AF), ∀E′ ∈σ(AF′), E ⊆E′)
then
(∀E ∈σ(AF), ∀E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E). such that AF ⪯N AF′, the following statement holds true: AF ⪯N AF′, the following statement holds true: PROOF. 1. By definition, ∀E ∈σ(AF), E′ ∈σ(AF′), E is a maximal conflict-free (w.r.t. set inclusion)
subset of AR and E′ is a maximal conflict-free (w.r.t. set inclusion) subset of AR′. Because
AF ⪯N AF′, it follows that, if E ⊆E′, we have the following cases:
2. (i) E = E’ or 1. By definition, ∀E ∈σ(AF), E′ ∈σ(AF′), E is a maximal conflict-free (w.r.t. set inclusion)
subset of AR and E′ is a maximal conflict-free (w.r.t. set inclusion) subset of AR′. Because
AF ⪯N AF′, it follows that, if E ⊆E′, we have the following cases: 2. (i) E = E’ or 3. (ii) E ⊂E′, which implies that ∃a ∈E′, such that a ̸∈E, a ∈AR′ \ AR, from which it follows
that E′ ̸⊆AR. 4
C
l
b (i)
d (ii) h f ll
i
h ld 3. (ii) E ⊂E′, which implies that ∃a ∈E′, such that a ̸∈E, a ∈AR′ \ AR, from which it follows
that E′ ̸⊆AR. 4. Consequently, by (i) and (ii) the following statement holds true: ntly, by (i) and (ii) the following statement holds true: ∀E ∈σ(AF), ∀E′ ∈σ(AF′),
if
E ⊆E′
then
E′ ̸⊆AR ∨E′ = E. Hence, the proposition holds true. □ □ However, we can prove that strong cautious monotony does not imply strong reference indepen-
dence. However, we can prove that strong cautious monotony does not imply strong reference indepen-
dence. PROPOSITION A.3 (Strong cautious monotony does not imply strong reference independence). (Strong cautious monotony does not imply strong reference independence). There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, such that the following statement does not hold true: There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, such that the following statement does not hold true: If
∀E ∈σ(AF), ({(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅
implies
∀E′ ∈σ(AF′), E ⊆E′) If
∀E ∈σ(AF), ({(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅
implies
∀E′ ∈σ(AF′), E ⊆E′) then (∀E ∈σ(AF), ∀E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E). (∀E ∈σ(AF), ∀E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E). Abstract argumentation and the rational man 696 PROOF. Let us provide a proof by counter-example. PROOF. Let us provide a proof by counter-example. PROOF. Let us provide a proof by counter-example. 1. Let AF = (AR, Attacks) = ({c, d}, {(c, d)}), AF′ = ({c, d, e}, {(c, d), (d, e), (e, e), (e, c)}) and
let us take stage semantics, denoted by σstage, as an example of a semantics whose extensions
are maximal conflict-free sets. Note that AF ⪯N AF′. 2. σstage(AF) = {{c}} and σstage(AF′) = {{c}, {d}}. 3. Given the only extension E = {c}, E ∈σstage(AF), {(a, b) | (a, b) ∈Attacks′, a ∈AR′\AR, b ∈
E} = ∅does not hold true and hence, ∀E′ ∈σstage(AF′), {(a, b) | (a, b) ∈Attacks′, a ∈
AR′ \ AR, b ∈E} = ∅
implies
E ⊆E′ holds true. 4. However, it does not hold true that ∀E ∈σ(AF), ∀E′ ∈σ(AF′), (E′ ̸⊆AR∨E′ = E). We have
proven the proposition. □ In a similar manner, we can prove that strong reference independence does not imply strong
cautious monotony. PROOF. We provide the following proof by counter-example. 1. Let AF = (AR, Attacks) = ({c, d, e}, {(c, d), (d, e), (e, c)}), 2. AF′ = ({c, d, e, f }, {(c, d), (d, e), (e, c), (f , d)}) and let us take stage semantics, denoted by
σstage, as an example of a semantics whose extensions are conflict-free sets. Note that AF ⪯N
AF′. 3. σstage(AF) = {{c}, {d}, {e}} and σstage(AF′) = {{e, f }}. g
g
4. Given the extension E ∈σstage(AF), E = {a}, {(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈
E} = ∅holds true. 5. Given E′ = {e, f } as the only extension in σ(AF′) it holds true that E′ ̸⊆AR; the result does
not violate the strong reference independence property. 5. Given E′ = {e, f } as the only extension in σ(AF′) it holds true that E′ ̸⊆AR; the result does
not violate the strong reference independence property. 6. However, given E′ = {e, f } as the only extension in σ(AF′) and given E = {c}, E ∈σstage(AF),
we have E ̸⊆E′, which violates strong cautious monotony. We have proven the proposition.□ 6. However, given E′ = {e, f } as the only extension in σ(AF′) and given E = {c}, E ∈σstage(AF),
we have E ̸⊆E′, which violates strong cautious monotony. We have proven the proposition.□ Let us now prove that strong rational monotony does not imply strong reference indepe Let us now prove that strong rational monotony does not imply strong reference independence. PROPOSITION A 5 PROPOSITION A.5 PROPOSITION A.4 (Strong reference independence does not imply strong cautious monotony). (Strong reference independence does not imply strong cautious monotony). There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, such that the following statement does not hold true: There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, such that the following statement does not hold true: If
(∀E ∈σ(AF), ∀E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E)
then ∀E ∈σ(AF), ({(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅
implies
∀E′ ∈σ(AF′), E ⊆E′) ∀E ∈σ(AF), ({(a, b) | (a, b) ∈Attacks′, a ∈AR′ \ AR, b ∈E} = ∅
implies
∀E′ ∈σ(AF′), E ⊆E′) PROOF. We provide the following proof by counter-example. PROOF. We provide the following proof by counter-example. where UE′
new =
E′∈σ(AF′)(E′ \ AR). where UE′
new =
E′∈σ(AF′)(E′ \ AR). PROOF. Let us provide a proof by counter-example. PROOF. Let us provide a proof by counter-example. 1. Let AF
= (AR, Attacks) = ({c, d}, {(c, d), (d, c)}) and let AF′
= (AR′, Attacks′) =
({c, d, e}, {(c, d), (d, c), (d, e), (e, d), (e, c)}). Let us take stage semantics, denoted by σstage,
as an example of a semantics whose extensions are maximal conflict-free sets. Note that
AF ⪯N AF′. 2. σstage(AF) = {{c}, {d}} and σstage(AF′) = {{d}, {e}}. 3. Given ∀E ∈σ(AF), {(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅does not hold true. Hence, {(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅
implies
E ⊆E′ holds true. However, ∃E ∈{{c}, {d}} such that it does not hold true that (∀E′ ∈σ(AF′), (E′ ̸⊆AR ∨E′ =
E)). We have proven the proposition. □ Finally, let us prove that strong reference independence does not imply strong rational monotony. PROPOSITION A 6 Finally, let us prove that strong reference independence does not imply strong rational monotony. P
A 6 Finally, let us prove that strong reference independence does not imply strong rational monotony. PROPOSITION A.5 Strong rational monotony does not imply strong reference independence). There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that 697 Abstract argumentation and the rational man AF ⪯N AF′, such that the following statement does not hold true: Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 ∀E′ ∈σ(AF′), E ⊆E′) then PROPOSITION A.6 (Strong reference independence does not imply strong rational monotony). (Strong reference independence does not imply strong rational monotony). There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, such that the following statement does not hold true: There exist an argumentation semantics σ whose extensions are maximal conflict-free sets (w.r.t. set inclusion) and argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, such that the following statement does not hold true: If
(∀E ∈σ(AF), ∀E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E))
then If
(∀E ∈σ(AF), ∀E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E))
then ∀E ∈σ(AF), ({(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅
implies
∀E′ ∈σ(AF′), E ⊆E′), ∀E ∈σ(AF), ({(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅
implies
∀E′ ∈σ(AF′), E ⊆E′), where UE′
new =
E′∈σ(AF′)(E′ \ AR). where UE′
new =
E′∈σ(AF′)(E′ \ AR). where UE′
new =
E′∈σ(AF′)(E′ \ AR). PROOF. Let us provide a proof by counter-example. PROOF. Let us provide a proof by counter-example. 1. Let AF = (AR, Attacks) = ({c, d, e}, {(c, d), (d, e), (e, c)}), 2. AF′ = ({c, d, e, f }, {(c, d), (d, e), (e, c), (f , d)}) and let us take stage semantics, denoted by
σstage, as an example of a semantics whose extensions are maximal conflict-free sets. Note
that AF ⪯N AF′. 3. σstage(AF) = {{c}, {d}, {e}} and σstage(AF′) = {{e, f }}. 4. ∀E′ ∈σ(AF′), it holds true that E′ ̸⊆AR. 5. However, given the extension E ∈σstage(AF), E = {c} it holds true that {(a, b) | (a, b) ∈
Attacks′, a ∈UE′
new, b ∈E} = ∅and ∃E′ ∈σ(AF′), such that E ̸⊆E′, which violates strong
rational monotony. □ □ Abstract argumentation and the rational man 698 If
(∀E ∈σ(AF), ∀E′ ∈σ(AF′), E ⊆E′)
then
(∀E ∈σ(AF), ∀E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E The proof follows from the proof of Proposition .7. Let us now provide the proof that weak rational monotony implies weak reference independence
for maximal admissible set-based semantics. Appendix B. Monotony and reference independence in maximal admissible set-
based semantics Let us prove that weak monotony implies weak reference independence and strong monotony
implies strong reference independence in the case of maximal admissible set-based semantics. ION B.1 PROOF. Let us provide a proof by contradiction. PROOF. Let us provide a proof by contradiction. 1. ∀E ∈σ(AF), E′ ∈σ(AF′) if E ⊆E′, then we have the following cases: 2. (i) E = E’ or 2. (i) E = E’ or 3. (ii) E ⊂E′. 3. (ii) E ⊂E′. ( )
4. We need to contradict (ii). From (ii), it follows that if E′ ⊆AR then ∃a ̸∈E, a ∈AR′ ∩AR,
such that E ∪{a} is admissible. 5. It follows that E is not a maximal admissible set (w.r.t. set inclusion) of AF. Contra The same proof applies to the proposition that strong monotony implies strong reference
independence in the case of maximal admissible set-based semantics. The same proof applies to the proposition that strong monotony implies strong reference
independence in the case of maximal admissible set-based semantics. COROLLARY B.2
For all argumentation semantics σ whose extensions are maximal admissible sets (w.r.t. set inclusion)
and every two argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, the following statement holds true: PROPOSITION B.1 (Weak monotony implies weak reference independence). For all argumentation semantics σ whose extensions are maximal admissible sets (w.r.t. set inclusion)
and every two argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), such that
AF ⪯N AF′, the following statement holds true: If
(∀E ∈σ(AF)
it holds true that
∃E′ ∈σ(AF′), E ⊆E′)
then
(∀E ∈σ(AF)
it holds true that
∃E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E). If
(∀E ∈σ(AF)
it holds true that
∃E′ ∈σ(AF′), E ⊆E′)
then
(∀E ∈σ(AF)
it holds true that
∃E′ ∈σ(AF′), E′ ̸⊆AR ∨E′ = E). PROPOSITION B.2 □ Case 2:
{(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅and ∃E′ ∈σ(AF′), such that E ⊆E′:
Because it holds true that ∃E′
∈
σ(AF′),
such that
E
⊆
E′ and because
all σ-extensions are maximal admissible sets (w.r.t. set inclusion), from the proof of
Proposition .7 it follows that weak reference independence is satisfied. We have proven
the proposition. □ PROPOSITION B.2 (Weak rational monotony implies weak reference independence). (Weak rational monotony implies weak reference independence). For every argumentation semantics σ whose extensions are maximal admissible sets (w.r.t. set
inclusion) and every two argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), For every argumentation semantics σ whose extensions are maximal admissible sets (w.r.t. set
inclusion) and every two argumentation frameworks AF = (AR, Attacks), AF′ = (AR′, Attacks′), Abstract argumentation and the rational man
69 699 such that AF ⪯N AF′, the following statement holds true: such that AF ⪯N AF′, the following statement holds true: F′, the following statement holds true: ∀E ∈σ(AF), (If
({(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅
implies
∃E′ ∈σ(AF′),
such that
E ⊆E′)
then (∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR ∨E′ = E))), (∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR ∨E′ = E))), (∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR ∨E′ = E))), where UE′
new =
E′∈σ(AF′)(E′ \ AR). where UE′
new =
E′∈σ(AF′)(E′ \ AR). where UE′
new =
E′∈σ(AF′)(E′ \ AR). ∈(
)
PROOF. Given any E ∈σ(AF), we have two cases for which weak rational monotony is satisfied. Given any E ∈σ(AF), we have two cases for which weak rational monotony is satisfied Case 1:
{(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} ̸= ∅: If {(a, b) | (a, b) ∈Attacks′, a ∈
UE′
new, b ∈E} ̸= ∅, then ∃E′ ∈σ(AF′),
such that
(E′ ̸⊆AR). Consequently, weak
reference independence is satisfied. Case 2:
{(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅and ∃E′ ∈σ(AF′), such that E ⊆E′:
Because it holds true that ∃E′
∈
σ(AF′),
such that
E
⊆
E′ and because
all σ-extensions are maximal admissible sets (w.r.t. set inclusion), from the proof of
Proposition .7 it follows that weak reference independence is satisfied. We have proven
the proposition. □ Case 2:
{(a, b) | (a, b) ∈Attacks′, a ∈UE′
new, b ∈E} = ∅and ∃E′ ∈σ(AF′), such that E ⊆E′:
Because it holds true that ∃E′
∈
σ(AF′),
such that
E
⊆
E′ and because
all σ-extensions are maximal admissible sets (w.r.t. set inclusion), from the proof of
Proposition .7 it follows that weak reference independence is satisfied. We have proven
the proposition. Appendix C. Uniqueness in argumentation semantics This appendix provides an overview of which argumentation semantics satisfies the uniqueness
principle. We can rely on an analysis provided by Baumann as the basis of our overview [8]. Note that we base the overview on the assumption that any argumentation framework is finite. Consequently, if, given an argumentation semantics, at least one extension is guaranteed given any
finite argumentation framework, unique skeptical extensions are guaranteed. Received 17 March 2020
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Supplemental Figures S1-S4 from Modeling Alveolar Soft Part Sarcoma Unveils Novel Mechanisms of Metastasis
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Supplemental Figure S1. Development of the mouse ASPS model. A, Transduction efficiency of eMCs
by the pMYs-ASPSCR1-TFE3 retrovirus. The positive fraction of infected
cells is shown. B, Electron microscopy of mouse ASPS tumor cells. The inset
shows abundant mitochondria (M) and lysosomes (L). C, Intra-tumor blood
vessels are positive for PDGFRB, a marker for hemangiopericytes. Supplemental Figure S1. Development of the mouse ASPS model. A, Transduction efficiency of eMCs
by the pMYs-ASPSCR1-TFE3 retrovirus. The positive fraction of infected
cells is shown. B, Electron microscopy of mouse ASPS tumor cells. The inset
shows abundant mitochondria (M) and lysosomes (L). C, Intra-tumor blood
vessels are positive for PDGFRB, a marker for hemangiopericytes. Development of the mouse ASPS model. A, Transduction efficiency of eMCs
by the pMYs-ASPSCR1-TFE3 retrovirus. The positive fraction of infected
cells is shown. B, Electron microscopy of mouse ASPS tumor cells. The inset
shows abundant mitochondria (M) and lysosomes (L). C, Intra-tumor blood
vessels are positive for PDGFRB, a marker for hemangiopericytes. 2 2 3
Supplemental Figure S2. Anti-FLAG immunostaining of tumor emboli covered with non-neoplastic
cells. A, Intracardiac tumor emboli. The inset shows FLAG-negative Supplemental Figure S2. Supplemental Figure S2. Anti-FLAG immunostaining of tumor emboli covered with non-neoplastic
cells. A, Intracardiac tumor emboli. The inset shows FLAG-negative Anti-FLAG immunostaining of tumor emboli covered with non-neoplastic
cells. A, Intracardiac tumor emboli. The inset shows FLAG-negative 3 epithelium-like cells on the surface. B and C, FLAG-negative flat cells were
also present in the intravasation of tumor at the primary lesion (B) and in
lung metastasis (C). D, In vivo imaging of ASPS infected with the
pMYs-ASPSCR1-TFE3-IRES-Luc retrovirus and transplanted into GFP tg
mice on a Balb/c background. 4 4 Supplemental Figure S3. eMCs transduced with ASPSCR1 fusion genes. A, Western blotting of 293T
cells transfected with pMYs vectors bearing the indicated fusion genes. Supplemental Figure S3. eMCs transduced with ASPSCR1 fusion genes. A, Western blotting of 293T
cells transfected with pMYs vectors bearing the indicated fusion genes. eMCs transduced with ASPSCR1 fusion genes. A, Western blotting of 293T
cells transfected with pMYs vectors bearing the indicated fusion genes. 5 Flag-tagged fusion proteins are indicated. B, Immunofluorescence of 293T
cells transfected with ASPSCR1 fusion genes. Nuclear localization of fusion
proteins was confirmed for all transfectants. C, Transduction efficiencies of
eMCs infected with mutant ASPSCR1 fusion retroviruses. 6 7 7 Supplemental Figure S4. Supplemental Figure S4. Genes involved in the phagosome maturation pathway are upregulated in
ASPS tumors and eMCs expressing ASPSCR1-TFE3. Upregulated genes are
indicated in red. The illustration was made by using the IPA software. 8 8
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https://openalex.org/W3138774926
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https://www.frontiersin.org/articles/10.3389/fpsyg.2021.554595/pdf
|
English
| null |
Similarities and Differences Between Eye and Mouse Dynamics During Web Pages Exploration
|
Frontiers in psychology
| 2,021
|
cc-by
| 15,131
|
Similarities and Differences Between
Eye and Mouse Dynamics During
Web Pages Exploration
Alexandre Milisavljevic 1,2,3*, Fabrice Abate 1, Thomas Le Bras 1, Bernard Gosselin 2,
Matei Mancas 2 and Karine Doré-Mazars 1
1 Vision Action Cognition Laboratory, Psychology Institute, Université de Paris, Boulogne-Billancourt, France, 2 Information,
Signal and Artificial Intelligence Laboratory, Numediart Institute, University of Mons, Mons, Belgium, 3 Research and
Development Department, Sublime Skinz, Paris, France Alexandre Milisavljevic 1,2,3*, Fabrice Abate 1, Thomas Le Bras 1, Bernard Gosselin 2,
Matei Mancas 2 and Karine Doré-Mazars 1 1 Vision Action Cognition Laboratory, Psychology Institute, Université de Paris, Boulogne-Billancourt, France, 2 Information,
Signal and Artificial Intelligence Laboratory, Numediart Institute, University of Mons, Mons, Belgium, 3 Research and
Development Department, Sublime Skinz, Paris, France The study of eye movements is a common way to non-invasively understand and analyze
human behavior. However, eye-tracking techniques are very hard to scale, and require
expensive equipment and extensive expertise. In the context of web browsing, these
issues could be overcome by studying the link between the eye and the computer
mouse. Here, we propose new analysis methods, and a more advanced characterization
of this link. To this end, we recorded the eye, mouse, and scroll movements of 151
participants exploring 18 dynamic web pages while performing free viewing and visual
search tasks for 20 s. The data revealed significant differences of eye, mouse, and
scroll parameters over time which stabilize at the end of exploration. This suggests the
existence of a task-independent relationship between eye, mouse, and scroll parameters,
which are characterized by two distinct patterns: one common pattern for movement
parameters and a second for dwelling/fixation parameters. Within these patterns, mouse
and eye movements remained consistent with each other, while the scrolling behaved the
opposite way. Edited by: Edited by:
Sumitava Mukherjee,
Indian Institute of Technology Delhi,
India Reviewed by:
Jacek Gwizdka,
University of Texas at Austin,
United States
Sukru Eraslan,
Middle East Technical University,
Turkey *Correspondence:
Alexandre Milisavljevic
alexandre.milisavljevic@
etu.parisdescartes.fr *Correspondence:
Alexandre Milisavljevic
alexandre.milisavljevic@
etu.parisdescartes.fr Keywords: eye movement, behavior, computer mouse, scroll, web page 1. INTRODUCTION Specialty section:
This article was submitted to
Cognitive Science,
a section of the journal
Frontiers in Psychology
Received: 22 April 2020
Accepted: 28 January 2021
Published: 24 March 2021 Specialty section:
This article was submitted to
Cognitive Science,
a section of the journal
Frontiers in Psychology Websites, and more particularly web pages, refer to a type of stimulus we potentially see every day. Such stimuli are rarely entirely visible, hence the fact that we cannot fully explore them using only
our eyes. That is one of the reasons web browsing on a desktop computer requires the use and
coordination of the eyes and the computer mouse. On the one hand, the eyes are used to explore
and extract information of interest, such as the location of items. On the other hand, the mouse
is used to interact with the content. This interaction can take multiple forms, including clicks,
scrolling, and drags and drops. While clicks and drags and drops allow the user to perform actions
on the visible content, scrolling drives which part of the web page is displayed. These characteristics
specific to web pages induce more complex behaviors, as well as more challenging issues to address. One particularly interesting aspect is how the mouse relates to the eyes. Received: 22 April 2020
Accepted: 28 January 2021
Published: 24 March 2021 Keywords: eye movement, behavior, computer mouse, scroll, web page ORIGINAL RESEARCH
published: 24 March 2021
doi: 10.3389/fpsyg.2021.554595 Citation: Web pages often follow a similar
template: a header with main sections of a website, a content
with left or right bar, and a footer at the end of the web page. Thus, users developed strategies to maximize their efficiency in
visual exploration (Buscher et al., 2009). Nielsen (2010) observed
that users tend to spend more time on the left part of a web
page than on the right one. He also observed this behavior
on right-to-left reading web pages. A more recent study from
Fessenden (2017) showed a similar behavior on search engine
result pages (SERP). Nielsen (2006) ran a usability experiment
during which he analyzed which part of a web page users
were looking at. He observed a recurring viewing pattern in
the shape of the F letter. People started their browsing at the
top-left corner of the web pages and read horizontally, then
they were scrolling down to read a second time horizontally
to finally scan the content vertically. Both factors have been
widely investigated during website exploration in order to better
understand user behavior and thus improve the usability of
web pages. For instance, Pan et al. (2004) showed differences
in visual exploration depending on the type of website, their
presentation order and the gender of the user. They did not find
any difference between a memorization and a free viewing task,
highlighting the importance of adapting a website to its targeted
audience. In his work, Tullis (2007) found that older users spent
more time looking at a page content, especially navigational
areas, compared to younger users. Additionally, Roth et al. (2013) showed that user expectations had an influence on visual
exploration, and, more particularly, less fixations were needed to
find items in expected locations compared to unexpected ones. To our knowledge, despite the fact that the use of the
computer mouse is well studied, its dynamic is rarely considered. Generally, research on the computer mouse focuses on how
mouse movements could reveal users’ intentions. Its availability
and its potential for scalability enable innovative applications,
such as authentication (Zheng et al., 2011), the prediction of the
users’ cognitive load (Rheem et al., 2018), the prediction of users’
intentions (Guo and Agichtein, 2010; Fu et al., 2017), or pattern
behavior analysis (Tzafilkou and Protogeros, 2018). One of the
most studied topics is the computer mouse movement patterns
commonly used by participants when browsing. Citation: Tzafilkou and
Protogeros (2018) reviewed six patterns: the straight pattern
(Griffiths and Chen, 2007), the hesitation pattern (Mueller and
Lockerd, 2001), the horizontal reading pattern (Rodden et al.,
2008), the vertical reading pattern (Rodden et al., 2008), the
random pattern (Ferreira et al., 2010), and the fixed pattern
(Griffiths and Chen, 2007). Whether it is necessary to describe mouse movement patterns
or their dynamic, mouse movements are not limited to moving
the mouse and include scrolling as well. However, contrary to
mouse movements, scrolling behavior has, to our knowledge, not
been closely examined. For instance, Liu et al. (2017) investigated
users’ strategies when navigating SERP through their scrolling
behavior. An SERP consists of a list of links corresponding to
a query entered by a user in a search engine. Liu et al. (2017)
analyzed the number of scrolls and their direction. In their work,
Braganza et al. (2009) evaluated user preferences depending
on the web page layout and the scrolling mechanism using
the number of scrolls and their total duration. More generally,
these studies show that the mouse is a convenient and cheap
way to infer users’ cognitive processes, such as intentions or
reading strategies. However, these studies mostly focus on users’
strategies and do not tackle quantitative analyses of mouse and
scroll parameters. Such extensive statistical description could
provide a baseline of typical behavior when exploring web pages
and could be used to assess more precisely strategies or any
other behavior. These studies clearly show an influence of bottom-up and top-
down factors. However, Tatler and Vincent (2008) and Anderson
et al. (2015) show that bottom-up influence was higher at the
beginning of visual exploration. Thus, both factors alternatively
influence visual exploration (Henderson, 2003; Torralba et al.,
2006). As such, Cronin et al. (2020) encouraged the need to focus
more on the dynamic of eye movements. They showed that the
study of global eye movement parameters could not necessarily
be used to distinguish different experimental conditions. To do
so, they compared fixation durations and saccade amplitudes
between a memorization task and an esthetic judgment task. While they did not find differences in the mean level analyses,
the use of temporal and distributional analyses allowed them to
discriminate the two tasks. Previous research already highlighted the dynamic of eye
movements (Unema et al., 2005; Pannasch et al., 2008; Pannasch
and Velichkovsky, 2009). Citation: Milisavljevic A, Abate F, Le Bras T,
Gosselin B, Mancas M and
Doré-Mazars K (2021) Similarities and
Differences Between Eye and Mouse
Dynamics During Web Pages
Exploration. Front. Psychol. 12:554595. doi: 10.3389/fpsyg.2021.554595 Eye movements have been extensively studied. For instance, we know that a fixation last in
average 250–350 ms (Mackworth and Morandi, 1967; Yarbus, 1967) and that visual exploration
is modulated by bottom-up and top-down factors regardless of the stimulus type (Yarbus,
1967; DeAngelus and Pelz, 2009; Helo et al., 2014; Itti and Borji, 2015). Bottom-up factors
are characterized by low-level features of the stimulus, such as luminance, contrast, or edges March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org Similarities and Differences Between Eye and Mouse Milisavljevic et al. (Tatler
and
Vincent,
2008),
while
top-down
factors
are
characterized by high-level properties representing cognitive
processes (Henderson and Hollingworth, 1999). It is generally
assumed that the interaction between bottom-up and top-down
factors influence how we orient our visual attention (Theeuwes
and Failing, 2020). In that sense, top-down factors are usually
addressed as factors influencing bottom-up ones and are not
considered as totally distinct factors (Theeuwes and Failing,
2020). Furthermore, Still and Masciocchi (2010) pointed out
that most of web-specific biases were top-down and were mainly
related to learned behaviors. Web pages often follow a similar
template: a header with main sections of a website, a content
with left or right bar, and a footer at the end of the web page. Thus, users developed strategies to maximize their efficiency in
visual exploration (Buscher et al., 2009). Nielsen (2010) observed
that users tend to spend more time on the left part of a web
page than on the right one. He also observed this behavior
on right-to-left reading web pages. A more recent study from
Fessenden (2017) showed a similar behavior on search engine
result pages (SERP). Nielsen (2006) ran a usability experiment
during which he analyzed which part of a web page users
were looking at. He observed a recurring viewing pattern in
the shape of the F letter. People started their browsing at the
top-left corner of the web pages and read horizontally, then
they were scrolling down to read a second time horizontally
to finally scan the content vertically. Both factors have been
widely investigated during website exploration in order to better
understand user behavior and thus improve the usability of
web pages. Citation: For instance, Pan et al. (2004) showed differences
in visual exploration depending on the type of website, their
presentation order and the gender of the user. They did not find
any difference between a memorization and a free viewing task,
highlighting the importance of adapting a website to its targeted
audience. In his work, Tullis (2007) found that older users spent
more time looking at a page content, especially navigational
areas, compared to younger users. Additionally, Roth et al. (2013) showed that user expectations had an influence on visual
exploration, and, more particularly, less fixations were needed to
find items in expected locations compared to unexpected ones. over time. Pannasch and Velichkovsky (2009) and Velichkovsky
et al. (2002) defined two visual exploration modes based on the
relationship between saccade amplitudes and fixation durations. The ambient mode corresponds to short fixations (<180 ms)
followed by saccades with an amplitude >5◦, while the focal
mode corresponds to long fixations (>180 ms) followed by
saccades with an amplitude of <5◦. Generally, visual exploration
begins in ambient mode before gradually switching to focal
mode (Velichkovsky et al., 2002; Pannasch and Velichkovsky,
2009). Our knowledge on these visual modes is growing but
still incomplete. For instance, we know that a fixation last in
average 250–350 ms (Mackworth and Morandi, 1967; Yarbus,
1967) and that visual exploration is modulated by bottom-up and
top-down factors regardless of the stimulus type (Yarbus, 1967;
DeAngelus and Pelz, 2009; Helo et al., 2014; Itti and Borji, 2015). A closer understanding of these two modes could help to better
grasp the dynamic of eye movements when looking at complex
stimuli, such as web pages. More specifically, in addition to eye
movements, it would also be of interest to use these two visual
modes to investigate the dynamic of mouse movements. (Tatler
and
Vincent,
2008),
while
top-down
factors
are
characterized by high-level properties representing cognitive
processes (Henderson and Hollingworth, 1999). It is generally
assumed that the interaction between bottom-up and top-down
factors influence how we orient our visual attention (Theeuwes
and Failing, 2020). In that sense, top-down factors are usually
addressed as factors influencing bottom-up ones and are not
considered as totally distinct factors (Theeuwes and Failing,
2020). Furthermore, Still and Masciocchi (2010) pointed out
that most of web-specific biases were top-down and were mainly
related to learned behaviors. Frontiers in Psychology | www.frontiersin.org 2.3. Stimuli In this experiment, 18 web pages (see example in Figure 1)
from 18 different websites were randomly presented to the
participants. The web pages had a width of 1,920 pixels and
their total height was between 5,000 pixels and 19,230 pixels
(M = 6, 405px, SD = ±2, 673px). Participants were allowed
to freely move the mouse, scroll, or click. However, hyperlinks
and content animations were deactivated, thus participants could
not leave the displayed web page. The presented web pages and
their topic were arbitrarily chosen, including blogs, front pages,
textual pages, articles (see example in Figure 1). We ensured
that each selected web page followed several criteria to minimize
biases. The first criteria was the language of the website. We
ensured stimuli were from French websites.The second criterion
was about the websites’ news content. Since this study was run
over several months, a web page could not have any content
referring to current events or content related to a season, date,
holiday, celebration, etc. As the third criterion, we checked that
the web pages did not have any external advertising. In contrast
to the first three criteria, which were respected on all web pages,
the following criteria were counterbalanced between web pages. As Bruyer et al. (1987) explained, faces are handled differently
by our brain during visual exploration. To this end, we made
sure that we keep a balance of faces between the web pages. We also made sure that a balance was maintained for images,
texts, general layout, and total length of the web page to have
stimuli with different content types and organization. Finally,
as described in the following paragraph, we gave targets already
present within the original web page. Thus, we checked the The goal of our study was to contribute to this growing area
of research by exploring the similarities and differences between
movement of the eyes and computer mouse on web pages. First, we introduced a new segmentation threshold in order to
differentiate two mouse movements or scrolls as precisely as
possible. Then, with this new segmentation, analyses from eye
movement methodology were applied to mouse movement and
scrolling parameters. This methodology allowed us to investigate
the influence of the tasks (free viewing and visual search) on eye,
mouse, and scroll parameters. Citation: They found that the amplitude of
saccades decreased while the duration of fixations increased March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 2 Similarities and Differences Between Eye and Mouse Milisavljevic et al. These limitations can also be found when it comes to the
relationship between the eye and the computer mouse. To this
day, one of the most studied web stimuli for investigating
this relationship is the SERP. On this type of web page, the
coordination between the eyes and the computer mouse is higher
for the vertical axis of the screen than for the horizontal axis
(Rodden and Fu, 2007; Guo and Agichtein, 2010). However, this
relationship remains uncertain, considering that the mouse could
be used as a means to mark a potential result previously located
with the eyes (Rodden et al., 2008). Furthermore, the amount
of time spent by a user on an SERP can affect the location of
the gaze and the mouse during the exploration (Huang et al.,
2012). Navalpakkam et al. (2013) designed a model to predict the
location of the eyes based on the mouse location and showed that
the correlation between the eyes and the mouse is nonlinear and
user dependent. More specifically, this correlation has been found
for time periods during which a user looked at an area of interest
(AOI) and when switched between AOIs. However, SERPs are
not representative of the web and remain transitional web pages
to access a content on a different website. As a matter of fact, users
spend a significant cumulative amount of time on SERPs, but
in short bursts of time. When focusing on common web pages,
the eyes and the mouse are also coordinated on the vertical axis,
and the scroll speeds influence the position of the eyes during
scrolling (Milisavljevic et al., 2018). The participant is looking at
the opposite part of the screen when scrolling at a high speed. Moreover, the presence of the cursor in a region of the screen
correlates with the probability that the participant is fixating on
this region (Chen et al., 2001). To better estimate if the eyes and
the mouse are coordinated, Boi et al. (2016) generalized the work
of Navalpakkam et al. (2013) by defining that the eyes and mouse
must be positioned over the same content. 2.3. Stimuli Beyond these global analyses, we
also considered the influence of time on the dynamic of each type
of movement through visual exploration modes. Citation: This new definition
allowed them to improve the predictive power of the models of
Guo and Agichtein (2010) and Huang et al. (2012) when applied
to classic web pages. Finally, when it comes to the coordination
of the eyes and scrolling, web pages are not of primary interest. That is why, to our knowledge, no studies tackle the coordination
between the two outside the reading field (Kumar et al., 2007;
Sharmin et al., 2013). Participants reported normal or corrected to normal vision
and were naive about the purpose of the study. They were
right-handed or accustomed to using a computer mouse
with the right hand. A majority were undergraduate students
from the psychology institute at the Université de Paris. Participants were compensated either by course credit or
a 15 euro gift card. All procedures performed in studies
involving
human
participants
were
in
accordance
with
the ethical standards of the institutional and/or national
research
committee
(local
Ethics
Committee
of
Paris
Descartes
University,
No. CER-PD:
2018-77)
and
with
the 1964 Helsinki declaration and its later amendments
or comparable ethical standards. All subjects gave written
informed consent. 2.2. Apparatus Eye movements were recorded using an Eye-Link 1000
Plus (SR Research Ltd., Canada) at a 1,000 Hz sampling
rate with 0.05◦precision. We recorded the right eye of the
participants with a 35 mm monocular lens. Mouse movements
were recorded with a standard USB optical mouse with
a 125 Hz polling rate. Stimuli were displayed on a 24.5
inch LCD computer screen with a 1,920 × 1,080 pixel
resolution and a 144 Hz refresh rate. The experiment was
run using Python 2.7 with Pylink from the manufacturer and
Chromium 64. 2.1. Participants We recruited 151 participants (127 females and 24 males)
aged between 18 and 56 (M
=
22.77, SD
=
±6.33). March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 3 Similarities and Differences Between Eye and Mouse Milisavljevic et al. FIGURE 1 | Example of website displayed during the experiment. number of targets available on the web page and their distribution
across the page. 2.4. Tasks Participants had to perform a total of nine free viewing tasks and
nine visual search tasks randomly distributed on the 18 websites
following a uniform distribution. Thus, each participant executed
one task per web page. The balance of tasks per web page was
ensured before any analyses. During the free viewing task, the
participants were instructed to explore the web page freely for
exactly 60 s. This duration was chosen after multiple trials and
errors to provide enough data for the study of long browsing. Thus, participants had enough time to fully explore the web
page. In the visual search task, participants were asked to find
a target in an arbitrarily maximum of 2 min. The participants
did not know how many targets there were but we informed
them that there were up to three targets, with at least one, per
web page. As previously defined, the targets were icons or images
present on the original web page. Moreover, the targets were
equally distributed between the top, middle, and bottom of the
web page, and could be found on the sides, or in the content,
header, or footer. 2.6.1. Data Cleaning Moreover, several studies focused specifically on scroll
segmentation (Braganza et al., 2009; Brady et al., 2018;
Milisavljevic et al., 2018). In their study into the scrolling
behavior, Braganza et al. (2009) determined that two scrolls
recorded within 1 s of each other were considered as a single
scroll. To set this threshold, they tried values ranging from 200
ms to 4 s, with increments of 100 ms. They did not find any major
differences between these timings, and consequently chose 1 s as a
threshold. In their study, Milisavljevic et al. (2018) defined a scroll
session as a set of continuous scroll events ended with a mouse
movement. On the topic of scrolling when reading, Brady et al. (2018) sampled a frame every 100 ms to check if the displayed
text had moved. If it had moved more than half a line between one
sentence and the next, it was counted as a scroll. Even though the
presented techniques try to segment scrolling or mouse events,
these techniques are mostly based on arbitrary thresholds. Thus,
our goal is to propose a better approach of mouse and scroll
events segmentation to provide more robust analyses. Data from 12 participants who did not finished the experimental
protocol due to calibration problems were discarded. Among
the remaining 139 participants (2,502 trials), due to problems
encountered
during
the
experiment,
such
as
calibration
problems, participants talking during a trial, external noise, etc.,
we removed 4.88% of all trials (122 trials). The remaining data
(2,380 trials) was then pre-processed and cleaned in three steps. The first step was only applied to the visual search task. The
two last seconds of recording were removed in order to deal
with the moment the participant looked at the keyboard when
pressing the space bar. In addition, and for the same reason,
residual fixations below the screen at the end of the exploration
were removed. Throughout the second step, blinks and fixations
under 100 ms around a blink were cleaned (Holmqvist et al.,
2011). During the third and final step, fixations with a visual
angle of more than 3◦from the screen’s border were deleted. Fixations outside the screen, but below the 3◦threshold, were
reset to the corresponding border of the screen. These three steps
led to deletions within all the trials. All 139 participants, and
95% of the initial trials (2,378 trials), were kept. 2.5. Procedure In a quiet room, with constant luminosity, the participants were
instructed to position their head on a chin rest in front of a
computer screen at a viewing distance of 57 cm. The experiment
then began with practice trials, one for each task. After this
phase, the participants’ right eye was calibrated at nine points
and this was repeated until the error value was below 1◦. Once
the calibration was successfully complete, the participants had to
click on the next trial with the mouse on a 3 × 6 table, as shown in
Figure 2. Then the instructions were displayed on a new screen
with a button to launch the trial. The position of this button was
randomly chosen in order to avoid bias related to the first fixation
commonly being at the same position as the button launching the
trial. Furthermore, to ensure the web page would have completely
loaded before the trial started a 3-s countdown was added to
the button launching the trial. The countdown only began after
the page entirely loaded, thus visual elements displayed after few
seconds could be avoided. During this phase, the participants
were informed of the presence of maximum three targets when
carrying out the visual search task. After clicking on the button,
the web page was displayed for 60 or 120 s, depending on the
task. During the visual search task, the participants had to click
on the targets when they founded them. If the image clicked was
one of the targets, a green rectangle surrounded the target to
indicate that one of the targets had been found. The participants
were instructed to press the space bar on the keyboard when they
thought they had found all the targets. After 1 min of the free
viewing task, and 2 min or after the space bar was pressed in
the visual search, the recording was stopped, and the 3 × 6 table
displayed at the beginning was displayed again. Between each
trial a 5-point calibration was performed. A 9-point calibration
was initiated after the ninth trial, or if any problems occurred
during the experiment. FIGURE 1 | Example of website displayed during the experiment. FIGURE 1 | Example of website displayed during the experiment. FIGURE 1 | Example of website displayed during the experiment. 2.5. Procedure March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 4 Similarities and Differences Between Eye and Mouse Milisavljevic et al. March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 2.6.2. Events Segmentation FIGURE 2 | Example of screen on which participants had to click the next
item to get the instruction. The white button indicates a website not yet visited,
the green button a website already visited, and the blue button the next
website to visit. Only the blue button was clickable. There are a number of well-established, and ever improving,
methods to label raw data from eye recordings. However,
mouse and scroll recordings lack such a method, specifically to
differentiate two close events. While it is easy to determine if
two mouse or scroll events separated by 2 or 3 s are indeed two
distinct events, doing the same operation for 2 events with, for
instance, <1 s in between is much difficult. In the literature, we can find multiple attempts to define a
threshold allowing the differentiation of idle time and movement
of the mouse. Since the mouse is a pointing device, a simple
threshold seems to be appropriate, contrary to eye movements
that are more complex. In their attempt to define a new
behavioral biometric technique based on mouse movements,
Gamboa and Fred (2004) differentiated two mouse movements
as a pause in the user’s interaction when the two consecutive
events were separated by more than 100 ms. In their work,
Reeder and Maxion (2006) arbitrarily considered a threshold of
3 s with to the user being silent and inactive (with both the
mouse and the keyboard) in order to propose a method to detect
user difficulties when using an interface. On the other hand,
Feher et al. (2012) empirically set this threshold to 500 ms to
categorize mouse movements and thus uniquely identify users. More recently, Seelye et al. (2015) studied cognitive impairment
using computer mouse movement patterns. They mentioned a
median idle time, which is the time spent idling or pausing
between mouse movements, of 310 ms. In the continuity of the
work of Gamboa and Fred (2004), Antal and Egyed-Zsigmond
(2019) used a threshold of 10 s to segment mouse movements
and used them to detect intruders on a computer. FIGURE 2 | Example of screen on which participants had to click the next
item to get the instruction. The white button indicates a website not yet visited,
the green button a website already visited, and the blue button the next
website to visit. Only the blue button was clickable. 2.6.1. Data Cleaning In total, 91.74%
of all records were retained for analyses. Finally, only the first
20 s were selected for this work, and 18 more trials were deleted
due to insufficient mouse moves or scrolling events (2,360 trials
remaining). It should be noted that eye movement analyses were
run on aggregated data, and scrolling and mouse events on raw
data. All analyses were carried out using Python 3.6. If we take a closer look at our previous attempt to segment
events, we defined a threshold based on the events number rather
than the time (Milisavljevic et al., 2018). This definition does
not take into account all parameters that come into play when
interacting using mouse or scroll. The main parameter is the fact
that, on a desktop, it is possible to move the mouse during a scroll. In such a case, a single scroll would be labeled as two different
scrolls. The bias will remain if the participant uses the browser
scroll bar, which allows the user to grab a bar on the right of the March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 5 Similarities and Differences Between Eye and Mouse Milisavljevic et al. browser and scroll by moving it up or down. Furthermore, Brady
et al. (2018) used a spatial threshold of 40 pixels to identify when a
user was scrolling, but this is applicable to mouse movements. In
addition to highlighting the need to use a time-based threshold,
all previously mentioned studies did not correctly handle stops
and micro-stops. A stop is a period of time during which the
user does not move the mouse or scroll. During this idle time,
the user explores the web page and processes it. But based on
this definition, a new question arises: what is the minimal length
of this period of time to give the user enough time to process
the stimulus and make the decision to keep moving, scrolling,
or stop entirely? In other terms, how can we differentiate micro-
stops from the movement itself? A micro-stop is an interruption
during the action which is long enough to allow the user to make
a decision, but this is not visible to the eye. 2.6.3. Variables After all cleaning processes, we ran our analyses on a wide range
of new parameters. In the state-of-the-art, the same types of
parameters are frequently used. For the use of the mouse, these
include curvature, trajectory, clicks, dwells, or the number of
movements (Zheng et al., 2011; Fu et al., 2017; Rheem et al.,
2018; Tzafilkou and Protogeros, 2018), and for scrolls, amplitude,
speed, and number (Braganza et al., 2009; Liu et al., 2017;
Milisavljevic et al., 2018). In comparison, eye-mouse studied
parameters are more related to their respective positions, but
are not limited to this factor. For instance, eye-mouse distance,
content hovered, lag, percentage of regions visited by both the
eyes and mouse, etc., have been used to study their relationship
(Chen et al., 2001; Rodden and Fu, 2007; Rodden et al., 2008; Guo
and Agichtein, 2010; Huang et al., 2012; Navalpakkam et al., 2013;
Boi et al., 2016). 2.6.4. Mouse and Scroll Overlap Participants were able to independently move the mouse
and scroll. Consequently, this led to overlaps between mouse
movements and scrolls. We found that this overlap occurred only
10% (SD = ±4.83%) of the total mouse movement time and
15% (SD = ±10.59%) of the total scrolling time. During these
overlaps, we observed mouse movements with an amplitude of
0.02◦(SD = ±0.02◦) and a duration of 240 (SD = ±195.53ms)
for a total duration of 570 (SD = ±430ms). As described,
during overlaps, movements represented a negligible part of
the exploration. Moreover, these overlaps followed three main
patterns: move–scroll, scroll–move, and move–scroll–move. The
move–scroll pattern refers to a scroll that began while already
moving the mouse. This pattern occurred 43% of the time and
was the most frequent. The second pattern we observed was
the scroll–move pattern. This pattern is the exact opposite: the
participant began to move the mouse while already scrolling. This pattern happened 25% of time. The move–scroll–move
pattern is when the participant scrolled within a single mouse
move. This was less common and occurred 21% of the time. Finally, the 11% remaining was exotic patterns, such as move–
scroll–move–scroll or move–scroll–move–scroll–move, which
represent 2% each. Due to the low frequency of overlaps between
scrolls and mouse movements, we can safely conclude that
these specific movements are residual movements or involuntary
micro-movements generated by the use of the mouse wheel. For this reason, we did not take overlaps into account in the
following analyses. 2.6.1. Data Cleaning To differentiate micro-
stops from movements, we looked at the study from Moher and
Song (2019) in which they compared behaviors between a 3D
reach tracker, a computer mouse, and a stylus. Among multiple
conditions, they measured the average response latency of 220
ms when displacing a target. This could be considered as the
minimum time to visualize a target’s new position and make the
decision to reorient the movement. Thus, a micro-stop could not
be <220 ms, and a stop below this threshold should be considered
as the continuity of the previous action. We used a unified
threshold to segment mouse movements and scrolls. We chose
a threshold of 300 ms to differentiate two distinct movements or
scrolls. This corresponds to the average visual fixation duration
in a scene viewing (Henderson and Hollingworth, 1998). Despite
the fact that visual fixations can be shorter than 300 ms, this does
not apply to ecological conditions and semantic-rich stimuli,
such as web pages. to mouse and scroll events. This coefficient is calculated by
averaging the differences in z-scores between the duration of
each fixation and the next saccade, as shown in Equation (1). A
negative value indicates that the fixation di is short and the next
saccade ai+1 is long (>5◦). In contrast, a positive value suggests
that the fixation di is long and the next saccade ai+1 is short (<5◦)
which corresponds to a focal mode. K = 1
n
X
n
di −µd
σd
−ai+1 −µa
σa
(1) (1) Milisavljevic et al. (2019) introduced two new variables to better
capture the dynamic of focal and ambient modes. While the K
coefficient did not discriminate between the different stimuli used
in their study, the number of switches between modes did. It
is for this reason that we are using these parameters to more
precisely describe the dynamic of the exploration for both the
eyes and mouse. Milisavljevic et al. (2019) introduced two new variables to better
capture the dynamic of focal and ambient modes. While the K
coefficient did not discriminate between the different stimuli used
in their study, the number of switches between modes did. It
is for this reason that we are using these parameters to more
precisely describe the dynamic of the exploration for both the
eyes and mouse. Frontiers in Psychology | www.frontiersin.org 3. RESULTS Post-hoc analyses were run using the pairwise Student’s t-test
with a Bonferroni correction. It should be noted that only mouse
and scroll movement parameters are presented in this section
(see Table 1 for dwell parameters). Contrary to a fixation that
provides information of current cognitive processes, a dwell
generally means that the mouse have not been used. Moreover,
the duration of a dwell is much longer than a fixation and
can easily last the equivalent of 10 fixations. This difference
of scale does not make it possible to determine what falls
within the scope of the cognitive process in progress, or the
simple nonuse of the mouse. However, movement parameters
remain comparable. 3.1.1. Number of Fixations and Saccades
Globally, participants made an average of 72 (SD = ±6.5)
fixations and saccades during the exploration of a website for 20
s. The task had an effect on the number of fixations and saccades
[F(1,138) = 424.29, p < 0.001] with less fixations and saccades
during the visual search (M = 68.4, SD = ±6.31) compared to
the free viewing task (M = 75.16, SD = ±7.08). We found a time
effect [F(3,414) = 27.86, p < 0.001], but there were no significant
differences between the first and second time-bins (t = 0.32, p >
0.05). However, there was a significant decrease in the number
of fixations and saccades between the second and third time-bins
(t = −4.84, p < 0.001), as well as between the third and fourth
time-bins (t = −2.85, p < 0.05). The interaction between the
time and task was also significant [F(3,414) = 3.29, p < 0.05]. The
main task effect was maintained for each time-bin (all p <0.001). In free viewing task, there were no significant differences between
the successive time-bins (all p >0.05). However, in visual search,
the only difference with the main time effect was the absence
of a reduction between the third and fourth time-bins (p >0.05)
(see Table 2). 3. RESULTS In this paper, we propose a more complete set of parameters
directly inspired from eye movement analyses. These parameters
include
dwell
duration,
movement
duration,
movement
amplitude, and number of events. It should be noted that
duration variables are expressed in seconds or milliseconds,
while amplitude variables are expressed in degrees of visual angle. Furthermore, in order to better characterize the dynamic of the
exploration through ambient and focal visual modes, we apply,
for the first time, the K coefficient defined by Krejtz et al. (2016) To study the similarities and differences between eye movements,
mouse movements, and scrolling, we ran two types of analyses. We first described eye, mouse, and scroll parameters globally,
to clearly define what a mouse or scroll movement was, and
summarized them in Table 1. Then, we examined the role of tasks
and time, by performing a 2 (free viewing and visual search) X 4
(0–5 s time-bin, 5–10 s time-bin, 10–15 s time-bin, and 15–20 March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 6 Similarities and Differences Between Eye and Mouse Milisavljevic et al. TABLE 1 | Global means and standard deviations of all studied variables (139 participants on 18 web pages for 20 s each). Eye
Mouse
Scroll
Mean
Std. Mean
Std. Mean
Std. Fixations/Dwells
% of time
85.76%
±1.72%
79.15%
±8.33%
83.42%
±5.32%
Avg. count
72.18
±6.5
8.71
±2.57
10.07
±1
Avg. duration
236.97 ms
±24.45 ms
2.49 s
±1 s
2.3 s
±0.71 s
Tot. duration
16.83 s
±0.33 s
15.68 s
±1.65 s
16.52 s
±2.33 s
Movements
% of time
14.24%
±1.72%
20.85%
±8.33%
16.58%
±5.32%
Avg. count
72.18
±6.5
6.04
±1.78
8.77
±2.04
Avg. duration
39.04 ms
±4.29 ms
768.24 ms
±342.55 ms
367.57 ms
±121.65 ms
Tot. duration
2.8 s
±0.34 s
4.13 s
±1.65 s
3.28 s
±1.39 s
Avg. amplitude
6.10◦
±0.67◦
0.27◦
±0.23◦
8.52◦
±2.35◦
Tot. amplitude
435.22◦
±52.74◦
1.6◦
±0.71◦
70.79◦
±19.70◦
Dynamic
Avg. K coeff. −0.13
±0.2
−0.35
±0.63
0.43
±0.45
Avg. nb. switches
33.15
±3.25
3.78
±0.89
3.63
±0.74
% time in ambient
42.82%
±6.81%
–
–
–
–
% time in focal
57.37%
±6.8%
–
–
–
– TABLE 1 | Global means and standard deviations of all studied variables (139 participants on 18 web pages for 20 s each). 3.1.1. Number of Fixations and Saccades s time-bin) repeated measures analyses of variance (ANOVAs). 3.1. Eye Movements Analysis We measured a stable distribution of fixations and saccades
across the different conditions. During the exploration of a
website, participants spent approximately 14% (SD = ±1.72%)
of the time making a saccade (see Table 1). Although this
proportion was maintained across the tasks, we found a task
effect on the distribution of fixations/saccades [F(1, 138) = 231.98,
p < 0.001]. Participants spent 13.6% (SD
=
±1.79%) of
the time making a saccade in the free viewing task and 15%
(SD = ±1.84%) during the visual search task. Furthermore,
we found a time effect [F(3, 414) = 685.59, p < 0.001] present
between the first and second time-bins (t
= −29.50, p <
0.001), and between the second and third time-bins (t = 8.98,
p
<
0.001), but not between the third and fourth time-
bins (t
=
−2.33, p
>
0.05). We also found a significant
interaction effect between task and time [F(1, 138) = 3.48, p <
0.05], and post-hoc analyses confirmed that main effects were
preserved (see Table 2). l sea
8
1
26
1
1
4
−
4
5 3.2. Mouse Analysis The participants spent 20.85% (SD = ±8.33%) of the time
moving the mouse during their exploration. We found a
significant task effect [F(1,138)
=
37.66, p
<
0.001], the
proportion of time spent moving the mouse was significantly
higher in the visual search task (M = 23.33%, SD = ±8.48%)
than in the free viewing task (M = 18.94%, SD = ±10.11%). We also observed a time effect [F(3,414) = 420.24, p < 0.001]
with a significant decrease between the first and second time-bins
(t = −24.14, p < 0.001), and between the second and third time-
bins (t = −3.25, p < 0.01). However, there was no significant
difference between the third and fourth time-bins (t = −1.68,
p > 0.05). There was a significant interaction between time and
task [F(3,414) = 7.75, p < 0.001]. The main task effect was
maintained excepted for the second time-bin (t = 1.2, p > 0.05). The main time effect was preserved in the free viewing, but not
entirely during the visual search task, there was no significant
difference between the second and third time-bins (p >0.05) (see
Table 3). 3.1.2. Fixation Duration We observed an average fixation duration of 236 ms (SD =
±24.45ms). The average fixation duration varied according to
the task [F(1,138) = 195.75, p < 0.001]. Fixations were shorter
during the free viewing task (M = 229ms, SD = ±24.59ms) than
during the visual search task (M = 247.17ms, SD = ±26.41ms). The average fixation duration significantly increased over time
[F(3,414) = 297.65, p < 0.001] up to the third time-bin. More
precisely, the first time-bin was significantly different from the
second time-bin (t = 20.91, p < 0.001), and this second time-
bin was significantly different from the third time-bin (t = 6.80, March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 7 search
T3V
T4V
Non-significant
85.90 ± 1.92
85.91 ± 1.95
[T3F-T4F, T3V-T4V]
16.98 ± 1.79
16.70 ± 1.74
[T1F-T2F, T2F-T3F, T3F-T4F, T1V-T2V,
T3V-T4V]
261.18 ± 33.71
262.53 ± 33.60
[T3F-T4F, T3V-T4V]
14.10 ± 1.92
14.09 ± 1.95
[T3F-T4F, T3V-T4V]
16.98 ± 1.79
16.70 ± 1.74
[T1F-T2F, T2F-T3F, T3F-T4F, T1V-T2V,
T3V-T4V]
41.58 ± 4.71
41.83 ± 5.21
[T3F-T4F, T3V-T4V]
6.82 ± 0.96
6.84 ± 1.01
All
−0.08 ± 0.31
−0.07 ± 0.29
All
8.53 ± 1.18
8.28 ± 1.24
[T3F-T3V, T3F-T4F, T2V-T3V, T3V-T4V]
43.75 ± 9.46
44.03 ± 8.92
[T3F-T4F, T3V-T4V]
57.10 ± 9.43
56.83 ± 8.88
[T3F-T4F, T3V-T4V] Similarities and Differences Between Eye and Mouse Milisavljevic et al. p < 0.001). However, the third time-bin was not significantly
different from the fourth (p >0.05. There was also an interaction
effect between task and time [F(3,414) = 3.29, p < 0.05], but
post-hoc analyses confirmed that main effects were preserved
(see Table 2). third time-bin (t = 4.33, p > 0.05). Similarly, the main time
effect was preserved for the free viewing task, but not in the visual
search task, during which there were no significant differences
between the second and third, and the third and fourth time-bins
(all p >0.05) (see Table 2). 3.1.4. Dominant Mode To summarize, we found a task and time effect on all the
variables of eye movements parameters. Most of the parameters
increased over time to then stabilize starting at the third
time-bin (after 10–15 s). More specifically, fixation-related
variables increased and movement-related variables decreased
over time. Moreover, ambient mode was predominant during
the exploration but progressively switched to focal mode as time
went by. Finally, to understand the dynamic of visual exploration, we
computed the K coefficient and its associated variables, as defined
by Krejtz et al. (2016) and Milisavljevic et al. (2019), and
described in the Materials and Methods section. Globally, we
found a dominance of the ambient mode with a K coefficient
below zero (M = −0.13, SD = ±0.2). There was a significant
difference between tasks [F(1,138) = 313.8, p < 0.001], which
indicated a higher dominance of the ambient mode in the visual
search task (M = −0.28, SD = ±0.23) than in the free viewing
task (M = −0.01., SD = ±0.21.) We also found a significant time
effect [F(3,414) = 579.66, p < 0.001]. The K coefficient, beginning
with negative values, got significantly closer to 0 between the first
and second time-bins (t = −27.10, p < 0.001), became positive
between the second and third time-bins (t = −10.23, p < 0.001),
but did not significantly change between the third and fourth
time-bins (t = 1.94, p > 0.05). Post-hoc analyses did not show a
significant interaction between the task and time [F(3,414) = 1.97,
p > 0.05] (see Table 2). 3.1.6. Visual Modes Proportions The participants spent, in total, 43% (SD = ±6.81%) of the time
in ambient mode. This proportion significantly varied according
to the task [F(1,138) = 358.75, p < 0.001]. It was higher in the
visual search task (M = 48.35%, SD = ±7.33%) than in the
free viewing task (M = 38.21%, SD = ±7.65%). There was
a significant time effect [F(3,414) = 638.94, p < 0.001]. The
proportion of time spent in ambient mode significantly decreased
between all successive time-bins: between the first and second
time-bins (t = −31.30, p < 0.001), between the second and
third time-bins (t = −9.32, p < 0.001), and between the third
and fourth time-bins (t = −1.44, p > 0.05). We also found a
significant interaction between the time and task [F(3,414) = 8.75,
p < 0.001], but post-hoc analyses confirmed that main effects
were preserved (see Table 2). We measured an average saccade amplitude of 6.1 ◦(SD
=
±0.67◦). We found a significant difference between the tasks
[F(1,138) = 1314.42, p < 0.001], saccade amplitudes were shorter
during the free viewing task (M = 5.08◦, SD = ±0.77◦) than
during the visual search task (M = 7.36◦, SD = ±0.77◦). We also
observed a time effect [F(3,414) = 378.60, p < 0.001] up to the
third time-bin. The average saccade amplitude decreased from
the first to the second time-bin (t = −21.27, p < 0.001), and
from the second to the third time-bin (t = −8.45, p < 0.001),
but not between the third and fourth time-bins (t = −1.55,
p > 0.05). However, there was no significant interaction between
the time and task [F(3,414) = 2.11, p > 0.05] (see Table 2). 3.1.5. Visual Modes Switches As described in the Methodology section, the number of visual
modes switches corresponds to how many times a participant
switched from ambient to focal and focal to ambient during a
trial. Participants switched between visual modes 33.15 (SD =
±3.25) times and this amount varied according to the task
[F(1,138) = 63.06, p < 0.001]. There were more switches in the
free viewing task (M = 34.26, SD = ±4.30) than in the visual
search task (M = 31.67, SD = ±3.22). There was also a time
effect [F(3,414) = 22.69, p < 0.001]. The number of visual mode
switches significantly increased between the first and second
time-bins (t = 8.05, p < 0.001), but significantly decreased
between the second and third time-bins (t = −4.05, p < 0.001). It was not, however, significantly different between the third and
fourth time-bins (t = −1.24, p > 0.05). Furthermore, we found a
significant interaction between the task and time [F(3,414) = 6.33,
p < 0.001]. The main task effect was maintained except for the 3.2.1. Number of Mouse Movements The participants did 6.04 (SD = ±1.78) movements on average. We found a task effect [F(1,138) = 73.45, p < 0.001] with more
mouse movements during the visual search task (M = 6.77,
SD = ±2.01) than during the free viewing task (M = 5.43,
SD = ±1.97). We found an influence of time [F(3,414) = 183.46,
p < 0.001] with a significant decrease between the first and March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 9 Similarities and Differences Between Eye and Mouse Milisavljevic et al. second time-bins (t = −14.34, p < 0.001), and between the
second and third time-bins (t = −4.70, p < 0.001). However,
there was no significant difference between the third and fourth
time-bins (t = −1.79, p > 0.05). We also found a significant
interaction between time and task [F(3,414) = 14.15, p < 0.001]. The main task effect was preserved excepted for the second time-
bin (p >0.05). In the free viewing task, the main time effect
was preserved, but in the visual search task this main effect
was maintained only between the first and second time-bins (p
<0.001) (see Table 3). TABLE 3 | Means and standard deviations of all studied variables as a function of tasks and time-bins for the mouse (139 participants on 18 web pages for 20 s each). Free Viewing
Visual Search
T1F
T2F
T3F
T4F
T1V
T2V
T3V
T4V
Non-significant
Fixations/Dwells
% of time (%)
63.68 ± 12.82
79.74 ± 13.30
82.44 ± 12.71
83.85 ± 12.24
60.14 ± 12.15
79.39 ± 11.25
77.84 ± 10.76
77.70 ± 10.53
[T2F-T2V, T3F-T4F, T2V-T3V, T3V-T4V]
Avg. count
3.52 ± 0.51
3.38 ± 0.73
3.22 ± 0.66
3.13 ± 0.66
3.71 ± 0.59
3.51 ± 0.79
3.54 ± 0.70
3.57 ± 0.78
[T3F-T4F, T2V-T3V, T3V-T4V]
Avg. duration (s)
1.00 ± 0.29
1.35 ± 0.43
1.45 ± 0.43
1.52 ± 0.43
0.89 ± 0.27
1.30 ± 0.39
1.25 ± 0.35
1.24 ± 0.35
[T3F-T4F, T2V-T3V, T3V-T4V]
Movements
% of time (%)
36.43 ± 12.79
22.32 ± 13.38
20.22 ± 12.96
18.31 ± 12.88
39.90 ± 12.19
22.97 ± 11.24
23.88 ± 11.01
23.96 ± 10.22
[T2F-T2V, T3F-T4F, T2V-T3V, T3V-T4V]
Avg. count
2.19 ± 0.44
2.04 ± 0.62
1.95 ± 0.62
1.79 ± 0.55
2.34 ± 0.48
2.12 ± 0.71
2.11 ± 0.59
2.15 ± 0.60
[T2F-T2V, T3F-T4F, T2V-T3V, T3V-T4V]
Avg. 3.2.3. Amplitude of Mouse Movements p
The participants performed mouse movements of 0.27◦(SD =
±0.23◦) on average. We found a significant differences between
the two tasks [F(1,138)
=
24.16, p
<
0.001]. The average
amplitude slightly decreased from the free viewing task (M =
0.26◦, SD = ±0.2◦) to the visual search task (M
= 0.3◦,
SD = ±0.3◦). We also found a time effect [F(3,414) = 235.57,
p < 0.001]. There was a significant decrease between the first and
second time-bins (t = −17.57, p < 0.001), but no significant
differences between the second and third time-bins (t = −2.42,
p > 0.05) or between the third and fourth time-bins (t = 0.22,
p > 0.05). We did not find any interaction effect [F(3,414) = 1.61,
p > 0.05] (see Table 3). 3.2.2. Duration of Mouse Movements 3.2.2. Duration of Mouse Movements
The participants moved the mouse for 768 ms (SD
=
±342.55ms) on average. We found a task effect [F(1,138) = 15.63,
p < 0.001] with significantly longer mouse movements in the
free viewing task (M = 772.68ms, SD = ±362.58ms) than
in the visual search task (M = 767.43ms, SD = ±386.39ms). Moreover, we found a time effect [F(3,414) = 269.83, p < 0.001]
with a significant decrease between the first and second time-
bins (t = −19.53, p < 0.001), but no significant difference
between the second and third time-bins (t
= −2.56, p >
0.05) or between the third and fourth time-bins (t = 0.74,
p > 0.05). We also found a significant interaction between
time and task [F(3,414)
=
3.69, p
<
0.05]. However, the
main task effect was preserved only for the two last time-bins
(all p <0.005), while the main time effect was only preserved
for the visual search task. During the free viewing task, we
observed significant differences between the first and second
time-bins, and between the second and third time-bins (all p
>0.05) (see Table 3). 3.2.1. Number of Mouse Movements duration (ms)
965 ± 399
570 ± 431
511 ± 358
517 ± 423
1 ± 000 , 452 572 ± 334
589 ± 328
592 ± 303
[T1F-T1V, T2F-T2V, T3F-T4F, T2V-T3V,
T3V-T4V]
Avg. amplitude (◦)
0.43 ± 0.27
0.15 ± 0.16
0.11 ± 0.12
0.13 ± 0.17
0.46 ± 0.31
0.18 ± 0.24
0.19 ± 0.16
0.20 ± 0.25
All
Dynamic
Avg. K coeff. −1.02 ± 0.74
−0.05 ± 0.71
0.34 ± 0.64
0.63 ± 0.79
−1.13 ± 0.81
−0.14 ± 0.99
0.08 ± 0.55
0.12 ± 0.64
All
Avg. switch nb. 1.61 ± 0.32
1.51 ± 0.37
1.45 ± 0.34
1.43 ± 0.37
1.65 ± 0.32
1.60 ± 0.40
1.60 ± 0.39
1.64 ± 0.38
[T1F-T1V, T3F-T4F,T2V-T3V, T3V-T4V] 3.2.4. Dynamic of Mouse Movements Here, K coefficient is used to better understand the mouse
movement dynamic. The K coefficient showed a dominance of
the ambient mode (M = −0.35, SD = ±0.63). We found
significant differences between tasks [F(1,138)
= 15.27, p <
0.001], which was slightly higher in the free viewing task (M =
−0.31, SD = ±0.58) than in the visual search task (M
=
−0.39, SD = ±0.77). There also was a significant time effect
[F(3,414) = 410.86, p < 0.001]. We found a significant increase
between all successive time-bins (all p <0.001). However, there
was no significant interaction effect [F(3,414) = 2.48, p > 0.05]
(see Table 3). March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 10 Similarities and Differences Between Eye and Mouse Milisavljevic et al. TABLE 4 | Means and standard deviations of all studied variables as a function of tasks and time-bins for scrolling (139 participants on 18 web pages for 20 s each). Free viewing
Visual search
T1F
T2F
T3F
T4F
T1V
T2V
T3V
T4V
Non-significant
Fixations/Dwells
Amount of time (%)
91.40 ± 3.51
86.19 ± 6.16
85.58 ± 5.96
84.58 ± 6.46
82.14 ± 8.23
73.54 ± 9.35
74.09 ± 8.66
72.56 ± 9.20
[T2F-T3F,T3F-T4F, T2V-T3V,T3V-T4V]
Avg. count
2.50 ± 0.45
3.24 ± 0.62
3.28 ± 0.66
3.35 ± 0.67
3.35 ± 0.73
4.36 ± 0.80
4.35 ± 0.72
4.29 ± 0.71
[T2F-T3F,T3F-T4F, T2V-T3V,T3V-T4V]
Avg. duration (s)
1.91 ± 0.29
1.50 ± 0.33
1.48 ± 0.31
1.44 ± 0.32
1.39 ± 0.39
0.95 ± 0.30
0.95 ± 0.26
0.93 ± 0.27
[T2F-T3F,T3F-T4F, T2V-T3V,T3V-T4V]
Movements
Amount of time (%)
8.68 ± 3.53
13.84 ± 6.18
14.46 ± 5.94
15.45 ± 6.43
17.90 ± 8.19
26.46 ± 9.35
25.91 ± 8.66
27.58 ± 9.57
[T2F-T3F,T3F-T4F, T2V-T3V,T3V-T4V]
Avg. count
1.50 ± 0.44
2.22 ± 0.62
2.26 ± 0.64
2.33 ± 0.67
2.35 ± 0.73
3.35 ± 0.81
3.32 ± 0.72
3.26 ± 0.68
[T2F-T3F,T3F-T4F, T2V-T3V,T3V-T4V]
Avg. duration (ms)
292 ± 125
305 ± 99
316 ± 104
333 ± 148
385 ± 172
397 ± 129
394 ± 141
429 ± 158
All
Avg. amplitude (◦)
5.88 ± 3.08
6.48 ± 3.08
6.46 ± 2.42
6.78 ± 2.56
9.29 ± 3.56
9.78 ± 2.87
10.12 ± 3.58
10.47 ± 3.62
[T2F-T3F,T3F-T4F, T2V-T3V,T3V-T4V]
Dynamic
Avg. K coeff. 3.2.5. Mode Switches 3.2.5. Mode Switches
On average, 3.78 (SD = ±0.89) switches occurred between
modes given by the K coefficient. There was a significant task
effect [F(1,138) = 70.08, p < 0.001], which was characterized by
a lower number of mode switches during the free viewing task
(M = 3.44, SD = ±1.04) than during the visual search task
(M = 4.19, SD = ±1.07). There was also a significant time
effect [F(3,414) = 109.86, p < 0.001]. The number of switches
significantly increased between the first and second time-bins
(t = 11.68, p < 0.001), and between the second and third time-
bins (t = 3.72, p < 0.005), but there was no significant difference
between the third and fourth time-bins (t = 1.42, p > 0.05). We also found a significant interaction between the time and
task [F(3,414) = 11.93, p < 0.001]. The main task effect was
preserved except for the first time-bin (p <0.05). Furthermore,
the main time effect was maintained for the free viewing task,
but, for the visual search task, the first and second time-bins were
significantly different (p <0.001), while remaining time-bins did
not have significant differences (all p >0.05) (see Table 3). On average, 3.78 (SD = ±0.89) switches occurred between
modes given by the K coefficient. There was a significant task
effect [F(1,138) = 70.08, p < 0.001], which was characterized by
a lower number of mode switches during the free viewing task
(M = 3.44, SD = ±1.04) than during the visual search task
(M = 4.19, SD = ±1.07). There was also a significant time
effect [F(3,414) = 109.86, p < 0.001]. The number of switches
significantly increased between the first and second time-bins
(t = 11.68, p < 0.001), and between the second and third time-
bins (t = 3.72, p < 0.005), but there was no significant difference
between the third and fourth time-bins (t = 1.42, p > 0.05). We also found a significant interaction between the time and
task [F(3,414) = 11.93, p < 0.001]. The main task effect was
preserved except for the first time-bin (p <0.05). Furthermore,
the main time effect was maintained for the free viewing task,
but, for the visual search task, the first and second time-bins were
significantly different (p <0.001), while remaining time-bins did
not have significant differences (all p >0.05) (see Table 3). 3.3. Scroll Analysis The participants, globally, spent 16.58% (SD = ±5.32%) of a
trial scrolling. There was a task effect [F(1,138) = 469.10, p <
0.001]. The proportion of time spent scrolling was higher in the
visual search task (M = 23.80%, SD = ±8.28%) compared
to the free viewing task (M = 10.86%, SD = ±4.87%). We
also found a time effect [F(3,414) = 239.92, p < 0.001]. There
was a significant increase between the first and second time-bins
(t = 20.74, p < 0.001), as well as between the third and fourth
time-bins (t = 3.70, p < 0.005), while there was no significant
differences between the second and third time-bins (t = 0.06,
p > 0.05). We found a significant interaction between the time
and task [F(3,414) = 11.94, p < 0.001]. The main task effect was
maintained for all time-bins (all p <0.001). However, the time
effect was not preserved. In both tasks, the first and the second
time-bins were significantly different (t = −20.5, p < 0.001), but
we did not find significant differences between other time-bins (p
>0.05) (see Table 4). 3.2.5. Mode Switches To summarize, we found a task and time effect for all the
mouse parameters. As found for eye movements, most of the
mouse parameters stabilized at the end of the exploration. Interestingly, the mouse parameters behaved similarly to
eye movements parameters. Finally, ambient mode was the
prevailing mode for mouse movements, but, as for the eyes,
progressively switched to the focal mode over time. 3.2.4. Dynamic of Mouse Movements 0.75 ± 0.39
0.96 ± 0.61
0.92 ± 0.77
0.88 ± 0.89
0.16 ± 0.51-0.06 , 0.54
−0.19 ± 0.56
−0.31 ± 0.68
[T2F-T3F,T3F-T4F, T3V-T4V]
Avg. switch nb. 1.24 ± 0.26
1.46 ± 0.32
1.46 ± 0.31
1.47 ± 0.31
1.47 ± 0.29
1.65 ± 0.36
1.65 ± 0.34
1.63 ± 0.34
[T2F-T3F,T3F-T4F, T2V-T3V,T3V-T4V] 3.3.2. Scroll Duration Scrolls lasted on average 367.57 (SD = ±121.65ms). We found a
task effect [F(1,138) = 205.20, p < 0.001]. Scroll was shorter in the
free viewing task (M = 328.64ms, SD = ±99.57ms) compared
to the visual search task (M = 417.24ms, SD = ±186.17ms). Additionally, we found a time effect [F(3,414) = 55.49, p < 0.001]. There was a significant increase between the first and second
time-bins (t = 9.34, p < 0.001), as well as between the third
and fourth time-bins (t = 3.39, p < 0.01). However, there was
no significant difference between the second and third time-bins
(t = 1, p > 0.05). We did not find any interaction [F(3,414) =
1.94, p > 0.05] (see Table 4). To summarize, we found a task and time effect for all scrolling
parameters. As with the eyes and mouse parameters, most of
the scrolling parameters stabilized at the end of the exploration. However, this evolution was in the opposite sense of that for
the eye and mouse movements. While the eye and mouse
fixation or dwelling parameters increased over time, scrolling
dwells decreased. Inversely, while the eye and mouse movement
parameters decreased over time, scrolling increased. As such,
the focal mode was predominant in the global exploration, but
tended to ambient mode over time. 4. CONCLUSION AND DISCUSSION A scroll was on average 8.52◦(SD = ±2.35) long. The task had
an influence on scroll amplitude [F(1,138) = 389.81, p < 0.001]. Scrolls were longer in the visual search task (M = 10.58◦, SD =
±3.12◦) than in the free viewing task (M = 6.91., SD = ±2.6.)
The time also had an influence [F(3,414) = 34.04, p < 0.001]. There was a significant increase between the first and second
time-bins (t = 9.44, p < 0.001), but not between the second and
third time-bins (t = 0.77, p > 0.05) or between the third and
fourth time-bins (t = 1.20, p > 0.05). There was a significant
interaction between the time and task [F(3,414) = 6.51, p <
0.001], but post-hoc analyses confirmed that main effects were
preserved (see Table 4). Since the seminal work of Buswell (1935), eye movements have
been extensively studied in a wide variety of conditions. From
viewing patterns (Yarbus, 1967) to average fixation durations
(Mackworth and Morandi, 1967), how eye movement parameters
behave are well-known. The knowledge of these basic parameters
led to more complex research aiming to infer cognitive processes
occurring during eye movements (Velichkovsky et al., 2002;
Unema et al., 2005; Pannasch et al., 2008). However, with
the stimuli diversity that aroused during the last decades, it
became crucial to extend and adapt this knowledge to new
stimuli types. That is why our study aims to provide a detailed
statistical description of eye movement parameters on ecological
web pages. Contrary to other stimuli such as natural images,
web pages allow the use of mouse movements and scrolls. As previously described, mouse movements are mostly studied
as patterns or trajectories (Rodden et al., 2008; Guo and
Agichtein, 2010; Tzafilkou and Protogeros, 2018) and scrolling
is sparsely studied (Braganza et al., 2009; Liu et al., 2017;
Milisavljevic et al., 2018). Although their respective parameters
are mentioned, to our knowledge, no quantitative analyses of
their parameters have been performed. Using the same approach
as for the study of eye movements, we intended to run such
analyses to describe mouse and scroll parameters. Thus, the
purpose of our study is to provide a statistics baseline of eye
movements, mouse movements, and scrolling parameters during
web pages exploration. 4.1. Eye Movement Parameters y
We first found a task effect for all eye variables that replicated
several studies in the literature (Yarbus, 1967; DeAngelus and
Pelz, 2009; Itti and Borji, 2015). Fixation-related variables were
higher in the free viewing task compared to the visual search
task, while movement-related variables were higher in the visual
search task. We also found a time effect on all variables. Fixation-related variables increased over time for both tasks while
movement-related variables decreased. Participants did fewer
fixations and saccades, but longer fixations and shorter saccades
over time (Unema et al., 2005). As a result, we observed a 3.3.1. Number of Scrolls During the trial, the participants scrolled on average 8.77 (SD =
±2.04) times. We found a task effect [F(1,138) = 512.15, p <
0.001]. We measured lower numbers in the free viewing task
(M = 6.62, SD = ±2.25) compared to the visual search task
(M = 11.44, SD = ±2.63). We also found a time effect [F(3,414) =
282.94, p < 0.001]. There was a significant increase between the
first and second time-bins (t = 24.37, p < 0.001). However, there
was no significant differences between the second and third time-
bins (t = 0.19, p > 0.05) or between the third and fourth time-
bins (t = −0.62, p > 0.05). There was a significant interaction March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 11 Similarities and Differences Between Eye and Mouse Milisavljevic et al. switches between the first and the second time-bins (t = −15.27,
p < 0.001), but no significant differences between the following
successive time-bins (all p >0.05). The interaction of the time
and task was also significant [F(3,414) = 4.60, p < 0.001], but
post-hoc analyses confirmed that main effects were preserved
(see Table 4). between the time and task [F(3,414) = 6.03, p < 0.001]. However,
post-hoc analyses showed that the main effects were maintained
(see Table 4). 3.3.4. Scrolling Dynamic In contrast to eye and mouse dynamics, scrolling dynamic was
dominated by the focal mode (M = 0.43, SD = ±0.45). There
was a task effect on the K coefficient [F(1,138) = 454.64, p <
0.001], which was significantly more indicative of the focal mode
in the free viewing task (M = 0.92, SD = ±0.67) than in the
visual search task (M = −0.17, SD = ±0.47). There was also
a time effect [F(3,414) = 5.58, p < 0.001], the K coefficient
significantly decreased between the first and second time-bins
(t = −4.29, p < 0.001), but did not between the following
successive time-bins (all p >0.05). We found an interaction
between the time and task [F(3,414)
=
39.55, p
<
0.001]. The main task effect was maintained (all p <0.001). However,
maintained during the free viewing task, the main time effect
was not maintained in the visual search task. We measured a
significant reduction between the first and second time-bins, and
the second and third time-bins (all p <0.05, but not between the
third and fourth time-bins (p >0.05) (see Table 4). 3.3.5. Modes Switches The participants switched between modes an average of 3.63
(SD = ±0.74) times. There was a significant task effect [F(1,138) =
257.59, p < 0.001]. The number of switches between modes
was significantly lower in the free viewing task (M = 2.99,
SD = ±0.94) than in the visual search task (M = 4.37, SD =
±1). We also found a significant time effect [F(3,414) = 109.40,
p < 0.001]. There was a significant decrease in the number of March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 12 Similarities and Differences Between Eye and Mouse Milisavljevic et al. FIGURE 3 | Relationship between fixation-related variables of the eyes, mouse and scroll. (A) Global z-scored averages. (B) z-scored averages over time in the free
viewing condition. (C) z-scored averages over time in the visual search condition. FIGURE 3 | Relationship between fixation-related variables of the eyes, mouse and scroll. (A) Global z-scored averages. (B) z-scored averages over time in the free
viewing condition. (C) z-scored averages over time in the visual search condition. FIGURE 4 | Relationship between saccade-related variables of the eyes, mouse and scroll. (A) Global z-scored averages. (B) z-scored averages over time in the free
viewing condition. (C) z-scored averages over time in the visual search condition. FIGURE 4 | Relationship between saccade-related variables of the eyes, mouse and scroll. (A) Global z-scored averages. (B) z-scored averages over time in the free
viewing condition. (C) z-scored averages over time in the visual search condition. Regarding scrolling, all parameters varied according to the
task. Comparably to eye and mouse movement parameters, we
found a task effect for all parameters. We also found a time
effect on all the variables, but dwell-related variables decreased
over time while scroll-related variables increased. However,
the stabilization of scroll parameters began earlier than for
mouse parameters (see Figures 3, 4). Although there were fewer
scroll movements than eye movements their frequency remained
slightly higher than that of mouse movements. Therefore, we
conducted analyses of dominant modes and found that, globally,
scrolling was in focal mode. However, when looking over time,
we observed that the focal mode was more dominant at the
beginning of the exploration and ambient mode at the end. 3.3.5. Modes Switches Since participants scrolled increasingly over time but did longer
eye fixations, they seemed to balance the natural emergence of
the focal mode of the eyes by scrolling to keep changing and
contextualizing the newly displayed content. global domination of ambient mode (i.e., short fixations with
long saccades), but over time the dominant mode progressively
switched to focal mode (i.e., long fixations with short saccades). This behavior could indicate that participants try to contextualize
the stimulus at the beginning of the exploration to then focus
more and more on content as time goes by. 4.2. Mouse Parameters Then we ran the same analyses on mouse movements and
scrolls. We found a task effect for all parameters of the mouse
exploration, except for the average amplitude and duration of
the mouse movements. As for the eye movements, dwell-related
variables were higher in the free viewing task compared to the
visual search task, while movement-related variables were higher
in the visual search task. Again, we found a time effect on
all variables. Comparably to eye movement parameters, dwell-
related variables increased over time and movement-related
variables decreased over time for both tasks. This behavior is
similar to that of eye movements and suggests strong similarities
between the two. Hence, we applied visual mode concepts to
mouse movements. However, it is worth noting that the number
of mouse movements was broadly lower to the number of eye
movements, so these results should be discussed with caution. Despite the difference in the number of events, we observed
similar behavior in the mouse dynamic, which began in ambient
mode to progressively switch to focal model over the course of
the exploration. 4.3. Similarities and Differences 4.3. Similarities and Differences
When studying eye, mouse, and scroll parameters, we observed
common tendencies over time. In order to study these
tendencies, we separated computed variables into two distinct
categories: variables related to movements and variables related
to fixations/dwells. Then, since focused on tendencies, the
relevant parameters were normalized between 0 and 1 to enable
the comparisons. This movement-fixation dichotomy is directly
inspired from how the visual cortex processes visual information. March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 13 Similarities and Differences Between Eye and Mouse Milisavljevic et al. The visual cortex is divided in two main pathways: ventral and
dorsal stream. Ventral stream carries information about object
recognition, while the dorsal stream is more related to visually
guided movements (Goodale and Milner, 1992). Since saccades,
mouse movements and scrolls are all visually guided movements,
they are analyzed together. However, while fixation is directly
involved in object recognition (ventral stream), it is not clear
whether a mouse or a scroll dwell is involved. The mouse remains
a tool used to browse a web page, and the implication of a
pause still needs further investigations. For the convenience of
the following analyses, we compare eye fixations with mouse and
scroll dwells. which confirms the interest of using mouse behavior to predict
eye behavior. Yet the interaction described here does not take
spatial coordinates into account that could be combined with
relationship parameters to better predict eye movements from
mouse events. Even though further studies are needed to confirm our results,
the relationship between eyes and mouse parameters seems
consistent over time. This may be related to similar processing
in the ventral and dorsal streams (Goodale and Milner, 1992). For instance, Stone and Gonzalez (2015) reported several studies
in which ventral and dorsal streams of congenitally blind
individuals were preserved during pointing and grasping tasks. Thus, we can assume that the important role of both streams
involved in hand movements and eye movements may explain
why the eyes and the mouse parameters behave similarly during
the exploration. However, this hypothesis does not address why
the scroll parameters behave oppositely. The opposite behavior
we observed for the scroll may be explained by the “the sensory
weighting hypothesis” (Ernst and Banks, 2002). 4.3. Similarities and Differences This theory states
that during a task involving sensory competition, here the
presence of both vision and haptic, we tend to rely on the optimal
one to complete the task. For instance, before reaching an object
whose position is unknown, we first need to look at it, but there
are occasions when we reach objects without looking at them
because we already know their exact position. In our case, the task
is to browse the page with or without a target. At the beginning
of the exploration, the optimal sensory input to fulfill this task
would be the eyes. As time goes by, we discover the web page
more and more until we browsed it entirely. The scroll would
gradually become the optimal way to browse the web page, since
fixation duration is increasing and saccade amplitude decreasing,
and the scroll would then replace large saccades. The first common tendency we observed is depicted in
Figure 3A . It shows a common pattern between the fixation-
related variables of the eyes and the mouse, and an opposite one
with the scroll. On the one hand, eye and mouse parameters
behaved similarly. Fixation or dwell durations, and percentages
of fixations/dwells, were at their lowest at the beginning of the
exploration and increased up to the end of exploration. For
instance, in the free viewing task, the average fixation duration
was 211.27 ms at the beginning of the exploration and increased
up to 241.28 ms, while the average mouse dwell lasted 1 s at
the beginning and increased up to 1.52 s (see Tables 2, 3 for
more details). On the other hand, scrolling behaved exactly the
opposite way. Scroll dwell was at its highest at the beginning
of the exploration and lasted 1.91 s in average during the first
time bin of the free viewing task and decreased overtime to
reach 1.44 s at the end of the exploration (see Table 4 for
more details). These observations are consistent in both the free
viewing and visual search tasks (Figures 3B,C). Yet we observed
a stabilization of mouse and scroll dwell durations starting from
the second time-bin. We observed a second tendency describing the opposite
pattern
for
movement-related
variables,
as
presented
in
Figure 4A. Eye and mouse movement variables decreased over
time and scroll variables increased. 4.3. Similarities and Differences Eye and mouse parameters
behaved in the opposite way to scroll parameters, just as with
fixation-related variables. Furthermore, this relationship was
maintained across both tasks (Figures 4B,C). For instance, we
observed an average saccade amplitude of 6.26◦and an average
mouse amplitude of 0.43◦at the beginning of exploration during
the free viewing task. Then both amplitudes have decreased to,
respectively, 4.47◦and 0.13◦at the end of exploration. Under
the same conditions, the scrolling amplitude increased from
5.88◦at the beginning of the exploration to 6.78◦at the end (see
Tables 2–4 for more details). Further research is necessary to better understand what
mechanisms are involved in the eyes and mouse coordination
during web pages exploration. For instance, we did not
differentiate scroll up from scroll down in our analyses. When
we scroll down, we usually discover the content for the first time. But a scroll up is necessary to re-examine an already seen area
of the web page. Differentiating the two directions might provide
finer results on what cognitive processes are involved. DATA AVAILABILITY STATEMENT The data analyzed in this study was obtained from the company
Sublime Skinz. Data cannot be distributed, remixed, adapted,
used to build upon, changed in any way or used commercially. Requests to access the datasets should be directed to Coralie
Petermann, coralie@sublime.xyz. Our results show a clear relationship between eye, mouse,
and scroll parameters. Previous studies have already shown the
spatial coordination of the eyes and mouse (Guo and Agichtein,
2010; Huang et al., 2012; Boi et al., 2016) and some coordination
between the eyes and scroll speed (Milisavljevic et al., 2018). However, here we show that this relationship is even deeper
than expected, and can be identified through analyzing eye,
mouse, and scroll parameters. Indeed, coordination is not only
between the eyes and the mouse, or, between the eyes and the
scroll, but clearly between all three. Our findings show, for
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effects of viewing task. Front. Psychol. 10:2915. doi: 10.3389/fpsyg.2019.02915 Liu, C., Liu, J., and Wei, Y. (2017). “Scroll up or down?: using wheel
activity as an indicator of browsing strategy across different contextual
factors,” in Proceedings of the 2017 Conference on Human Information
Interaction and Retrieval - CHIIR ’17
(Oslo: ACM Press), 333–336. doi: 10.1145/3020165.3022146 DeAngelus, M., and Pelz, J. B. (2009). Top-down control of eye movements: Yarbus
revisited. Vis. Cogn. 17, 790–811. doi: 10.1080/13506280902793843 Ernst, M. O., and Banks, M. S. (2002). Humans integrate visual and
haptic information in a statistically optimal fashion. Nature 415, 429–433. doi: 10.1038/415429a Mackworth, N. H., and Morandi, A. J. (1967). The gaze selects informative details
within pictures. Percept. Psychophys. 2, 547–552. ETHICS STATEMENT The studies involving human participants were reviewed
and approved by Research Ethics Board of Paris Descartes
University (Comité d’éthique de la Recherche de l’université
Paris Descartes). The patients/participants provided their written
informed consent to participate in this study. March 2021 | Volume 12 | Article 554595 14 Similarities and Differences Between Eye and Mouse Milisavljevic et al. FUNDING AM, TL, MM, BG, and KD-M conceived and designed the
study. AM, FA, and TL contributed to the data collection. AM
and FA conducted all analyses. AM, FA, and KD-M wrote the
manuscript. All authors contributed to the article and approved
the submitted version. AM, TL, MM, BG, and KD-M conceived and designed the
study. AM, FA, and TL contributed to the data collection. AM
and FA conducted all analyses. AM, FA, and KD-M wrote the
manuscript. All authors contributed to the article and approved
the submitted version. This work was supported by the French Research and Technology
Association (ANRT, Association Nationale de la Recherche et de
la Technologie) for a CIFRE doctorate program between Sublime
Skinz, Université de Paris and University of Mons from 2016 to
2019 (Grant Number 2016/0957). REFERENCES doi: 10.3758/BF03210264 Feher, C., Elovici, Y., Moskovitch, R., Rokach, L., and Schclar, A. (2012). User identity verification via mouse dynamics. Inform. Sci. 201, 19–36. doi: 10.1016/j.ins.2012.02.066 Milisavljevic, A., Bras, T. L., Mancas, M., Petermann, C., Gosselin, B., and Doré-
Mazars, K. (2019). “Towards a better description of visual exploration through
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the effects of web object placement on different types of websites. Int. J. Hum. Comput. Stud. 71, 228–235. doi: 10.1016/j.ijhcs.2012.09.001 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Seelye, A., Hagler, S., Mattek, N., Howieson, D. B., Wild, K., Dodge,
H. H., et al. (2015). Computer mouse movement patterns: a potential
marker of mild cognitive impairment. Alzheimer’s Dement. 1, 472–480. doi: 10.1016/j.dadm.2015.09.006 Copyright © 2021 Milisavljevic, Abate, Le Bras, Gosselin, Mancas and Doré-Mazars. 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) and the copyright owner(s) 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. Frontiers in Psychology | www.frontiersin.org REFERENCES Copyright © 2021 Milisavljevic, Abate, Le Bras, Gosselin, Mancas and Doré-Mazars. 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) and the copyright owner(s) 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. Sharmin, S., Špakov, O., and Räihä, K.-J. (2013). “Reading on-screen text with gaze-
based auto-scrolling,” in Proceedings of the 2013 Conference on Eye Tracking
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24–31. doi: 10.1145/2509315.2509319 March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 16
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Gait asymmetry, and bilateral coordination of gait during a six-minute walk test in persons with multiple sclerosis
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bioRxiv (Cold Spring Harbor Laboratory)
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cc-by
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Gait asymmetry, and bilateral
coordination of gait
during a six‑minute walk test
in persons with multiple sclerosis
N Meir Plotnik1,2,3*, Joanne M. Wagner4, Gautam Adusumilli5, Amihai Gottlieb1 &
Robert T. Naismith5 Gait impairments in persons with multiple sclerosis (pwMS) leading to decreased ambulation and
reduced walking endurance remain poorly understood. Our objective was to assess gait asymmetry
(GA) and bilateral coordination of gait (BCG), among pwMS during the six-minute walk test (6MWT),
and determine their association with disease severity. We recruited 92 pwMS (age: 46.6 ± 7.9; 83%
females) with a range of clinical disability, who completed the 6MWT wearing gait analysis system. GA was assessed by comparing left and right swing times, and BCG was assessed by the phase
coordination index (PCI). Several functional and subjective gait assessments were performed. Results
show that gait is more asymmetric and less coordinated as the disease progresses (p < 0.0001). Participants with mild MS showed significantly better BCG as reflected by lower PCI values in
comparison to the other two MS severity groups (severe: p = 0.001, moderate: p = 0.02). GA and PCI
also deteriorated significantly each minute during the 6MWT (p < 0.0001). GA and PCI (i.e., BCG)
show weaker associations with clinical MS status than associations observed between functional and
subjective gait assessments and MS status. Similar to other neurological cohorts, GA and PCI may be
important parameters to assess and target in interventions among pwMS. Multiple sclerosis (MS) is a degenerative, progressive, autoimmune disease of the central nervous system, often
resulting in a continuous deterioration of walking1. Hence, gait parameters, e.g. cadence, step-length, step-time,
are impaired as compared to those measured in abled bodied individuals1–4. This gait deterioration has been
demonstrated as a decline in the ability to walk long distances, based on the 6-min or 500-m walk tests5,6. One
critical component of walking impairment is gait variability. Gait variability tends to change throughout the MS
disease course, with greater variability in the higher levels of disability7,8. Furthermore, gait variability is associ-
ated with increased fall risk9. Human gait requires a high degree of symmetry and coordination. Gait asymmetry is associated with reduced
walking velocity10–12 and increased energy expenditure13. Spinal cord injury is frequent in persons with multiple
sclerosis (pwMS), noted in 83% by MRI and up to 99% at autopsy14,15. Spinal cord injury is associated with lower
extremity sensorimotor deficits and impaired ambulation. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ movements by alternating activity between groups of flexor and extensor muscles. CPGs are modulated by large
fiber sensory afferents, along with supraspinal input20. Damage within the spinal cord may limit afferent (PC)
and efferent (CST) input to the CPGs and thus, contributes to impaired BCG21,22. Thus, it is also hypothesized
that MS is characterized by impaired BCG.f y
p
In the present study we assess GA by the evaluating how leg movements differ while walking (comparing
swing times between the legs and symmetry of swing duration)23. Left–right stepping coordination is assessed by
the phase coordination index (PCI), a measure of the accuracy and consistency of the phase relationship between
the step timing of the left and right legs23. Only three reports directly assessed PCI or GA among pwMS. Gian-
francesco et al. compared between cane users (n = 6) with non-users (n = 5) and attributed lower values of PCI
and GA (i.e., better coordination and lower asymmetry) to the former group24. Kasser et al. compared PCI values
before and after an acute aerobic exercise in pwMS and reported no significant change25. However, the relation to
other clinical parameters, such as disease severity, was not assessed in these papers. Recently, Shema‑Shiratzky
et al.26 measured GA among pwMS during the 6-min walk test (6MWT) and reported its relation to disease sever-
ity, however, only mild and moderate pwMS were assessed and PCI values were not measured in their study. Thus,
gait difficulties in left–right stepping coordination and GA in PwMS, measured by temporal gait metrics (cycle
to cycle performance), has not yet been fully described. In the present work we study these gait features using
the 6MWT which is widely accepted as a valid outcome to assess fitness and endurance in a clinical context5,27. Study rationale. Characterizing GA and coordination impairments in MS, and their relations with func-
tional (and subjective) gait assessments and with disease severity, would provide a more comprehensive picture
about gait impairments in this cohort. GA and coordination impairments may prove useful in defining treat-
ment targets and efficacy assessments for improving gait in MS. Participants. Participants. A total of 92 pwMS were recruited through the John L. Trotter MS Center at Washington
University School of Medicine. This cross-sectional study was approved by the local Human Research Protection
Office/Institutional Review Board of the Washington University in St. Louis. All participants signed an informed
consent prior to entering the study and all methods were performed in accordance with the relevant guidelines
and regulations. Inclusion criteria were: > 18 years of age; relapsing–remitting MS (RRMS), secondary-progres-
sive MS (SPMS), or primary-progressive MS (PPMS); evidence of cervical spinal cord disease by symptoms or
signs (e.g. Lhermitte’s sign, upper extremity weakness, upper extremity sensory symptoms), T2 lesions on clini-
cal cervical MRI, and > 180 days post-relapse. Exclusion criteria: confounders which could affect ambulation
outside the spinal cord (e.g. poor vision) pregnancy, lower extremity orthopedic conditions that limit ambula-
tion, morbid obesity, cardiac pacemaker, metallic implant, and claustrophobia. Participants were then grouped
by disease severity, i.e. mild (EDSS 0–2.5, n = 60), moderate (EDSS 3–4, n = 26) and severe (EDSS 4.5–6.5, n = 6). Experimental protocol. The participants wore a six Opal motion sensor-based gait analysis system
(APDM, Portland, Oregon, USA). The 6MWT5 was performed along a 50 foot walkway (i.e., participants turned
around cones at each end of the 50 foot walkway). Spatiotemporal parameters of gait were collected by sensors
worn on the ankles, wrists, lower back and chest, and transmitted to a wireless access device for storage on a
mobile computer. Additional assessments included the Timed 25-foot Walk Test (T25FWT)28, self-perceived
walking limitation [12-item MS walking scale (MSWS-12v2)]29 and balance confidence (Activities-specific bal-
ance confidence scale (ABC)30. Gait cycle timing related outcome measures. Outcomes were calculated based on ‘heel strike’ and
‘toe off’ timing obtained from the straight-line walking segments (180° turns at end of walkway were omitted): (1)
Gait variability [%]: was defined by Stride time variability7,23: coefficient of variation (CV) of the mean value
of the stride time (multiplied by 100 to obtain percentage). We termed this parameter stride CV. www.nature.com/scientificreports/ Assessing the relation of these parameters with
disease severity could shed light on the progression of the MS and its effect on gait and risk of falls.h y
g
p
gf
g
The objective of this study was to characterize GA and BCG in pwMS, during a long-distance walk (i.e., the
6MWT), and to assess their relationship with established gait assessments and other clinical parameters for
pwMS. The study is driven by two hypotheses: (1) Human gait requires a high degree of symmetry and coordi-
nation, both of which are potentially altered in MS. (2) The type of gait patterns in pwMS (as reflected by gait
asymmetry, coordination and variability outcomes) will be correlated with the level of impairment on key MS
clinical outcomes, such as walking endurance as reflected by the distance covered during the 6MWT, and self-
reported walking limitations. Gait asymmetry, and bilateral
coordination of gait
during a six‑minute walk test
in persons with multiple sclerosis
N It was previously reported that walking velocity in
pwMS was reduced when in vivo diffusion tensor imaging (DTI) of the cervical spinal cord reveals myelin and
tissue injury within posterior columns (PC) and lateral corticospinal tracts (CST)16. Since CST injury in pwMS
is asymmetric17, we hypothesize that MS will be associated with increased gait asymmetry, since asymmetric
lesions in the spinal white matter lesions have been shown to correspond to asymmetric motor function18.f p
p
y
Gait coordination is the ability to maintain a consistent phase-dependent cyclical relationship between differ-
ent body segments or joints in both spatial and temporal domains19. In humans, the control of right-left stepping,
or bilateral coordination of gait (BCG), is hypothesized to be mediated by central pattern generators (CPGs)
within lumbrosacral spinal locomotor centers13. CPGs are local neuronal circuits that generate rhythmic stepping 1Center of Advanced Technologies in Rehabilitation, Sheba Medical Center, 5265601 Ramat Gan,
Israel. 2Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv,
Israel. 3Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel. 4Department of Physical Therapy and
Athletic Training, Saint Louis University, St. Louis, USA. 5Department of Neurology, Washington University in
St. Louis, St. Louis, USA. *email: meir.plontik@sheba.health.gov.il Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 www.nature.com/scientificreports/ (
; p
),
p
y
g
g
(2)
GA[%] = 100*|ln(R_SW/L_SW)|, where R_SW and L_SW stand for the mean value of right and lef
swing time, respectively23.i (1)
Gait variability [%]: was defined by Stride time variability7,23: coefficient of variation (CV) of the mean value
of the stride time (multiplied by 100 to obtain percentage). We termed this parameter stride CV. Stride CV
was calculated separately for the left and the right legs and since values of the right and left legs were highly
correlated (r = 0.989; p < 0.001), we present data only for the right leg.t Results Demographic and clinical data of the sample appear in Table 1. Of the 92 participants, 60 were classified as having
mild disability, 26 as moderate disability, and 6 as severe disability. As expected, significant group differences were
found for age (F(2,89) = 10.8, p < 0.001, η2 = 0.19) and disease duration (F(2,89) = 3.39, p < 0.03, η2 = 0.07). Post-hoc
comparisons indicate younger age and shorter disease duration associated with mild disability. In light of these
group differences, in the following analyses, we entered age and disease duration as covariates. Group (MS severity) and time effects on distance covered, gait variability, coordination
and asymmetry. Significant group differences were found for distance covered (F(2,87) = 20.7, p < 0.0001,
η2 = 0.32), gait variability (i.e., Stride CV, F(2,86) = 13.9, p < 0.0001, η2 = 0.24), PCI (F(2,86) = 9.3, p < 0.0001, η2 = 0.17),
and GA (F(2,86) = 12.0, p < 0.0001, η2 = 0.21; see Fig. 1 for post-hoc group to group comparisons).h ( ,
)
p
η
g
p
g
p
g
p
p
Figure 2 depicts three examples of data sets from three participants, one from each severity level group. These
examples are representative of the group differenced described above. Stride-to-stride time variability is larger
for the more severely affected participants, as expressed by the larger range of values of the stride times in the
participants with moderate and severe MS. Left–right stepping coordination (i.e., BCG) as expressed by the large
variability in φ values (i.e., reduced consistency), and with values fairly ‘distant’ from the ‘ideal’ value of 180° is
impaired in the participant with moderate MS (φ = 173.0 ± 4.2°) as compared to the participant with mild MS
(φ = 179.4 ± 2.7°), and even worse in the participant with the severe MS (φ = 187.3 ± 11.3°; see PCI values for all
participants in Fig. 2). Functional and objective outcome measures. Functional and objective outcome measures. Distance covered during the 6MWT (ft) (reflective of gait speed). Time (sec’) to complete the T25FWT. Transformed Score on the MSWS-12v233 (higher score reflect perception of being more limited). Transformed Score on the ABC (higher scores reflect more confidence in preforming balance capabilities
related activities). Gait variably GA PCI and distance covered were calculated and measured for each minute of the 6MWT trail Distance covered during the 6MWT (ft) (reflective of gait speed). t
Time (sec’) to complete the T25FWT. Transformed Score on the MSWS-12v233 (higher score reflect perception of being more limited). Transformed Score on the ABC (higher scores reflect more confidence in preforming balance capabilities
related activities). (
gl
p
p
g
)
Transformed Score on the ABC (higher scores reflect more confidence in preforming balance capabilities
related activities). Gait variably, GA, PCI and distance covered were calculated and measured for each minute of the 6MWT trail Statistical analysis. For Gait variability, GA, PCI and distance covered, we performed a Repeated meas-
ures ANOVA treating each 1-min interval (‘time’ effect) as a within subject level during the 6MWT, and the three
severity subgroups (‘group’ effect) as a between-subject factor. When violations of the assumption of sphericity
were observed, Greenhouse–Geisser estimates were used to correct the degrees of freedom. The Bonferroni
method was used to correct for the post-hoc comparisons. p
p
One-way between-groups ANOVAs and Bonferroni’s post-hoc tests compared the functional and objective
utcome measures (apart of distance covered) among the MS severity subgroups.fi p
g
y
g
p
Demographic and clinical factors which differed significantly between these groups (i.e. Age, Disease dura-
tion) were added as covariates in both of these analyses.f y
Effect sizes were reported as Eta squared and Cohen’s d. Effect sizes were reported as Eta squared and Cohen’s d. f
Correlation analyses between distance covered, stride CV, GA and PCI and the functional and subjective gait
assessments were performed (Pearson) for the entire cohort and within each severity group. For the correlation
analysis, we used the distance covered, stride CV, GA and PCI values measures and calculated over the entire
6MWT. Participants. Stride CV
was calculated separately for the left and the right legs and since values of the right and left legs were highly
correlated (r = 0.989; p < 0.001), we present data only for the right leg.t (1)
Gait variability [%]: was defined by Stride time variability7,23: coefficient of variation (CV) of the mean value
of the stride time (multiplied by 100 to obtain percentage). We termed this parameter stride CV. Stride CV
was calculated separately for the left and the right legs and since values of the right and left legs were highly
correlated (r = 0.989; p < 0.001), we present data only for the right leg.t (
; p
),
p
y
g
g
(2)
GA[%] = 100*|ln(R_SW/L_SW)|, where R_SW and L_SW stand for the mean value of right and left leg
swing time, respectively23.i p
p
y
g
g
(2)
GA[%] = 100*|ln(R_SW/L_SW)|, where R_SW and L_SW stand for the mean value of right and left leg
swing time, respectively23.i g
p
y
(3)
Phase coordination index (PCI; [%]—for the quantification of BCG): A full description and derivation of
the PCI metric is detailed elsewhere23. Briefly, PCI is a metric that combines the accuracy and consistency
of stepping phases generation with respect to the value of 180°, which represents the ideal anti-phased
left–right stepping. Lower PCI values (also transformed to percentile unit; %) reflect a more consistent and
more accurate phase generation, while higher values indicate a more impaired BCG23,31,32. Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 www.nature.com/scientificreports/ Total (n = 92)
Mild (n = 60)
Moderate (n = 26)
Severe (n = 6)
Age in years, mean ± SD (range)
46.6 ± 10.9 (22–66)
43.2 ± 10.4 (22–66)
53.0 ± 8.1 (33–65)
54.1 ± 11.2 (34–63)
EDSS, median (range)
2.0 (2.0–6.5)
2.0 (0–2.5)
3.0 (3–4)
5.5 (5.0–6.5)
Female, n (%)
76 (83%)
49 (82%)
21 (81%)
6 (100%)
Race (Caucasian, African–American,
Asian), n (%)
83/8/1 (90, 9, 1)
53/6/1 (88, 10, 2)
24/2/0 (92, 8, 0)
6/0/0 (100, 0, 0)
Type of MS (RR/SP/PP), n (%)
88/2/2 (96%, 2%, 2%)
59/0/1 (98%, 0%, 2%)
23/2/1 (88%, 8%, 4%)
6/0/0 (100%, 0%, 0%)
Years since diagnosis, mean ± SD (range)
10.7 ± 7.9 (1–37)
9.2 ± 7.0 (1–30)
13.8 ± 7.8 (2–31)
12.3 ± 12.6 (2–37) Table 1. Demographic and clinical data of the cohort. Participants. EDSS expanded disability status scale, RR relapsing
remitting, SP secondary progressive, PP primary progressive. Results Similarly, the increased GA exhibited by the participants with moderate and severe MS is
clearly demonstrated by the separation in the scatter of right (red) and left (green) swing times.if y
y
p
gt
g
g
Significant time (i.e., potential fatigue) effects were found for PCI (F(5,430) = 5.6, p < 0.0001, η2 = 0.06, due to
a general decrease in coordination (i.e., larger PCI values) over time, in addition to a significant interaction
between severity group and time, F(10,430) = 4.9, p < 0.0001, η2 = 0.1, due to a larger increase in PCI values among
the severe MS group. Further, significant time effects were also found for GA (F(5,430) = 4.9, p < 0.0001, η2 = 0.05,
due to a general increase in asymmetry across groups, and in this case as well, a significant interaction was
yielded between severity group and time, F(10,430) = 5.3, p < 0.0001, η2 = 0.11, due to a larger increase in asymmetry
among the severe MS group. Unexpectedly, time effect was not demonstrated with regards to distance covered,
F(5,435) = 0.7, p = 0.6, η2 = 0.008, or Gait variability (i.e., Stride CV, F(5,430) = 1,8, p = 0.09, η2 = 0.02) nor interactions
between fatigue (i.e., time effect) and severity group regarding these measures. Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 www.nature.com/scientificreports/ Figure 1. Mean values of distance covered, Stride time CV, GA and PCI during 6MWT in pwMS with different
disease severity (see key). Bars represent mean values calculated based on the per-minute measures (see
“Methods”). Statistically significant differences in post-hoc group-to-group comparisons are marked above with
horizontal brackets. Means and standard deviations are as followed: The severe disability group had greater
asymmetry (GA: 22.6 ± 3.5) than the mild (GA: 3.9 ± 1.1) or moderate (GA: 7.1 ± 1.7) disability groups, and
more impaired PCI (10.2 ± 1.5) than the mild disability group (3.6 ± 0.5). The moderate disability group had
worse PCI (6.2 ± 0.5) compared to the mild disability group, and better PCI than the severe disability group. Gait Asymmetry did not significantly differ between the mild vs. moderate disability groups (p = 0.47). Results Distance
covered significantly differed among all three MS severity groups (Severe: 141.6 ± 17.2, Moderate: 211.3 ± 8.3,
Mild: 253.4 ± 5.4) and Gait Variability also significantly differed among all three MS severity groups (Severe:
4.5 ± 0.4, Moderate: 2.7 ± 0.2, Mild: 1.9 ± 0.1). Upper limit of the abscissa is 360 s’. Figure 1. Mean values of distance covered, Stride time CV, GA and PCI during 6MWT in pwMS with different
disease severity (see key). Bars represent mean values calculated based on the per-minute measures (see
“Methods”). Statistically significant differences in post-hoc group-to-group comparisons are marked above with
horizontal brackets. Means and standard deviations are as followed: The severe disability group had greater
asymmetry (GA: 22.6 ± 3.5) than the mild (GA: 3.9 ± 1.1) or moderate (GA: 7.1 ± 1.7) disability groups, and
more impaired PCI (10.2 ± 1.5) than the mild disability group (3.6 ± 0.5). The moderate disability group had
worse PCI (6.2 ± 0.5) compared to the mild disability group, and better PCI than the severe disability group. Gait Asymmetry did not significantly differ between the mild vs. moderate disability groups (p = 0.47). Distance
covered significantly differed among all three MS severity groups (Severe: 141.6 ± 17.2, Moderate: 211.3 ± 8.3,
Mild: 253.4 ± 5.4) and Gait Variability also significantly differed among all three MS severity groups (Severe:
4.5 ± 0.4, Moderate: 2.7 ± 0.2, Mild: 1.9 ± 0.1). Upper limit of the abscissa is 360 s’. The role of gait speed: post hoc analyses. Since significant correlations were found between walking
speed (distance covered) and PCI, GA and stride CV (see Table 2 below), we performed three additional repeated
measures analyses to test for PCI, GA and stride CV group differences while adding the distance covered (i.e.,
walking speed) as a covariate. While time effects remained significant for all three analyses, the analyses also
revealed that for both gait variability and PCI, the group effect did not remain statistically significant (Stride CV,
F(2,85) = 1.5, p = 0.21, η2 = 0.03, PCI (F(2,85) = 0.9, p = 0.38, η2 = 0.02), and the group effect for GA remained statisti-
cally significant (F(2,85) = 4.2, p = 0.01, η2 = 0.09). y
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We also performed additional analyses to determine the relationship within a subject between variability in
walking speed and variability in gait parameters. Results Stride CV, GA and PCI were analyzed, for each minute in the
6MWT, expressed by the Pearson correlation coefficient (rp, see Fig. 3). See full account on rp analyses in the
Supplementary material 1).f To control for the effects of these intrinsic associations, we performed three additional repeated measures
analyses to test group effects for PCI, GA and stride CV while adding the relevant rp (i.e., for PCI, GA and Stride
CV). The new analyses revealed that for Stride CV, PCI, and GA, the group effects remained significant (Stride
CV, F(2,85) = 14.0, p < 0.0001, η2 = 0.24, PCI (F(2,85) = 9.5, p < 0.0001, η2 = 0.18, GA (F(2,85) = 10.3, p < 0.0001, η2 = 0.19). Functional and subjective gait assessments. Figure 4 depicts groups’ performance on the functional
and subjective gait assessments. Eighty-one participants completed the T25FWT (52 form the mild group, 23
from the moderate group and six from the severe group). Group effect was statistically significant (F(4,76) = 13.86,
p < 0.001, η2 = 0.42), demonstrating that participants with milder MS severity walk faster.if p
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p
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Self-perceived walking limitations and balance confidence worsened with disease severity. Group effect was
statistically significant for the MSWS-12v2 (F(4,87) = 14.48, p < 0.001, η2 = 0.40) and for the ABC test (F(4,87) = 7.49, Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 www.nature.com/scientificreports/ p < 0.001, η2 = 0.25), as participants with milder MS severity reporting lower self-perceived gait limitations and
Figure 2. Three examples of data sets from three participants. Series of stride times (Top), left –right stepping
phases (Middle), and swing times, (Bottom) are plotted for a 42 year old female patient with mild disease
severity (left: EDSS = 2; 6MWT distance = 1,514 ft; MSWWS-12v2 = 12; ABC = 98), a 59 year old female patient
with moderate disease severity (middle: EDSS = 3.0, 6MWT distance = 1,319 ft; MSWWS-12v2 = 38; ABC = 66),
and a 48 year old female with severe disease severity (right: EDSS 6.5; 6MWT distance = 406 ft; MSWS12v2 = 76;
ABC = 49). Red and green dots at the lower panels represent right and left swing times, respectively. Table 2. Pearson correlations between clinical status, gait parameters and functional and subjective gait
assessments. Results EDSS Expanded Disability Status Scale, CV coefficient of variation, PCI phase coordination index,
GA gait asymmetry, MSWS-12v2 12-item multiple sclerosis walking scale, ABC activities-specific balance
confidence scale, T25FWT Timed 25-Foot Walk Test. **p ≤ 0.01. EDSS
Distance covered
MSWS-12v2
ABC
T25FWT
Stride CV
0.44**
−0.57**
0.53**
−0.47**
0.60**
PCI
0.42**
−0.54**
0.43**
−0.39**
0.50**
GA
0.35**
−0.42**
0.29**
−0.31**
0.46**
Distance covered
−0.59**
−0.60**
0.56**
−0.84**
MSWS-12v2
−0.59**
−0.72**
0.61**
ABC
−0.49**
−0.53**
T25FWT
0.60**
Clinical status
Gait parameters
Functional and subjective measures Figure 2. Three examples of data sets from three participants. Series of stride times (Top), left –right stepping
phases (Middle), and swing times, (Bottom) are plotted for a 42 year old female patient with mild disease
severity (left: EDSS = 2; 6MWT distance = 1,514 ft; MSWWS-12v2 = 12; ABC = 98), a 59 year old female patient
with moderate disease severity (middle: EDSS = 3.0, 6MWT distance = 1,319 ft; MSWWS-12v2 = 38; ABC = 66),
and a 48 year old female with severe disease severity (right: EDSS 6.5; 6MWT distance = 406 ft; MSWS12v2 = 76;
ABC = 49). Red and green dots at the lower panels represent right and left swing times, respectively. Figure 2. Three examples of data sets from three participants. Series of stride times (Top), left –right stepping
phases (Middle), and swing times, (Bottom) are plotted for a 42 year old female patient with mild disease
severity (left: EDSS = 2; 6MWT distance = 1,514 ft; MSWWS-12v2 = 12; ABC = 98), a 59 year old female patient
with moderate disease severity (middle: EDSS = 3.0, 6MWT distance = 1,319 ft; MSWWS-12v2 = 38; ABC = 66),
and a 48 year old female with severe disease severity (right: EDSS 6.5; 6MWT distance = 406 ft; MSWS12v2 = 76;
ABC = 49). Red and green dots at the lower panels represent right and left swing times, respectively. Table 2. Pearson correlations between clinical status, gait parameters and functional and subjective gait
assessments. EDSS Expanded Disability Status Scale, CV coefficient of variation, PCI phase coordination index
GA gait asymmetry, MSWS-12v2 12-item multiple sclerosis walking scale, ABC activities-specific balance
confidence scale, T25FWT Timed 25-Foot Walk Test. **p ≤ 0.01. Results EDSS
Distance covered
MSWS-12v2
ABC
T25FWT
Stride CV
0.44**
−0.57**
0.53**
−0.47**
0.60**
PCI
0.42**
−0.54**
0.43**
−0.39**
0.50**
GA
0.35**
−0.42**
0.29**
−0.31**
0.46**
Distance covered
−0.59**
−0.60**
0.56**
−0.84**
MSWS-12v2
−0.59**
−0.72**
0.61**
ABC
−0.49**
−0.53**
T25FWT
0.60**
Clinical status
Gait parameters
Functional and subjective measures EDSS
Distance covered
MSWS-12v2
ABC
T25FWT
Stride CV
0.44**
−0.57**
0.53**
−0.47**
0.60**
PCI
0.42**
−0.54**
0.43**
−0.39**
0.50**
GA
0.35**
−0.42**
0.29**
−0.31**
0.46**
Distance covered
−0.59**
−0.60**
0.56**
−0.84**
MSWS-12v2
−0.59**
−0.72**
0.61**
ABC
−0.49**
−0.53**
T25FWT
0.60** EDSS
Distance covered
MSWS-12v2
ABC
T25FWT
Stride CV
0.44**
−0.57**
0.53**
−0.47**
0.60**
PCI
0.42**
−0.54**
0.43**
−0.39**
0.50**
GA
0.35**
−0.42**
0.29**
−0.31**
0.46**
Distance covered
−0.59**
−0.60**
0.56**
−0.84**
MSWS-12v2
−0.59**
−0.72**
0.61**
ABC
−0.49**
−0.53**
T25FWT
0.60**
Clinical status
Gait parameters
Functional and subjective measures Table 2. Pearson correlations between clinical status, gait parameters and functional and subjective gait
assessments. EDSS Expanded Disability Status Scale, CV coefficient of variation, PCI phase coordination index
GA gait asymmetry, MSWS-12v2 12-item multiple sclerosis walking scale, ABC activities-specific balance
confidence scale, T25FWT Timed 25-Foot Walk Test. **p ≤ 0.01. EDSS
Distance covered
MSWS-12v2
ABC
T25FWT
Stride CV
0.44**
−0.57**
0.53**
−0.47**
0.60**
PCI
0.42**
−0.54**
0.43**
−0.39**
0.50**
GA
0.35**
−0.42**
0.29**
−0.31**
0.46**
Distance covered
−0.59**
−0.60**
0.56**
−0.84**
MSWS-12v2
−0.59**
−0.72**
0.61**
ABC
−0.49**
−0.53**
T25FWT
0.60**
Clinical status
Gait parameters
Functional and subjective measures Table 2. Pearson correlations between clinical status, gait parameters and functional and subjective gait
assessments. EDSS Expanded Disability Status Scale, CV coefficient of variation, PCI phase coordination index,
GA gait asymmetry, MSWS-12v2 12-item multiple sclerosis walking scale, ABC activities-specific balance
confidence scale, T25FWT Timed 25-Foot Walk Test. **p ≤ 0.01. Table 2. Pearson correlations between clinical status, gait parameters and functional and subjective gait
assessments. EDSS Expanded Disability Status Scale, CV coefficient of variation, PCI phase coordination index,
GA gait asymmetry, MSWS-12v2 12-item multiple sclerosis walking scale, ABC activities-specific balance
confidence scale, T25FWT Timed 25-Foot Walk Test. **p ≤ 0.01. Table 2. Pearson correlations between clinical status, gait parameters and functional and subjective gait
assessments. EDSS Expanded Disability Status Scale, CV coefficient of variation, PCI phase coordination index,
GA gait asymmetry, MSWS-12v2 12-item multiple sclerosis walking scale, ABC activities-specific balance
confidence scale, T25FWT Timed 25-Foot Walk Test. **p ≤ 0.01. Table 2. Pearson correlations between clinical status, gait parameters and functional and subjective gait
assessments. Results EDSS Expanded Disability Status Scale, CV coefficient of variation, PCI phase coordination index,
GA gait asymmetry, MSWS-12v2 12-item multiple sclerosis walking scale, ABC activities-specific balance
confidence scale, T25FWT Timed 25-Foot Walk Test. **p ≤ 0.01. p < 0.001, η2 = 0.25), as participants with milder MS severity reporting lower self-perceived gait limitations and
higher balance confidence. Group to group post-hoc comparisons are detailed in Fig. 4. p < 0.001, η2 = 0.25), as participants with milder MS severity reporting lower self-perceived gait limitations and
higher balance confidence. Group to group post-hoc comparisons are detailed in Fig. 4. Correlation analyses. Statistically significant correlations were found between distance covered, PCI, GA
and stride CV and clinical, functional and subjective outcomes (i.e., EDSS score, distance covered, MSWS12,
T25WT and ABC) for the whole cohort (rp =|0.29| − |0.60|, p < 0.01; see Table 2 for details). PCI, GA and stride
CV showed stronger correlations when evaluating data from pwMS participants with more severe disease (see Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 www.nature.com/scientificreports/ Figure 3. Examples of Intrinsic relations between gait coordination and gait speed. PCI values calculated
for each minute of the 6MWT plotted against distance covered (equivalent to gait speed as shown on the top
horizontal axis) for 24 years old male person with MS with EDDS score 2 (green triangles), and 55 years old
female person with MS with EDDS score 1 (red circles). The numbers indicate the minute number of the 6MWT
from which data were extracted to calculate the values for this point. The Pearson correlation coefficients
are indicated for both cases (top right) and linear trend lines (dashed) are plotted (see more details in the
Supplementary material 1. Figure 3. Examples of Intrinsic relations between gait coordination and gait speed. PCI values calculated
for each minute of the 6MWT plotted against distance covered (equivalent to gait speed as shown on the top
horizontal axis) for 24 years old male person with MS with EDDS score 2 (green triangles), and 55 years old
female person with MS with EDDS score 1 (red circles). The numbers indicate the minute number of the 6MWT
from which data were extracted to calculate the values for this point. The Pearson correlation coefficients
are indicated for both cases (top right) and linear trend lines (dashed) are plotted (see more details in the
Supplementary material 1. Figure 4. Results Mean values of ABC scores, MSWS12 V2 scores, and T25FWT seconds to complete in pwMS with
different disease severity (see key). Statistically significant differences in post hoc group to group comparisons
are marked above the horizontal brackets. Means, standard deviations and effect sizes are as followed: The
severe disability group reported less balance confidence (ABC score: 50.9 ± 18.0), more self-perceived walking
limitation (MSWS12 V2 score: 69.4 ± 9.9), and performed worse on the T25FWT seconds to complete (7.5 ± 2.2)
than the moderate group (ABC score: 70.3 ± 16.3, d = 1.12; MSWS12 V2 score: 41.2 ± 22.5, d = 1.6; T25FWT
seconds to complete: 12.3 ± 3.8, d = 1.45) and compared to the mild group (AB C score: 85.4 ± 18.2, d = 1.9;
MSWS12 V2 score: 16.4 ± 20.4, d = 3.29; T25FWT seconds to complete: 6.8 ± 1.4, d = 1.9). Furthermore, the
moderate severity group also reported less balance confidence, more self-perceived walking limitation, and
performed worse on the T25FWT than the mild group (ABC, a). igure 4. Mean values of ABC scores, MSWS12 V2 scores, and T25FWT seconds to complete in pwMS with fif Figure 4. Mean values of ABC scores, MSWS12 V2 scores, and T25FWT seconds to complete in pwMS with
different disease severity (see key). Statistically significant differences in post hoc group to group comparisons
are marked above the horizontal brackets. Means, standard deviations and effect sizes are as followed: The
severe disability group reported less balance confidence (ABC score: 50.9 ± 18.0), more self-perceived walking
limitation (MSWS12 V2 score: 69.4 ± 9.9), and performed worse on the T25FWT seconds to complete (7.5 ± 2.2)
than the moderate group (ABC score: 70.3 ± 16.3, d = 1.12; MSWS12 V2 score: 41.2 ± 22.5, d = 1.6; T25FWT
seconds to complete: 12.3 ± 3.8, d = 1.45) and compared to the mild group (AB C score: 85.4 ± 18.2, d = 1.9;
MSWS12 V2 score: 16.4 ± 20.4, d = 3.29; T25FWT seconds to complete: 6.8 ± 1.4, d = 1.9). Furthermore, the
moderate severity group also reported less balance confidence, more self-perceived walking limitation, and
performed worse on the T25FWT than the mild group (ABC, a). Table 3 for details). Discussion
S
f Summary of findings. This study which assessed gait asymmetry and impaired BCG among pwMS with
spinal cord injury (i.e., excluding those with clear brainstem or cerebellar signs, see “Methods”) during relatively
long-distance walk (i.e., 6MWT) observed a relationship with disease severity and functional and subjective gait
assessments. In this cohort, gait was more asymmetric and less coordinated as the disease progressed. While
impairments in these functions are probably related to loss of spinal structures affecting CPGs outcomes (see
below), potential impacted supra spinal regions (e.g., cortex, basal ganglia) may also contribute to deficits in GA
and BCG (e.g.31). Participants with severe MS had larger GA values as compared to participants from the moderate and mild
groups. Participants with mild MS showed significantly better BCG as reflected by lower PCI values in compari-
son to the other two MS severity groups.if Significant time effect (i.e., minute by minute evaluation of parameters) was observed for PCI and GA, indi-
cating within testing deteriorations in bilateral coordination and in gait symmetry. This deterioration was more
evident in participants with more severe symptomology (i.e., significant group × time interaction). Time was
not a statistically significant effect for distance covered (i.e., gait speed) and stride CV. A possible interpretation
is that maintaining gait coordination (PCI) and symmetry (GA) is not maintained as well as maintaining gait
rhythmicity (i.e., low gait variability—stride CV) and speed among pwMS when walking without stopping for
several minutes. Cross-subjects analyses show that PCI and GA measured in participants with MS were highly inter-correlated
nd correlated with gait variability, functional gait performance and subjective gait and balance assessments. The role of gait speed. The 6MWT measures average gait speed by distance covered over a period of six
minutes. When using overall distance covered (i.e. averaged walking speed) as a covariate, MS severity level (i.e.,
‘group’ effect) was not statistically significant for stride CV and PCI. However, using this parameter as covariate
was too robust as it is based on the overall distance covered (and not per each minute). Notably, our measurement methodologies allowed us to partition the data to sub-segments, 1 min long each,
and to assess the effect of time. This analyses revealed the potential intrinsic (i.e., within subject) association
between gait speed and gait parameters (e.g., Fig. 3, and Supplementary material 1) by calculating correlation
coefficients based on six data points sets. Results For example, PCI was strongly correlated with GA within the group with moderate MS
(rp = 0.86; p < 0.01) and severe MS (rp = 0.99; p < 0.01), but not as strongly correlated within the group with mild
MS (rp = 0.36; p < 0.01). Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 www.nature.com/scientificreports/ Table 3. General and severity group Pearson correlations between the gait parameters. CV coefficient o
variation, PCI phase coordination index, GA gait asymmetry. *p ≤ 0.05. **p ≤ 0.01. Stride CV
PCI
PCI
0.70**
0.57**
0.61**
0.84**
GA
0.62**
0.83**
0.32*
0.50**
0.83**
0.36**
0.86**
0.99**
Gait parameters
Mild MS group
Moderate MS group
Severe MS group Table 3. General and severity group Pearson correlations between the gait parameters. CV coefficient of
variation, PCI phase coordination index, GA gait asymmetry. *p ≤ 0.05. **p ≤ 0.01. Discussion
S
f We used these correlation coefficients as covariates and determined that
after controlling for how gait speed is related with gait parameters (stride CV, PCI and GA) for each individual,
‘group’ effect remain significant (i.e., CV, PCI and GA are affected by disease severity in pwMS).h The literature lacks consensus with regards to the association between gait speed and GA, PCI or Gait Variabil-
ity. Studies report conflicting findings regarding each association with the literature supporting positive, negative
and no correlations between gait speed and GA34, gait speed and PCI35,36, and gait speed and gait variability37–39. Within subject effects were seen on PCI but not on GA when gait was deliberately slowed40. Relation to previous findings. PCI and GA values in the current study observed in the severe group were
consistent with the PCI and GA values reported in Gianfrancesco et al.24 measured among pwMS with severe MS
(EDSS 4–5.5) while walking unassisted at a preferred walking speed. Despite the use of a different gait analysis
system by these investigators, GA and PCI appear to be robust measures characterizing the gait of pwMS. Recently, Shema‑Shiratzky et al.26 reported the results of a study in which various gait domains (e.g., pace,
rhythm, variability, symmetry, and complexity) were measured and compared among mild- and moderate pwMS
who performed the 6MWT while wearing body-fixed sensors. They did not recruit participants with severe
MS nor reported on PCI values. In addition, they did not use gait speed as a covariate in their analysis of the
gait domains. Their results show, similarly to the current findings, an association between gait deterioration,
patient-reported gait disability and disease severity. However, in our study, symmetry had worsened during the
6MWT and variability did not while in Shema‑Shiratzky et al.’s study, variability had worsened, and symmetry
did not change. Since we included participants with EDSS score lower than 2 and above 6, (not included by
Shema-Shiratzky et al.) it might be the case that trends were missed or enhanced as a result of this truncation. Future studies may clarify this possibility. y
y
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While a control group was not included in the present study, prior studies32,33 suggest gait parameters in
healthy individuals to be similar to our mild pwMS cohort. www.nature.com/scientificreports/ p
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Few studies have reported upon correlations between clinical, functional and subjective measures among
pwMS. Goldman et al.5 reported high correlation between the 6MWT and subjective quality of life and walking
quality. Socie et al.3 reported high correlations between disease severity and three measures of gait variability
(e.g., r = 0.51 between step length and EDSS values). Learmonth et al.47 reported high correlations (r >|0.62|)
between EDSS and 6MWT distance, time to complete the T25FW and subjective walking quality. All these
findings point to a positive relation between objective gait measures and subjective assessments of performance
and quality of life. Relation between GA, BCG and MS. We found a strong correlation between GA and PCI (c.f., Table 3). However, BCG differs from gait symmetry. BCG reflects the level of coordination between the ongoing stepping
movements of both legs (i.e. the phase-dependent relationship between right and left heel strike over a number
of steps)23,31. Because people can have different swing durations for each leg with preservation of the phase rela-
tionship between the legs, an asymmetrical gait is not necessarily an uncoordinated gait23,48. We propose that the
PCI-GA correlation found in the present study may reflect the global impact of the pathogenicity on the ability
to generate symmetric as well as coordinated gait.h g
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The symmetrical gait commonly observed during normal walking can be attributed to the symmetric func-
tion of the central pattern generators (CPGs—the assumed neuronal substrate underlying rhythmical stepping
movement of the lower limbs). Anti-phased stepping is an expression of the coordination between CPGs on
both sides of the spine, most likely connected by commissural fibers21. Damage to the CST in pwMS16 most likely
impact these neuronal substrates49. Thus, we hypothesize that pwMS with asymmetrical CST are more likely to
have an asymmetrical and less coordinated gait compared to pwMS with symmetrical CST, PC, or no injury. These hypotheses, and how spinal pathophysiological pathways are interacting with effects of supra spinal lesions
to alter BCG and GA functions may be tested in future studies also involving imaging, including assessments
of spinal gray matter integrity. Limitations, implications of findings and future directions. While providing data from relatively
large cohort of pwMS, the small number of participants (n = 6, ~ 7%) in the severe group should be acknowl-
edged as it poses limitation on the external validity of our findings. www.nature.com/scientificreports/ pwMS cohort (45.4 ± 3.6 years; n = 20 vs 43.2 ± 10.4; n = 60, respectively). GA values were not reported on that
study. Despite similar PCI values, the total distance covered by the present mild cohort (~ 1,520 ft) was smaller
than an able-bodied control group with similar age range (~ 2,300 ft41). This suggests that PCI may not be the
most sensitive or earliest parameters to coincide with walking impairment, and that deficits in walking endur-
ance may precede impairment in the bilateral coordination of gait. In our study, pwMS with moderate disease
severity exhibited PCI values of ~ 6.2% (Fig. 1), larger than the PCI values observed in able-bodied participants
from a comparable age group (PCI = 4.3%42). Thus, the PCI may differentiate pwMS from able-bodied persons
as the disease progresses. p
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PCI values observed among participants from the severe MS group were similar to an elderly population. One
study of healthy individuals in their eight decade of life demonstrated that their PCI was approximately 10%,
comparable to the severe MS cohort PCI of 10.2% at a median age of 54 years35. This observation illustrates the
high degree of impaired coordination of gait in ambulatory pwMS with severe clinical disability. These data, taken
together with a previous report of lower PCI and GA values (i.e., better coordination and lower asymmetry) in
pwMS using a cane vs no cane24, suggest the use of an assistive device may improve the BCG in similar pwMS. g
gg
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PCI and GA were documented in other cohorts: stroke patients31, persons with Parkinson’s disease (PD)23, 43
and elderly fallers43. Similar to the results of the present study, PCI and GA were found to be associated with
severity of the PD symptoms. Specifically, patients who suffer from the freezing of gait symptom exhibited higher
values of GA44 and PCI32, indications for asymmetric gait, and impaired BCG.h Gait variability in pwMS has been assessed in many studies (see45 for review). This gait parameter was found
to be associated to disease severity, in agreement with the present results. It is worth noting that not all studies
use the same parameter to describe gait variability. For instance, Kalron7 and Socie et al.3 used Step Length, Time
and Width CV, Kaipust et al.46 used Stride Length and Step Width CV. Discussion
S
f PCI values calculated using a similar gait analysis
system were comparable (3.9 ± 13%) to able-bodied controls with an age range similar to the age range of the mild Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 www.nature.com/scientificreports/ References https://
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y
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9. Krasovsky, T. & Levin, M. F. Review: Toward a better understanding of coordination in healthy and poststroke gait. Neurorehabil
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4. Gianfrancesco, M. A. et al. Speed- and cane-related alterations in gait parameters in individuals with multiple sclerosis. Gait Posture
33, 140–142. https://doi.org/10.1016/j.gaitpost.2010.09.016 (2011). g
j g
25. Kasser, S. L., Jacobs, J. V., Sibold, J., Marcus, A. & Cole, L. Employing body-worn sensors to detect changes in balance and mobility
after acute aerobic exercise in adults with multiple sclerosis. Int. J. MS Care (2019).i t
p
6. Shema-Shiratzky, S. et al. Deterioration of specific aspects of gait during the instrumented 6-min walk test among people with
multiple sclerosis. J. Neurol. www.nature.com/scientificreports/ According to the literature of gait studies
with the participation of pwMS, the relative portion of participants with more severe symptomology varies. In
some studies it is relatively small or absent (e.g.2,3), in few others the proportion of this sub group was higher
than in the presents study (~ 15%5). Noteworthy, while we used EDDS score 4.5–6 as a criteria for the sever
group, different studies set different criteria (e.g.7,24). Future studies should investigate a larger cohort of pwMS
with severe disability. y
We propose that stride CV, GA and PCI are important parameters to assess among pwMS, since they seem
to deteriorate with disease progression. Stride CV, GA and PCI can be objectively assessed during home moni-
toring. While functional tests and subjective questionnaires require trained personnel, current light wearable
sensors technologies (e.g.50,51) allow an objective assessment of gait parameters like those at the focus of the
present study. Longitudinal studies will be required to determine whether changes over time in these (and other
gait parameters) have predictive diagnostic value regarding the progression of MS. Subtle impairments in gait
performances have been demonstrated as prodromal signs for worsening due to PD42,52. Further, gait coordina-
tion and gait asymmetry can be defined as targets for interventions aiming to improve gait performance53–55. The
efficacy of similar interventions in the context of fall prevention in MS56 is still to be determined. fi
In conclusion, GA and BCG are worse in pwMS who have more disability and disease progression, compared
to those with mild to moderate disability. Longitudinal studies would help determine the rate of change in these
parameters, and whether early changes have a predictive ability on disability and quality of life. Studies of physical
therapy programs designed to target GA and BCG in pwMS may have potential to improve ambulation and falls. Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 www.nature.com/scientificreports/ Received: 19 December 2019; Accepted: 22 June 2020 Received: 19 December 2019; Accepted: 22 June 2020 References References
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tion at Sheba Medical Center for technical support. This research has been supported by NIH K12 HD055931
(Wagner, PI), and NMSS PP1940 (Wagner, PI). This publication was made possible by Grant Number UL1
RR024992 from the National Center for Research Resources (NCRR), a component of the National Institutes of
Health (NIH) and NIH Roadmap for Medical Research. This research was also supported in part by NIH Grants
CO6 RR020092 and RR024992 (Washington University Institute of Clinical and Translational Sciences—Brain,
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Dynamic Trajectory of Long-Term Cognitive Improvement Up to 10 Years in Young Community-Dwelling Stroke Survivors: A Cohort Study
|
Frontiers in neurology
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cc-by
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ORIGINAL RESEARCH
published: 12 February 2019
doi: 10.3389/fneur.2019.00097 Dynamic Trajectory of Long-Term
Cognitive Improvement Up to 10
Years in Young Community-Dwelling
Stroke Survivors: A Cohort Study
Eva Elgh 1 and Xiaolei Hu 2* 1 Department of Psychology, Umeå University, Umeå, Sweden, 2 Department of Community Medicine and Rehabilitation,
Umeå University, Umeå, Sweden Background and objective: The trajectories of long-term and domain-specific
cognitive alterations over a decade after stroke are largely unknown. This study aims
to investigate the dynamic alterations of domain-specific cognitive performance among
young stroke survivors over 10 years after their first stroke. Methods: A prospective cohort study was carried out on 38 young stroke survivors
(aged 18–65 at stroke onset) living in the community at 10 years after their first stroke. The
cognitive outcomes were assessed repeatedly at 1 week, 7 months, and 10 years after
their first stroke on the sub-domains: process speed (Symbol search and Coding from
WAIS, TMT-A), visual attention (Bells test), visuospatial function (Block design from WAIS,
RCFT), executive function (TMT-B, verbal fluency), verbal function [Letter fluency (FAS)
from D-KEFS and CD], working memory (Digit Span from WAIS), immediate memory
(RCFT and CD), and delayed memory (RCFT and CD). Global cognition was evaluated
with Mini mental state examination at the two later time-points. Keywords: cognition, cognitive improvement, cognitive impairment, stroke, young adults, longitudinal study Edited by:
Swathi Kiran,
Boston University, United States Edited by:
Swathi Kiran,
Boston University, United States
Reviewed by:
Erin Godecke,
Edith Cowan University, Australia
Aristeidis H. Katsanos,
University of Ioannina, Greece
*Correspondence:
Xiaolei Hu
xiaolei.hu@umu.se Reviewed by:
Erin Godecke,
Edith Cowan University, Australia
Aristeidis H. Katsanos,
University of Ioannina, Greece Reviewed by:
Erin Godecke,
Edith Cowan University, Australia
Aristeidis H. Katsanos,
University of Ioannina, Greece Results: We found a delayed significant improvement of working memory with total
recovery 10 years after participants’ stroke. Visuospatial function recovered already at
7 months and remained stable at 10-year follow-up. Process speed demonstrated a
significant decrease at 10 years compared to 7 months after stroke onset, a decrease
which could be compensated by enhancements of other cognitive domains. No further
deterioration was found in verbal function, immediate-, and delayed memory, and
executive function during 10-year follow-up. Global cognition improved by on average
two points between 7 months and 10 years. Education level and fatigue showed low to
moderate positive correlations with cognitive improvements. Specialty section:
This article was submitted to
Stroke,
a section of the journal
Frontiers in Neurology
Received: 23 October 2018
Accepted: 24 January 2019
Published: 12 February 2019 Specialty section:
This article was submitted to
Stroke,
a section of the journal
Frontiers in Neurology Received: 23 October 2018
Accepted: 24 January 2019
Published: 12 February 2019 Conclusions: The concordance of cognitive improvements between domain-specific
and global cognitions strongly suggest that some young stroke survivors do improve
their cognitive outcome over a 10-year period following their first stroke. This finding fills
a gap of knowledge with respect to the dynamic trajectory of post-stroke cognition, with
important implications in clinical practice. Abbreviations: NPA, Neuropsychological assessment; WAIS-IV, Wechsler Adult
Intelligence Scale-IV; RCFT, Rey complex figure test and recognition trial; CD,
Claeson-Dahl; FAS, a part of the test battery Delis-Kaplan Executive Function
System (D-KEFS), named letter fluency; MMSE, Mini mental state examination;
mRS, Modified Rankin Scale. Citation: Elgh E and Hu X (2019) Dynamic
Trajectory of Long-Term Cognitive
Improvement Up to 10 Years in Young
Community-Dwelling Stroke Survivors:
A Cohort Study. Front. Neurol. 10:97. doi: 10.3389/fneur.2019.00097 February 2019 | Volume 10 | Article 97 1 Frontiers in Neurology | www.frontiersin.org Cognitive Improvement Among Stroke Survivors Elgh and Hu Study Design This study was a single-center prospective cohort study
of stroke survivors with 10-year follow-up. It was carried
out
at
the
Department
of
Neurological
Rehabilitation,
University Hospital of Northern Sweden. Ethical approval
was obtained from the regional Ethical Review Board in Umeå,
Sweden, D-nr 2015/144-31. The following cognitive domains were examined: process
speed (Symbol search and Coding from WAIS, TMT-A), visual
attention (Bells test), visuospatial function (Block design from
WAIS, RCFT), executive function (TMT-B, verbal fluency), verbal
function (Letter fluency (FAS) from D-KEFS and CD), working
memory (Digit Span from WAIS), immediate memory (RCFT and
CD), and delayed memory (RCFT and CD) (Table 2). Cognitive Function Assessments Cognitive Function Assessments
NPAs at 10-year follow-up were administrated between 2015
and 2016. The follow up assessments were completed by four
assessors who were blinded to the previous assessments. The
entire test battery took approximately 2–3 h, with an extra
30-min break with refreshments in the middle. To ensure
comparability, the selection of tests at follow-up was based on
the tests that had been used at the first assessment within 1
year after stroke. Notably, Wechsler Adult Intelligence Scale
(WAIS)-R and WAIS-III was replaced by WAIS-IV at 10-
year follow-up due to practical reasons. Previous validation
studies have shown that WAIS-IV has the same construction
as WAIS-III/R (13), with very high correlation between
subscales (r = 0.82–94) (14). Swedish norms for WAIS-
IV were used as control (14). In addition, Mini mental
state examination (MMSE) was carried out immediately prior
to NPAs. The aim of the current study was to investigate dynamic
alterations
of
the
domain-specific
and
global
cognitive
performances among young stroke survivors over 10 years
after their first stroke. Recruitment and Participants All young patients (>18 years to 65 years) who had suffered
a first-ever stroke between January 2004 and December 2007
with neuropsychological assessment within the first year after
stroke, at Stroke Centrum, University Hospital of Northern
Sweden were contacted and provided informed and written
consent via letter and telephone for recruitment to the INTRODUCTION study. Exclusion criteria were severe dementia, severe aphasia,
and severe comorbidity/non-community-dwelling, recurrence of
stroke/TIA and other physical and/or psychiatric disease after
first-ever stroke. Stroke
is
the
second
most
common
cause
of
acquired
cognitive impairment and is strongly associated with increased
disability, dependency, institutionalization, and mortality (1). Thrombolysis, improvements on rehabilitation, and secondary
stroke prevention during the latest two decades have significantly
reduced stroke mortality, which prolongs life expectancy
with extended disability-adjusted life years (DALYs) among
stroke survivors, particularly younger ones (2). While many
young stroke survivors may live several decades after stroke
onset, knowledge regarding the long-term outcomes and
longitudinal care after stroke remain poorly investigated. Cognitive impairment as one of the most “invisible disabilities”
has often been overlooked among stroke survivors during follow-
up, despite its significant impact on their lives. There was a total of 425 first-ever young stroke patients in
the Riksstroke registry and medical journals between January
1st, 2004 and December 31st, 2007 within the catchment
area. The Riksstroke registry has a proximately cover-rate of
86%. Neuropsychological assessments (NPAs) were performed
for 108 of the young stroke patients within 1 year after
their stroke. The reason for the initial NPA was unknown
to us but possibly due to patients’ capability to be assessed
and access to a psychologist at that time. NPAs were
often carried out within 1 week after stroke debut. Some
of the participants were assessed again at approximately
seven (IQR 2–10) months after stroke. After a thorough
recruitment process, 38 stroke survivors participated in the 10-
year follow-up with written consent, while 49 declined due
to various reasons (Figure 1). All participants were native
Swedish citizens. g
Currently, the few studies regarding the longitudinal effect of
stroke on cognition have shown mixed results, demonstrating
either a trend toward deterioration (3–5), stability (6, 7), or
improvements (8–11) over time. Notably, these previous studies
are based mostly upon simple global cognitive assessment instead
of comprehensive assessment with a neuropsychological test
battery (12). Two high-quality studies with sufficient sample size
have shown cognitive decline at 6 and 10 years after stroke,
but assessed only cognition at one single time-point (3, 4). These studies failed to demonstrate dynamic cognitive alterations
after stroke, despite acute and late post-stroke cognitive declines
often being reported (12). Therefore, trajectories of long-term
and domain-specific cognitive alterations among stroke survivors
over a decade are largely missing. Basic Demographic and Clinical
Characteristics Baseline characteristics of study participants, stroke survivors
who declined participation and all first-ever young stroke
patients within the catchment area under the same period are
presented in Table 1. There were no significant differences with
respect to any characteristic between participants and decliners
as well as between first-ever stroke survivors with and without
NPA at the early stage. However, there were significantly less
ischemic strokes but more hemorrhagic strokes among patients
with NPAs (P < 0.01). FIGURE 1 | Flow diagram of inclusion process. At 10-year follow-up (Table 1), the mean age of participants
was 63.8 (SD 10.6) years. All participants were living in the
community. Most participants (n = 34, 34/37, 92%) had no
or only slight disability (mRS = 0–2), the remaining three
participants had moderate disability (mRS = 3). More than
two thirds of participants (n = 21, 21/29) were overweight
(BMI >25). Approximately one third of participants (n = 15,
15/36) had anxiety problems (BAI >8), four participants
(4/36, 11%) had moderate anxiety and two participants (2/36,
5%) suffered severe anxiety (BAI >25). Eleven participants
(11/38, 29%) were depressed (BDI-II >13), with three having
moderate (3/38, 8%) and one severe (1/38, 3%) depression
(BDI-II >28). Almost half of the participants (n = 17, 17/37,
46%) had received education for more than 12 years. Four
participants were still working full time (4/37, 10%); and
six participants were working part time at 10-year follow-up. No significant difference with respect to basic characteristics
was observed between men and women at 10-year follow-
up (Table 1). FIGURE 1 | Flow diagram of inclusion process. Data Presentation and Statistical Analysis
Demographic characteristics are presented as mean ± SD,
number
with/without
number
of
cases
(%)
or
median
[25–75%
interquartile
ranges
(IQR)]
as
appropriate. Baseline characteristics in patients were compared using
a
Student
t-test,
Fisher’s
exact
test,
or
Chi-square
test
when appropriate. Other Outcome Measurements One month prior to the scheduled appointment for NPA,
the participants received various questionnaires to complete
at home and return on the NPA occasion. Depression,
anxiety, and fatigue were assessed with the Beck Depression
Inventory-II (BDI-II), Beck Anxiety Inventory (BAI), and Fatigue
assessment scale (FAS). Baseline data were collected from
the Riksstroke registry and medical records. Participants also
provided information regarding their education level, weight,
height, and employment status. February 2019 | Volume 10 | Article 97 Frontiers in Neurology | www.frontiersin.org 2 Cognitive Improvement Among Stroke Survivors Elgh and Hu then a Wilcoxon matched-pairs signed rank test was used
between two time-points. Each p-value was adjusted to account
for multiple comparisons using Dunn’s multiple comparisons
test. A Spearman correlation test was used to obtain the analysis
correlation coefficient. Statistical analyses were performed using
GraphPad Prism software version 6.0., with a P < 0.05 being
considered significant. FIGURE 1 | Flow diagram of inclusion process. Frontiers in Neurology | www.frontiersin.org Improved Working Memory Over 10-Year
After Stroke Compared to results at 1-week post-stroke, no significant
enhancements on working memory were observed at 7 months
after stroke onset as assessed by WAIS-Digit span (14). However,
significant improvements were demonstrated at 10-year follow-
up in Digit span total score (10.72, 95%CI— 9.24 to 12.21,
P < 0.0001) (Figure 2A and Table 2) and in Digit span backward
alone (2.14, 95%CI— 1.39 to 2.89, P < 0.0001) (Figure 2B and
Table 2) compared to 1 week after stroke onset as well as 7-month
follow-up (9.42, 95%CI— 7.44 to 11.41, P < 0.0001, respectively
1.85, 95%CI— 0.89 to 2.8, p = 0.006). The digit span total
score among participants reached a similar level as no-stroke
individuals at age 55–64 years (14). Digit span forward showed Data from NPA are presented in the medium raw scores
with 25–75% IQR because of the limited and varying number
of participants at the early stage. The NPAs at the early
stage were presented at two time-points (i.e., 1 week and
7 months, with some missing values). No adjustment was
made on missing values. Numbers of raw scores on each
assessment /time-point are clearly presented in Table 2. A
repeated measures ANOVA (Friedman test) or non-repeated
measures ANOVA (Kruskal-Wallis test) was chosen for non-
parametric multiple comparisons between three time-points, February 2019 | Volume 10 | Article 97 Frontiers in Neurology | www.frontiersin.org 3 Cognitive Improvement Among Stroke Survivors Elgh and Hu TABLE 1 | Demographic and clinical characteristics of all first-ever young stroke survivors within the catchment area between 2004 and 2007 and all participants at
10-years follow-up. Improved Working Memory Over 10-Year
After Stroke TABLE 1 | Demographic and clinical characteristics of all first-ever young stroke survivors within the catchment area between 2004 and 2007 and all participants at
10
f ll haracteristics of all first-ever young stroke survivors within the catchment area between 2004 and 2007 and all participants at TABLE 1 | Demographic and clinical characteristics of all first-ever young stroke survivors within the catchment area between 2004 and 2007 and all participants at
10-years follow-up. PATIENT CHARACTERISTICS AT STROKE ONSET Improved Visuospatial Function Already at
Subacute Phase (7 Months After Stroke) Improved Visuospatial Function Already at
Subacute Phase (7 Months After Stroke)
The visuospatial function, assessed with WAIS Block design,
showed a significant improvement (14.36, 95%CI— 10.78
to 17.95, p = 0.0002) already at 7 months after stroke
onset compared to the results at 1-week post-stroke. The
improvements remained at 10-year follow-up (9.96, 95%CI—
6.46 to 13.47, p = 0.003) (Figure 2D and Table 2) and
reached the normative level compared to non-stroke peers
(14). However, there were no significant changes in RCFT-copy
test (Table 2). Improved Visuospatial Function Already at
Subacute Phase (7 Months After Stroke)
The visuospatial function, assessed with WAIS Block design,
showed a significant improvement (14.36, 95%CI— 10.78
to 17.95, p = 0.0002) already at 7 months after stroke
onset compared to the results at 1-week post-stroke. The
improvements remained at 10-year follow-up (9.96, 95%CI—
6.46 to 13.47, p = 0.003) (Figure 2D and Table 2) and
reached the normative level compared to non-stroke peers
(14). However, there were no significant changes in RCFT-copy
test (Table 2). Improved Visuospatial Function Already at
Subacute Phase (7 Months After Stroke)
The visuospatial function, assessed with WAIS Block design,
showed a significant improvement (14.36, 95%CI— 10.78
to 17.95, p = 0.0002) already at 7 months after stroke
onset compared to the results at 1-week post-stroke. The
improvements remained at 10-year follow-up (9.96, 95%CI—
6.46 to 13.47, p = 0.003) (Figure 2D and Table 2) and
reached the normative level compared to non-stroke peers
(14). However, there were no significant changes in RCFT-copy
test (Table 2). Improved Visuospatial Function Already at
Subacute Phase (7 Months After Stroke) a significant improvement already at 7-month follow-up (sub-
acute phase) and remained stable at 10-year follow-up (Figure 2C
and Table 2). Immediate memory assessed by RCFT (15) and CD (16)
showed no significant changes between 7-month and 10-year
follow-ups (Table 2). Frontiers in Neurology | www.frontiersin.org Improved Working Memory Over 10-Year
After Stroke Characteristics
First-ever young stroke survivor
with NPA
First-ever young stroke survivor
Recruitment 10 years follow-up
All with NPA
(n = 108)
All
without NPA
(n = 317)
p-value
Declined
(n = 49)
Included
(n = 38)
p-value
PATIENT CHARACTERISTICS AT STROKE ONSET
Mean age ± SD
54.9 ± 7.9
53.9 ± 9.1
0.58
54.7 ± 8.6
55.8 ± 8.4
0.18
Men/Women
33/16
19/19
0.13
70/38
207/110
>0.99
Civil status
(Live alone/live with somebody/unknown)
8/39/2
7/30/1
0.91
22/82/3a
85/219/13
0.33
STROKE CHARACTERISTICS AT STROKE ONSET
Ischemia (%)
35 (71%)
30 (79%)
0.47
81 (75%)
191 (86%)b
0.007*
Hemorrhage (%)
10 (20%)
6 (16%)
0.78
18 (17%)
24 (13%)b
0.009*
Unknown (%)
4 (8%)
2 (5%)
0.69
9 (8%)
4 (1%)b
0.001*
RISK FACTORS AT STROKE ONSET
Atrial fibrillation (%)
2 (4%)
4 (11%)
0.40
6 (6%) a
19 (6%)
>0.99
Hypertension (%)
19 (39%)
11 (29%)
0.37
36 (34%) a
117 (37%)
0.56
Diabetes mellitus (%)
6 (12%)
4 (11%)
>0.99
12 (11%) a
39 (12%)
0.86
Smoking (%)
16 (33%)
9 (24%)
0.47
30 (28%) a
72 (22%)
0.30
CHARACTERISTICS AT 10 YEARS-FOLLOW-UP
Total
Female
Male
Age (mean ± SD)
63.8 ± 10.6
61.8 ± 9.9
65.7 ± 11.3
0.14
Years between follow-up and stroke onset (mean ± SD)
10.5 ± 0.9
10.6 ± 0.9
10.4 ± 0.8
0.40
Median of mRS (25–75% percentile)
1 (0–2)a
1 (0–2)
0 (0–1.3)
0.21
Median of BMI
(25–75% percentile)
26 (22–28)c
28 (23–30)
26 (22–27)
0.08
Median of BAI
(25–75% percentile)
6 (2–11)
10 (3–17)
4 (1–7)
0.09
Median of BDI-II
(25–75% percentile)
9 (4–14)
10 (6–16)
7 (2–11)
0.15
EDUCATION (NUMBER OF CASES %)
9 years
7 (19%)
3 (16%)
4 (22%)
0.69
12 years
12 (32%)
8 (42%)
4 (22%)
0.30
>12 years
17 (46%)
8 (42%)
9 (50%)
0.75
FULLTIME EMPLOYMENT (NUMBER OF CASES %)
Before stroke
28 (76%)a
14 (74%)
14 (77%)
>0.99
2 years after stroke
11 (32%)d
5 (29%)
6 (35%)
>0.99
10 years after stroke
4 (11%)a
1 (5%)
3 (17%)
0.34
*P < 0.05 was considered as significance. aMissing 1 patient in the group. b30% missing value. c24% missing value. d10% missing value. ns, no significant difference; NPA,
neuropsychological assessment. Improved Working Memory Over 10-Year
After Stroke Similar to immediate memory, delayed
memory remained at the same levels when RCFT- delayed
recall and recognition as well as CD- retention and recognition
were examined at 7 months and 10 years after first-ever stroke
(Table 2). Notably, only a few participants (n = 1 or 4) were tested
in the memory domain at 1 week after stroke onset (Table 2). February 2019 | Volume 10 | Article 97 Frontiers in Neurology | www.frontiersin.org 4 Cognitive Improvement Among Stroke Survivors Elgh and Hu TABLE 2 | Neuropsychological assessment scores from 1 week up to 10 years after stroke onset. Cognitive
domain
Assessments
Time after stroke onset
1 week
7 months
10 years
p-value
Raw score
N
Raw score
N
Raw score
N
Global cognition
MMSE
27 (25–29)
17
29 (27–30)
38
0.01*
Processing speed
WAIS-Symbol searching
27 (23–37)
3
28 (20.5–35)a
21
23 (16.8–27)b
38
0.03*(a: b)
WAIS-Coding
43 (31–46)c
23
57 (42–69)d
23
49 (35.5–56.5)
37
0.007*(c: d)
TMT–A
46 (35–56)
27
40 (26–65)
11
37 (29.5–50)
37
0.27
Visual attention
Bells test
34 (30.5–35)
29
34 (32.5–34.5)
21
33.5 (31.8–35)
38
0.87
Verbal fluency
D-KEFS-FAS
35 (25.3–41.8)
24
38 (27.8–59.8)
8
34 (25.8–48)
38
0.65
CD
See below
Immediate
memory
RCFT-IM
14 (10.3–17.8)
4
14 (10.6–21.8)
14
12 (7.8–16)
34
0.32
CD-weighted score
230
1
138 (29–196)
19
127.5
(99.5–181.5)
38
0.47
Delayed memory
RCFT-delayed recall
12.5
(8.9–19.1)
4
14.3
(10.8–20.6)
14
10.5 (6.6–16)
36
0.14
RCFT-recognition
19 (15.8–20.8)
4
20 (19–20)
13
19 (18.3–21)
36
0.68
CD-retention
57
1
71 (50–100)
19
70 (56–86.5)
38
0.80
CD-recognition
10
1
10 (9–10)
19
10 (9–10)
38
0.39
Executive function
TMT-B
101 (67–134)
27
72 (54–126)
11
81 (62–116)
37
0.17
D-KEFS-FAS
See above
Visuospatial
function
WAIS-Block design
27 (15–35)e
27
39 (34–51)f
19
35.5(28.8–48)g
38
0.0003*(e: f)
0.004*(e: g)
RCFT-copy
31 (29.3–33.9)
4
31 (29–34.3)
14
28.5 (24.7–31.8)
36
0.11
Working memory
WAIS-Digit span (F)
7 (6–8)h
30
8 (7–10)i
26
8 (7–9)j
38
0.03*(h: i)
0.02*(h: j)
WAIS-Digit span (F + B)
13 (11–14)k
29
13 (11.75–18)l
26
24 (20–27)m
38
<0.001*(k: m)
<0.001*(l:m)
WAIS-Digit span (B)
6 (4–7)n
29
5.5 (4–8)o
26
8 (6–9.25)p
38
<0.001*(n: p)
0.006*(o:p)
Data presented as median (25–75% percentile). *P < 0.05 was considered as significance. Stable Attention, Verbal Fluency, and
Executive Function During 10-Year
Follow-Up The global cognition at 10-year follow-up assessed by MMSE
showed a median score of 29 with 25–75% percentile (27–30). Compared to the results at 7 months after stroke onset, with a
median score of 27 with 25–75% percentile (25–29), there was a
weak but significant improvement in global cognition (p = 0.02)
among participants (Table 2). Attention examined with Bells test (17) showed similar
results from 1 week, 7 months up to 10 years after stroke
(Table 2). Verbal fluency and executive function also remained
stable
between
1-week
to
10-year
follow-ups
(Table 2)
when tested with D-KEFS-Verbal fluency (18) and TMT-B
(19, 20). Improved Working Memory Over 10-Year
After Stroke ns, no significance; Nr, number of participants; WAIS-IV, Wechsler Adult Intelligence Scale-
IV; RCFT, Rey Complex Figure Test and recognition trial; CD, Claeson-Dahl; FAS: a part of the test battery Delis-Kaplan Executive Function System (D-KEFS), named letter fluency. F, Forward; B, backward; MMSE, Mini Mental Scale Examination; TMT, Trail making test; IM, Immediate Memory. TABLE 2 | Neuropsychological assessment scores from 1 week up to 10 years after stroke onset. Data presented as median (25–75% percentile). *P < 0.05 was considered as significance. ns, no significance; Nr, number of participants; WAIS-IV, Wechsler Adult Intelligence Scale-
IV; RCFT, Rey Complex Figure Test and recognition trial; CD, Claeson-Dahl; FAS: a part of the test battery Delis-Kaplan Executive Function System (D-KEFS), named letter fluency. F, Forward; B, backward; MMSE, Mini Mental Scale Examination; TMT, Trail making test; IM, Immediate Memory. Dynamic Process Speed Fatigue (n = 19) demonstrated a low to moderate but significant
positive correlation to working memory that is forward and
backward digit span [r = 0.51, 95%CI (0.06–0.79), p = 0.03] and
backward digit span [r = 0.49, 95%CI (0.03–0.78), P = 0.03]
(Figures 3A,B), and visuospatial function [r = 0.48, 95%CI
(0.01–0.77), p = 0.04]. Education (n = 37) showed a low but
significant positive correlation with working memory, that is
forward and backward digit span [r = 0.46, 95%CI (0.15–0.69), Process
speed
assessed
with
a
WAIS-Symbol
search
demonstrated a significant decrease (6.33, 95%CI— 3.65
to 9.02, p = 0.03) after 10 years compared to the 7-
month
follow-up. However,
WAIS-Coding,
not
TMT-A,
demonstrated
a
significant
speed
enhancement
at
sub-
acute phase but returned to the former level at the chronic
stage (Table 2). February 2019 | Volume 10 | Article 97 Frontiers in Neurology | www.frontiersin.org 5 Cognitive Improvement Among Stroke Survivors Elgh and Hu FIGURE 2 | Improvement of working memory (A–C) and visuospatial function (D) over 10-year follow-up. Dotted lines in (A,D) indicate respective normative data
(mean ± SD). *P < 0.05; **P < 0.01; ***P < 0.001; ****P< 0.0001. FIGURE 2 | Improvement of working memory (A–C) and visuospatial function (D) over 10-year follow-up. Dotted lines in (A,D) indicate respective normative data
(mean ± SD). *P < 0.05; **P < 0.01; ***P < 0.001; ****P< 0.0001. p = 0.004] and backward digit span [r = 0.44, 95%CI (0.12–0.67),
p = 0.007] (Figures 3C,D) as well as forward digit span [r = 0.38,
95%CI (0.05–0.63), p = 0.02]. and executive function, remained relatively stable over 10 years
after initial post-stroke declines. Processing speed was the only
domain in the cohort that demonstrated a significant decrease in
one of three subtests at 10-year follow-up compared to 7-month
follow-up. Education level and fatigue, but not stroke sub-type,
age, and gender, showed low to moderate positive correlations
with cognitive improvements among participants. No significant differences were observed in the working
memory and visuospatial function when comparing different
stroke sub-type (ischemic vs. hemorrhagic infarct) at 10-year
follow-up (Figure 4). Notably, only six participants suffered
hemorrhagic infarct in the cohort. The most striking finding in the current study was significant
improvement of working memory in all Digit span assessments
at 10 years after stroke. Dynamic Process Speed No such late cognitive recovery among
stroke survivors has previously been reported according to
our knowledge, although good functional outcome is common
among young stroke survivors (21). This surprising result is truly
encouraging because working memory plays an important role
in everyday functions. The amelioration of working memory
capacity among the participants could implicate a potential
enhancement of other complex cognitive functions (22), such as
reading comprehension, learning ability, and executive function
(9) in their daily lives. This role is in fact supported by basic
characteristic data in the present cohort, where more than
90% of participants were independent in their daily activities
(mRS = 0–2) and one fourth of participants remained working
part or full time at 10 years after stroke. In fact, improvement
of working memory in the present cohort reached a similar
level to their non-stroke peers (14), suggesting that a true
recovery of working memory does occur among these young
stroke survivors. There were no correlations demonstrated between cognitive
improvements and age, gender, anxiety, or depression among
participants at 10-year follow-up. Frontiers in Neurology | www.frontiersin.org DISCUSSION The current study provides a unique longitudinal cognitive
evaluation of young stroke survivors using a comprehensive
neuropsychological test battery repetitively from acute (1-
week), subacute (7-month) to chronic (10-year) stages after
stroke onset. To our knowledge, no such valuable cognitive
data have been reported previously. We found significant
improvements and recoveries with respect to working memory,
visuospatial function, and global cognition, after initial post-
stroke cognitive decline among the participants at 10 years after
stroke. Visuospatial function improved already at 7 months post-
stroke but working memory was enhanced mainly at the chronic
phase, 10 years after stroke. The remaining cognitive domains,
such as attention, immediate and delayed memory, and verbal February 2019 | Volume 10 | Article 97 Frontiers in Neurology | www.frontiersin.org 6 Cognitive Improvement Among Stroke Survivors Elgh and Hu Elgh and Hu
Cognitive Improvement Among Stroke Survivors
FIGURE 3 | Correlations between working memory and fatigue (A,B) as well as education (C,D). FIGURE 4 | No significant difference when comparing between ischemic and hemorrhagic stroke on working memory (A–C) and visuospatial function (D) at 10-year
follow-up. FIGURE 3 | Correlations between working memory and fatigue (A,B) as well as education (C,D). FIGURE 3 | Correlations between working memory and fatigue (A,B) as well as education (C,D). FIGURE 4 | No significant difference when comparing between ischemic and hemorrhagic stroke on working memory (A–C) and visuospatial function (D) at 10-year
follow-up. FIGURE 4 | No significant difference when comparing between ischemic and hemorrhagic stroke on working memory (A–C) and visuospatial function (D) at 10-year
follow-up. February 2019 | Volume 10 | Article 97 7 Frontiers in Neurology | www.frontiersin.org Cognitive Improvement Among Stroke Survivors Elgh and Hu Our results are however contradictory to two previous studies
with relatively large samples (515 and 277 stroke survivors,
respectively) showing cognitive decline at 6 or 10 years after
stroke (3, 4). The weaknesses of these previous studies are their
cognitive assessments at only a single time-point, meaning that
they were unable to detect dynamic cognitive improvements
over time after stroke. Compared to the Digit span we used
to examine working memory, Schaapsmeerder et al. employed
the paper and pencil memory scanning test (PPMST) to assess
working memory (3) and no specific working memory was
assessed in another previous study (4). DISCUSSION A possible explanation
could be that Coding demands cognitive processes other than
Symbol search, such as visual short-term memory, visuo-motor
coordination, and attention (14). The pure decline of processing
speed may be compensated by other cognitive improvements
mentioned above. The decline of processing speed on Symbol
search may be an expected age-related change (27) when the
participants aged, with a mean age of 54–64 years during 10-
year follow-up in the current study. Beside stroke, age alone has
important impact on cognitive performance due to structural
changes in the aged brain (27, 28). Compared to working memory, visuospatial function assessed
with Block design demonstrated another temporal profile. There
was an early significant recovery to a similar level as their peers
(14) already at sub-acute phase and it remained stable at 10-year
follow-up. Although this result is in line with previous studies
(10, 24), the different recovery trajectories in varying domains
are intriguing. Notably, no significant alterations were observed
over time in RCFT-copy test as well as Bell-test, results which
could be explained by a ceiling effect on these tests already at the
acute stage. This result may indicate a certain difficulty of NPA
required at the acute phase in order to detect the improvement
over time. Global cognition measured by MMSE also improved by on
average two points between 7-month and 10-year follow-ups
in the present study. Together with previous studies showing
global cognitive recovery from 3 weeks to 1 year after stroke
onset (8, 9, 11, 12), the current results suggest that cognitive
recovery may begin early after initial cognitive decline, and
continue to improve over a long period of time (i.e., more
than a year or years after stroke). Improvements of global
cognition are supported by neuropsychological data because
most cognitive domains measured by the neuropsychological
test battery showed either marked improvements or stable
results over time. The concordance between global and domain-
specific cognitions suggest strongly that stroke survivors may
improve their cognitive outcome over a long time at a
young age. Beside long-term recoveries on working memory and
visuospatial function, our results demonstrated that other
cognitive domains such as verbal fluency, immediate and
delayed memory, and executive function were relatively stable
over 10 years. This is important knowledge for clinicians and
patients because a significant amount of previous data could
only point out the cognitive decline after stroke at certain
time-points (3, 4). DISCUSSION The discrepancy of these
two assessment tools and characteristics of different samples may
have contributed to the differences in the results. In addition, a
relatively higher education level (almost half of the participants
had a university degree) in our cohort could also partially
explain the improvements of cognitive outcomes at the 10-year
post-stroke, due to the fact that we and others (14, 23) found
strong evidence that education plays a significant positive role in
working memory. many young stroke survivors and clinicians because they provide
evidence counteracting the previous assumption regarding stroke
survivors acquiring a faster rate of cognitive decline over the
years (4), at least among younger stroke survivors. As stated
previously, different assessments and sample characteristics, such
as age and education, may explain some discrepancies between
the studies. The
reason
for
the
cognitive
improvements
in
the
current study was not investigated at this stage. Because no
organized cognitive rehabilitation was supplied after the initial
hospitalization in this cohort, participants’ struggle to be active
and/or live independently in their daily lives may presumably be
a crucial element for their cognitive improvements. These daily
activities could possibly trigger activity-induced neuroplasticity
(25) to further ameliorate cognitive recovery. Furthermore, a
better collateral blood flow with a more pronounced neuronal
plasticity as well as absence/less of neurodegenerative pathology
may facilitate cognitive enhancement in young stroke survivors
(21, 26). Therefore, active participation in daily activity should
be taken into account when cognitive rehabilitation is planned in
clinical settings. g
y
Intriguingly, Digit span forward (which considers pure short-
term memory and rote learning) had improved already at sub-
acute phase after stroke; but the more challenging Digit span
backward and Digit span total score reflected real working
memory recovered later at the chronic stage. This result is highly
encouraging because it indicates that a delayed improvement
of working memory does occur at least among these younger
community-dwelling stroke survivors at chronic phase. This
finding suggests that more complicated functions, such as
working memory, may require longer time (more than a year/
years) to recover. This prolonged improvement of working
memory after stroke illustrated in this study provides important
implications for cognitive rehabilitation, driving, and care
planning in clinical practice. In contrast, process speed examined by a Symbol search
showed significant decline at 10-year follow-up but not process
speed measured with Coding and TMT-A. Frontiers in Neurology | www.frontiersin.org February 2019 | Volume 10 | Article 97 DISCUSSION Our current results suggest no further
deterioration in overall cognition among these young stroke
survivors over 10 years’ time, despite the initial declines after
stroke. This result may imply that our participants had in
fact slightly improved their cognition to compensate for age-
related declines. Our results provide important information to A small but significant positive association was found
between level of education and working memory. This result
is in line with early evidence that working memory capacity
is correlated with learning outcomes in literacy and numeracy
(14, 29). These education effects may be also mediated by
age (23). However, different stroke sub-type did not influence
cognitive recovery in our cohort with notably very limited
sample size. Furthermore, stroke localization could play a
very important role in cognitive recovery. Unfortunately, February 2019 | Volume 10 | Article 97 Frontiers in Neurology | www.frontiersin.org 8 Cognitive Improvement Among Stroke Survivors Elgh and Hu the
clinical
characteristics
in
the
cohort
were
collected
from the Riksstroke registry, where stroke localization was
not registered. population, the current study suggests mainly that cognitive
recovery does occur during both sub-acute and chronic phases
after first-ever stroke, at least among some young stroke survivors
who are living in the community. We are surprised about the positive association between
fatigue and improved cognitive function. This relation could
possibly be explained by the fact that fatigue was assessed
by answering questionnaires at home prior to NPA, and
thus mainly reflected fatigue level in participants’ daily lives. Presumably, those participants who performed well in the
NPA and struggle in daily life have to use more energy to
achieve better functional outcomes with fatigue as a drawback. This presumption may suggest various reasons behind the
fatigue at different stages after stroke. Therefore, awareness
of the importance of balance between activity and rest
over time should be enhanced in both stroke survivors and
medical staff. This study demonstrated that significant cognitive recoveries
on visuospatial function and working memory do occur at sub-
acute and chronic stages after initial post-stroke cognitive decline
in young community-dwelling adults after first-ever stroke. No
further deterioration in overall cognition occurred among these
young stroke survivors over 10 years’ time after stroke. The
age-related decline of processing speed at 10-year follow-up
could be compensated by other cognitive improvements and
stabilizations over time. DISCUSSION This finding fills a gap of knowledge
concerning the dynamic trajectory of post-stroke cognition over
10 years after stroke. The current results may supply strong
clinical implications on rehabilitation and care planning in
clinical practice. The
current
study
has
several
unique
strengths. We
demonstrated repeatedly domain-specific cognitive outcomes by
NPAs at acute, sub-acute, and chronic phases over 10 years after
stroke onset, providing us with a distinctive opportunity to detect
longitudinal cognitive alterations over time. Moreover, trajectory
of post-stroke cognition was examined by both MMSE and
NPAs in the same cohort. It provides not only more comparative
information between global- and domain-specific cognition
but also more comprehensive information regarding multiple
cognitive domains: process speed, visual attention, as well as
visuospatial, executive, and verbal functions, and in addition
working, immediate, and delayed memory types. Furthermore,
all assessors were blind to the previous assessments in order to
reduce possible sources of bias. FUNDING However, some methodological limitations need to be
addressed. First, we are aware of the small number of participants
and some variations of the number of participants at early time-
points in the current study. The current findings need to be
replicated in other studies with a larger sample size. Second, no
cognitive data were collected for those who denied participating
in the study. One may assume that the deniers may have
worse clinical outcomes. However, demographic characteristics
did not show any significant differences between participants
and deniers, indicating that selection bias was unlikely during
the follow-ups. Third, our results were generalized only from
community-dwelling participants without aphasia. Nevertheless,
rather than generalizing the findings to the entire young stroke This study was supported by Västerbotten County Council
and Umeå University (ALF Foundation), the Swedish Stroke
Foundation (Stroke Riksförbundet), and the Northern Swedish
Stroke Fund (Strokeforskning I Norrland Insamlingsstiftelse). This study was supported by Västerbotten County Council
and Umeå University (ALF Foundation), the Swedish Stroke
Foundation (Stroke Riksförbundet), and the Northern Swedish
Stroke Fund (Strokeforskning I Norrland Insamlingsstiftelse). ACKNOWLEDGMENTS We would like to thank all participants and assessors for their
patience and time, rehabilitation coordinator Catharina Elback
for her excellent recruitment work and Professor Per Wester for
supplying demographic data from the Riksstroke Registry. AUTHOR CONTRIBUTIONS EE
contributed
to
study
conception,
sample
collection,
interpretation of data, and revising the manuscript, provided
final approval of the version to be published; agreed to be
accountable for all aspects of the work. XH contributed to study
conception, supervision of the acquisition, interpretation of
data, drafting, and revised the manuscript including the figures,
tables, and references and provided final approval of the version
to be published and agreed to be accountable for all aspects of
the work. 4. Levine DA, Galecki AT, Langa KM, Unverzagt FW, Kabeto MU, Giordani
B, et al. Trajectory of cognitive decline after incident stroke. JAMA (2015)
314:41–51. doi: 10.1001/jama.2015.6968
5. Delavaran H, Jonsson AC, Lovkvist H, Iwarsson S, Elmstahl S, Norrving B,
et al. Cognitive function in stroke survivors: a 10-year follow-up study. Acta
Neurol Scand. (2017) 136:187–94. doi: 10.1111/ane.12709
6. Kohler M, Kliegel M, Kaduszkiewicz H, Bachmann C, Wiese B, Bickel H, et al.
Effect of cardiovascular and metabolic disease on cognitive test performance
and cognitive change in older adults. J Am Geriatr Soc. (2012) 60:1286–91.
doi: 10.1111/j.1532-5415.2012.04032.x
7. Dik MG, Deeg DJ, Bouter LM, Corder EH, Kok A, Jonker C. Stroke
and
apolipoprotein
E
epsilon4
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doi: 10.1161/01.STR.31.10.2431 4. Levine DA, Galecki AT, Langa KM, Unverzagt FW, Kabeto MU, Giordani
B, et al. Trajectory of cognitive decline after incident stroke. JAMA (2015)
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and
apolipoprotein
E
epsilon4
are
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for
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et al. Cognitive function in stroke survivors: a 10-year follow-up study. Acta
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conducted in the absence of any commercial or financial relationships that could
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TX: The Psychological Corporation (2001). 19. Mitrushina M, Boone KB, Razani J, D’Elia LF. Handbook of Normative Data
for Neuropsychological Assessment. New York, NY: Oxford University Press
(2005). Copyright © 2019 Elgh and Hu. This is an open-access article distributed under the
terms of the Creative Commons Attribution License (CC BY). Frontiers in Neurology | www.frontiersin.org February 2019 | Volume 10 | Article 97 REFERENCES The use, distribution
or reproduction in other forums is permitted, provided the original author(s) and
the copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
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PRODUCTION OF FOAMED HEAT INSULATION MATERIALS FROM COPPER ORE MILL TAILINGS
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Vestnik Tomskogo gosudarstvennogo arhitekturno-stroitelʹnogo universiteta
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cc-by
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ПОЛУЧЕНИЕ ПОРИСТОГО
ТЕПЛОИЗОЛЯЦИОННОГО МАТЕРИАЛА
ИЗ ХВОСТОВ ОБОГАЩЕНИЯ МЕДНОЙ РУДЫ Статья посвящена актуальной экологической проблеме рационального использова-
ния природных ресурсов путем вовлечения в производство техногенных отходов. Рас-
сматривается технология получения пористого материала на основе хвостов обогащения
медной руды. Цель исследования – оценка пригодности данных отходов для производ-
ства теплоизоляционного материала. Химический состав хвостов и его высокая диспер-
сия указывают на потенциальную пригодность для синтеза фритты без полного плавле-
ния шихты. Синтез фритты осуществляется из двухкомпонентной шихты, состоящей на
80 % из отходов и 20 % кальцинированной соды. Температура синтеза не превышает
900 °С, чем выгодно отличается от традиционного стекловарения, протекающего при
1500 °С. Экспериментальным путем установлено, что оптимальную поровую структуру
с высокой степенью однородности и средним размером пор не более 1 мм имеют образ-
цы, полученные при температуре вспенивания 850 °С с выдержкой 10 мин. Полученный
пористый материал плотностью 250 кг/м3 имеет повышенную прочность (до 1,7 МПа). Ключевые слова: медные хвосты; низкотемпературный синтез фритты;
прочность; пористый материал. Для цитирования: Казьмина О.В., Семке А.П., Беляева И.В., Семухин Б.С. Получение пористого теплоизоляционного материала из хвостов обогащения
медной руды // Вестник Томского государственного архитектурно-строитель-
ного университета. 2019. Т. 21. № 1. С. 159–168. O.V. KAZMINA1, A.P. SEMKE1, I.V. BELYAEVA1, B.S. SEMUKHIN2,
1National Research Tomsk Polytechnic University,
2 Tomsk State University of Architecture and Building УДК 666.1.022.8 УДК 666.1.022.8 Вестник ТГАСУ Т. 21, № 1, 2019 Вестник ТГАСУ Т. 21, № 1, 2019 159 PRODUCTION OF FOAMED HEAT INSULATION
MATERIALS FROM COPPER ORE MILL TAILINGS This paper focuses on ecological and environmental management problems involving the
industrial wastes. The paper proposes the production method for foamed materials on made
from copper ore tailings. The goal of the study is to evaluate the industrial waste applications
in the production of heat-insulating materials. The chemical composition of wastes and its high
dispersion indicates to its potential use in the frit synthesis without complete melting of the
mixture. Frit is synthesized from a two-component blend consisting of 80 % waste and 20 %
caustic ash. The synthesis temperature does not exceed 900 °С which is a better alternative to
the conventional glassmaking temperature of 1500 °С. It is shown that the optimum porous,
uniform structure with the average pore size is not over 1 mm forms in samples prepared at
a foaming temperature of 850 °С followed by a 10-minute curing. The resulting materials has
a density of 250 kg/m3 and increased strength up to 1.7 MPa. Keyword: copper or mill tailing; low-temperature frit synthesis; strength; foamed
material. Казьмина О.В., Семке А.П., Беляева И.В., Семухин Б.С., 2019 160 О.В. Казьмина, А.П. Семке, И.В. Беляева и др. For citation: Kazmina O.V., Semke A.P., Belyaeva I.V., Semukhin B.S. Polu-
chenie poristogo teploizolyatsionnogo materiala iz khvostov obogashcheniya mednoi
rudy [Production of foamed heat insulation materials from copper ore mill tailings]. Vestnik Tomskogo gosudarstvennogo arkhitekturno-stroitel'nogo universiteta – Jour-
nal of Construction and Architecture. 2019. V. 21. No. 1. Pp. 159–168. Известно, что в результате добычи и переработки горных пород образу-
ется значительное накоплению различных отходов, которые, как правило,
складируют в отвалах, хвостохранилищах или шлаконакопителях. Такой под-
ход к решению вопросов утилизации отходов представляет серьезную опас-
ность в экологическом плане, особенно это касается многотоннажных про-
мышленных отходов, таких как металлургические шлаки, ежегодный выход
которых в мире составляет сотни миллионов тонн. По некоторым данным,
мировая добыча горнорудного сырья и топлива значительно превысила
150 млрд т в год [1–3]. Поэтому задачи переработки техногенного сырья пред-
ставляют собой одну из важнейших проблем современного мира и являются
чрезвычайно актуальными. р
у
Использование техногенного сырья в качестве компонента шихты для
производства строительных материалов является одним из основных направ-
лений переработки отходов. В этом плане разработка пористых теплоизоля-
ционных материалов на основе техногенного и некондиционного сырья рас-
сматривается как эффективный метод снижения издержек производства. По-
лучение теплоизоляционных материалов с использованием техногенного
сырья – одно из интенсивно развивающихся направлений исследований [4–6]. PRODUCTION OF FOAMED HEAT INSULATION
MATERIALS FROM COPPER ORE MILL TAILINGS Особенно актуальны вопросы получения экологически безопасных и долго-
вечных эффективных теплоизоляторов, к которым относятся пеностекольные
материалы. Разработкой технологии и расширения сырьевой базы для их про-
изводства занимаются ученые различных стран [7, 8]. В основном эти иссле-
дования направлены на использование различных видов стеклобоя, в частно-
сти стекла электронно-лучевых трубок, экранов компьютеров и т. п. Цель работы – исследовать пригодность хвостов обогащения медной
руды для получения гранулированного пористого теплоизоляционного мате-
риала при температурах, не превышающих 900 °С. Для этого решались следующие задачи: исследование пригодности хво-
стов обогащения медной руды в качестве основного компонента шихты для
низкотемпературного (при температурах ниже 950 °С) синтеза фритты; разра-
ботка состава пенообразующей смеси на основе синтезированной фритты;
определение оптимальных параметров температурного режима вспенивания;
исследование основных свойств гранулированного пористого материала. По внешнему виду хвосты обогащения медной руды представляют со-
бой мелкозернистую пробу светло-бежевого цвета. По химическому составу
отходы представлены пониженным содержанием основного стеклообразу-
ющего оксида SiO2 и повышенным содержанием Al2O3, Fe2O3, а также окси-
дов щелочных и щелочноземельных металлов. Согласно результатам рент-
генофлуоресцентного анализа отходы не соответствуют марке кварцевого
песка ПС-250 (ГОСТ 22551–77), который используют для производства пе-
ностекла, консервной тары и бутылок из полубелого стекла, изоляторов, Получение пористого теплоизоляционного материала 161 труб, аккумуляторных банок (табл. 1). Оксиды, которые входят в состав
стекла в качестве основных компонентов, такие как SiO2, Al2O3, CaO, MgO,
K2O, Na2O, необходимо учитывать при расчете состава шихты. Важным по-
казателем является стабильность химического состава отходов. Установле-
но, что отклонения по содержанию основных оксидов не превышают 0,2 %,
что указывает на их высокую стабильность, обусловленную усреднением
состава в процессе переработки. Результаты спектрального анализа пробы
отходов показали присутствие следующих элементов, содержание которых
не превышает 0,1 масс. %: As – 0,0006 %; Co – 0,0102 %; Ni – 0,0066 %; Mn –
0,098 %. Радиологические измерения, проведенные с помощью радиометра-
дозиметра, показали, что радиационная активность не превышает допусти-
мые нормы естественного фона и составляет 0,07 мкзв/ч. Эффективная ак-
тивность составляет 241 Бк/кг, что также не превышает уровень безопасно-
сти для строительных материалов (370 Бк/кг). Т б
1 Табл
Химический состав отходов и кварцевого песка марки ПС-250 ц
Химический состав отходов и кварцевого песка марки ПС-250
Сырье
Содержание оксидов, масс. PRODUCTION OF FOAMED HEAT INSULATION
MATERIALS FROM COPPER ORE MILL TAILINGS %
SiO2
Al2O3
Fe2O3
CaО
MgO Na2O K2O Cr2O3 TiO2 SO3
Кварцевый
песок
(ПС-250)
95,00 4,00
0,25
–
–
–
–
–
–
–
Проба 1
68,18
17,16
3,85
3,04
1,81
3,47 1,67 0,15 0,49 0,18
Проба 2
68,58
16,91
3,77
2,99
1,77
3,51 1,63 0,14 0,50 0,19
Средний
состав
68,38
17,04
3,81
3,02
1,79
3,49 1,65 0,14 0,50 0,18 Минералогический состав хвостов обогащения представлен двумя ми-
нералами – кварцем и альбитом. По результатам количественного рентгено-
структурного анализа, проведенного при использовании программы Match!,
содержание кварца и альбита составляет 60 и 40 % соответственно. По гранулометрическому составу отход относится к тонкодисперсным
материалам. Согласно результатам лазерной дифракции (рис. 1), исследуемый
отход на 90 % представлен частицами размером менее 55 мкм, при этом 50 %
зерен имеют размер менее 10 мкм. Насыпная плотность исследуемого отхода
составляет 807 кг/м3, истинная плотность – 1980 кг/м3. Выбор химического состава фритты обусловлен следующими требовани-
ями. В составе фритты должно быть достаточное количество стеклообразовате-
ля (60–75 масс. %), к которым относится SiO2, и относительно высокое количе-
ство модификаторов (13–22 масс. %), представленных оксидами щелочных ме-
таллов. Количество расплава, образующегося при температуре до 900 °С,
должно составлять более 70 %, что установлено по ранее полученным данным
[9]. Низкотемпературный синтез фритты осуществляется при температуре
не менее 0,8 от температуры ликвидуса, не превышая 900 °С. При таких темпе-
ратурах во фритте сохраняется кристаллическая фаза, количество которой
не должно превышать 25 % [10]. Кроме того, количество окисляющего компо- О.В. Казьмина, А.П. Семке, И.В. Беляева и др. 162 нента (в виде SO3), который необходим для протекания реакций вспенивания,
должно быть не менее 0,15 %. Важным условием выбора является также низкая
кристаллизационная способность стеклофазы, т. к. неуправляемая кристаллиза-
ция негативно сказывается на качестве готового материала. Рис. 1. Гранулометрический состав отходов Рис. 1. Гранулометрический состав отходов Пересчитанный трехкомпонентный состав отхода попадает на диаграмме
состояния системы Na2O-Al2O3-SiO2 в область альбита и имеет температуру пол-
ного плавления 1050 °С. Рассчитанная кривая плавкости отхода показывает, что
содержание расплава 70 % достигается при температуре свыше 1000 °С, что ука-
зывает на необходимость добавления легкоплавкого компонента, в качестве ко-
торого выбрана кальцинированная сода с содержанием основного вещества
Na2СO3 в количестве 99 масс. %. Шихта, отвечающая всем вышеперечисленным
требованиям, состоит из отхода (80 %) и кальцинированной соды (20 %)
и обеспечивает следующий состав фритты, масс. %: SiO2 – 60,88; Na2O – 16,23;
Al2O3 – 15,21; Fe2O3 – 3,40; CaO – 2,69; MgO – 1,59. PRODUCTION OF FOAMED HEAT INSULATION
MATERIALS FROM COPPER ORE MILL TAILINGS Фигуративная точка состава
также находится в поле альбита и имеет температуру полного плавления 880 °С. Кривая плавкости данного состава показывает, что при температуре плавления
смеси 730 оС содержание расплава составляет 70 %. Вспенивание силикатного расплава с техническим углеродом проходит
при температуре 800–900 °С. Известно, что для получения пористых материалов
типа пеностекла вязкость расплава в интервале температур вспенивания должна
находиться в пределах 105–107 Пас. Предварительную оценку вязкостных харак-
теристик фритты проводили по модулю вязкости (Мв), значение которого должно
находиться в пределах 1,6–1,9. Значение модуля вязкости состава фритты состав-
ляет 2,1, что выше оптимального. При этом необходимо учитывать, что вязкость
расплава, полученного при плавлении реальной шихты, будет ниже за счет при-
сутствия в отходе примесей, которые не учитываются в формуле
2
2
3
2
3
2
2
в
SiO
Al O
Fe O
CaO
MgO
K O
Na O
М
M
2M
2M
M
M
2M
2M
, где
2
SiO
M
,
2
3
Al O
M
,
2
3
Fe O
M
,
CaO
M
,
MgO
M
,
2
K O
M
,
2
Na O
M
– количество оксидов крем-
ния, алюминия, железа, кальция, магния, калия, натрия соответственно, масс. %. где
2
SiO
M
,
2
3
Al O
M
,
2
3
Fe O
M
,
CaO
M
,
MgO
M
,
2
K O
M
,
2
Na O
M
– количество оксидов крем-
ния, алюминия, железа, кальция, магния, калия, натрия соответственно, масс. %. 163 Получение пористого теплоизоляционного материала Для рассчитанного состава фритты при использовании программы SciGlass
Professional на диаграмме Na2O-SiO2-Al2O3 построены линии температур, соот-
ветствующие вязкости расплава 106 Пас, согласно которым установлено, что со-
став фритты обеспечивает необходимую вязкость в интервале температур вспе-
нивания 800–900 °С. Таким образом, теоретические расчеты показывают, что состав модель-
ной шихты обеспечивает необходимое количество расплава (более 70 %) при
температуре до 900 °С и отвечает требованиям вязкости расплава, значение
которой находится в пределах 105–107 Пас при температурах вспенивания
800–900 °С. Поэтому далее исследования проводили на составе шихты, состоя-
щей на 80 % из хвостов обогащения и 20 % из кальцинированной соды. В каче-
стве газообразователя использован технический углерод с удельной поверхно-
стью 16000 м2/кг и насыпной плотностью 320 кг/м3. Технология получения пористого теплоизоляционного материала со-
стоит из двух основных этапов, которые отражены в ранее полученных ре-
зультатах [11]. Вначале путем термообработки исходной шихты на основе
отхода синтезируется низкотемпературная фритта. PRODUCTION OF FOAMED HEAT INSULATION
MATERIALS FROM COPPER ORE MILL TAILINGS Далее фритта измельчается
с добавлением газообразователя, смесь гранулируется и вспенивается до гото-
вого продукта. Технология позволяет поэтапно оптимизировать структуру
и свойства готового материала. Для этого на первом этапе решается задача
синтеза фритты с заданными характеристиками, управлять которыми можно
за счет рецептурных и технологических факторов. На втором этапе осуществ-
ляется управление основными показателями макроструктуры материала. Результаты дифференциального термического анализа (ДТА) (рис. 2)
показали, что нагрев шихты выбранного состава до 1000 оС сопровождается
эндоэффектами, которые отвечают за удаление гигроскопической воды
(100 °С), плавление эвтектик и двойных солей (768 и 877 °С). Эндотермиче-
ский эффект незначительной интенсивности при 575 °С соответствует поли-
морфному превращению кварца. Основные потери массы приходятся на тем-
пературный интервал 500–750 °С, что соответствует реакциям силикатообра-
зования. При достижении температуры 800 °С термогравиметрическая кривая
выходит на горизонталь, что указывает на полное связывание Na2CO3 и за-
вершение реакций силикатообразования. При температуре 768 °С появляется
расплав, что способствует ускорению процесса образования силикатов. Таким образом, термообработка исследуемой шихты при температуре
до 900 °С обеспечит полное завершение процессов силикатообразования, что
является необходимым условием для стеклообразования. Для подтверждения
проведен количественный рентгенофазовый анализ фритты, полученной при
температуре 800, 850, 900 °С. По результатам экспериментально полученных
данных установлено, что на всех рентгенограммах наблюдается аморфное га-
ло, указывающее на присутствие стеклофазы, а также максимумы отражения,
отвечающие за кристаллическую фазу (рис. 3), представленную остаточным
кварцем (d = 3,342 нм; 2Θ = 26,7°) и альбитом (d = 3,18 нм; 2Θ = 28,0°). Уве-
личение температуры обработки шихты с 800 до 900 °С приводит к увеличе-
нию количества стеклофазы с 75 до 85 %. По результатам количественного
рентгенофазвового анализа установлено различное содержание стеклофазы О.В. Казьмина, А.П. Семке, И.В. Беляева и др. 164 в зависимости от температуры синтеза фритты и времени выдержки. С увели-
чением температуры синтеза с 800 до 1000 °С при изотермической выдержке
от 5 до 15 мин количество стеклофазы увеличивается с 75 % (800 °С, 10 мин)
до 83 % (1000 °С, 10 мин). Рис. 2. TG, DTG кривые двухкомпонентной шихты с соотношением компонентов: от-
ход – 80 масс. % и кальцинированная сода – 20 масс. %
Рис. 3. Рентгенограмма фритты, полученной при температуре:
1 – 900 °С; 2 – 850 °С; 3 – 800 °С (К – кварц; А – альбит)
Пенообразующая смесь готовилась из измельченной фритты (удельная по-
верхность 5000 см2/г) путем перемешивания с техническим углеродом в количе-
стве 0,5 масс. %. PRODUCTION OF FOAMED HEAT INSULATION
MATERIALS FROM COPPER ORE MILL TAILINGS Гранулирование проводили на тарельчатом грануляторе с ис-
К
К
К
А
А
А
Потери массы, мг
Тепловой поток, Вт/г
Температура, °С
1
2
3 Потери массы, мг
Тепловой поток, Вт/г
Температура, °С Рис. 2. TG, DTG кривые двухкомпонентной шихты с соотношением компонентов: от-
ход – 80 масс. % и кальцинированная сода – 20 масс. %
Потери массы, мг
Тепловой поток, Вт/г
Температура, °С Потери массы, мг Рис. 2. TG, DTG кривые двухкомпонентной шихты с соотношением компонентов: от-
ход – 80 масс. % и кальцинированная сода – 20 масс. % Рис. 3. Рентгенограмма фритты, полученной при температуре:
1 – 900 °С; 2 – 850 °С; 3 – 800 °С (К – кварц; А – альбит)
К
К
К
А
А
А
1
2
3 Рис. 3. Рентгенограмма фритты, полученной при температуре:
1 – 900 °С; 2 – 850 °С; 3 – 800 °С (К – кварц; А – альбит)
К
К
К
А
А
А
1
2
3 Рис. 3. Рентгенограмма фритты, полученной при температуре:
1 – 900 °С; 2 – 850 °С; 3 – 800 °С (К – кварц; А – альбит) Пенообразующая смесь готовилась из измельченной фритты (удельная по-
верхность 5000 см2/г) путем перемешивания с техническим углеродом в количе-
стве 0,5 масс. %. Гранулирование проводили на тарельчатом грануляторе с ис- Получение пористого теплоизоляционного материала 165 пользованием в качестве связки 15% раствора жидкого стекла. Полученные гра-
нулы высушивались на воздухе и вспенивались при температуре 800–900 °С. Установлено, что из фритты, синтезированной при 800 °С, несмотря на достаточ-
ное количество стеклофазы, не получается качественный пористый материал. Расплав данного состава обладает повышенной вязкостью, что обусловлено зна-
чительным количеством кристаллической фазы (25 %). Вспенивание при темпе-
ратуре 800 °С приводит к появлению пористой структуры только в поверхност-
ном слое, внутри гранулы материал выглядит монолитным. Фритта, синтезиро-
ванная при 850 °С, также не обеспечивает получение равномерной пористой
структуры. Поэтому дальнейшие исследования проводились на фритте, синтези-
рованной при температуре 900 °С. На термограмме данной фритты не наблюда-
ется явно выраженных экзотермических эффектов, что указывает на отсутствие
процессов кристаллизации. Эндотермический эффект при 674 °С соответствует
плавлению фритты. Потери массы, связанные с выделение остаточного СО2,
не превышают 2 %, что говорит о достаточно полном протекании процессов си-
ликатообразования на первой стадии синтеза фритты. пользованием в качестве связки 15% раствора жидкого стекла. Полученные гра-
нулы высушивались на воздухе и вспенивались при температуре 800–900 °С. PRODUCTION OF FOAMED HEAT INSULATION
MATERIALS FROM COPPER ORE MILL TAILINGS Установлено, что из фритты, синтезированной при 800 °С, несмотря на достаточ-
ное количество стеклофазы, не получается качественный пористый материал. Расплав данного состава обладает повышенной вязкостью, что обусловлено зна-
чительным количеством кристаллической фазы (25 %). Вспенивание при темпе-
ратуре 800 °С приводит к появлению пористой структуры только в поверхност-
ном слое, внутри гранулы материал выглядит монолитным. Фритта, синтезиро-
ванная при 850 °С, также не обеспечивает получение равномерной пористой
структуры. Поэтому дальнейшие исследования проводились на фритте, синтези-
рованной при температуре 900 °С. На термограмме данной фритты не наблюда-
ется явно выраженных экзотермических эффектов, что указывает на отсутствие
процессов кристаллизации. Эндотермический эффект при 674 °С соответствует
плавлению фритты. Потери массы, связанные с выделение остаточного СО2,
не превышают 2 %, что говорит о достаточно полном протекании процессов си-
ликатообразования на первой стадии синтеза фритты. Количество кристаллической фазы в пористом материале зависит от режи-
ма вспенивания. В зависимости от температуры вспенивания (от 850 до 900 °С)
и выдержки при данной температуре (от 5 до 15 мин) содержание кристалличе-
ской фазы уменьшается до 10 % (900 °С, 10 мин) (рис. 4). Рис. 4. Количество стеклофазы в образцах, полученных при различной температуре
вспенивания с изотермической выдержкой 5, 10 и 15 мин
81
82
83
84
85
86
87
88
89
90
800 оС
850 оС
900 оС
5 мин
5 мин
5 мин
10 мин
10 мин
15 мин
15 мин
Температура вспенивания, °С
Содержание стеклофазы, %
800 °
850 °
900 ° Рис. 4. Количество стеклофазы в образцах, полученных при различной температуре
вспенивания с изотермической выдержкой 5, 10 и 15 мин При различном температурном режиме вспенивания получено три типа
макроструктур, отличающихся по среднему размеру пор и степени неоднородно-
сти. Оптимальной поровой структурой является тип с высокой степенью одно-
родности (Сн < 10 %) и со средним размером пор не более 1 мм. Образцы c такой
макроструктурой имеют наибольшую прочность при относительно низкой плот-
ности. Установлено, что высокую степень однородности имеют образцы, полу-
ченные при температуре вспенивания 850 °С с выдержкой 10 и 15 мин. Образцы, О.В. Казьмина, А.П. Семке, И.В. Беляева и др. 166 полученные при температуре 800 и 900 °С, имеют неравномерную структуру
с присутствием мелких и крупных пор соответственно. На рис. 5 представлена
макроструктура образцов, синтезированных с различной изотермической про-
должительностью вспенивания при 850 °С. Оптимальным режимом является
температура вспенивания 850 °С с выдержкой 10 мин. а
б
в
Рис. 5. PRODUCTION OF FOAMED HEAT INSULATION
MATERIALS FROM COPPER ORE MILL TAILINGS Макроструктура образцов, полученных при температуре вспенивания 850 °С
с выдержкой (20):
а – 5 мин; б – 10 мин; в – 15 мин в б а б в а Рис. 5. Макроструктура образцов, полученных при температуре вспенивания 850 °С
с выдержкой (20):
а – 5 мин; б – 10 мин; в – 15 мин От режима вспенивания также зависят физико-механические свойства го-
товых образцов, полученных на основе низкотемпературной фритты (табл. 2). БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. Abd Rashid R.Z., Mohd Salleh H., Ani M.H., Yunus N.A., Akiyama T., Purwanto H. Reduction
of low grade iron ore pellet using palm kernel shell // Renew Energy. 2014. 63. Р. 617–623. 2. Adiansyah J.S., Rosano M., Vink S., Keir G. A framework for a sustainable approach to mine
tailings management: Disposal strategies // J. Clean. Prod. 2015. 108. P. 1050–1062. g
g
p
g
3. Ali M.M., Agarwal S.K., Pahuja A. Potentials of copper slag utilisation in the manufacture of
ordinary Portland cement // Adv. Cem. Res. 2013. 25. P. 208–216. y
4. Liao Y.C., Huang C.Y. Effects of CaO addition on lightweight aggregates produced from water
reservoir sediment // Constr. Build. Mater. 2011. 25. P. 2997–3002. 5. Mueller A., Schnell A., Ruebner K. The manufacture of lightweight aggregates from recycled
masonry rubble. Constr. Build. Mater. 2015. 98. P. 376–387. 5. Mueller A., Schnell A., Ruebner K. The manufacture of lightweight aggregates from recycled
masonry rubble. Constr. Build. Mater. 2015. 98. P. 376–387. 6. Volland S., Brötz J. Lightweight aggregates produced from sand sludge and zeolitic rocks //
Constr. Build. Mater. 2015. 85. P. 22–29. 7. Makowski C., Finkl C.W., Rusenko K. Suitability of Recycled Glass Cullet as Artificial Dune
Fill along Coastal Environments // J. Coastal Res. 2013. 29 (4). P. 772–782. g
8. Chul-Tae Lee. Production of alumino-borosilicate foamed glass body from waste LCD glass //
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l f I d
t i l
d E
i
i
Ch
i t
2013 V 19 Р 1916 1925 8. Chul-Tae Lee. Production of alumino-borosilicate foamed glass body from waste LCD glass
Journal of Industrial and Engineering Chemistry. 2013. V. 19. Р. 1916–1925. 9. Казьмина О.В., Верещагин В.И. Физико-химическое моделирование состава пеностекло-
кристаллических материалов // Физика и химия стекла. 2015. Т. 41. № 1. С. 166–172. 10. Kazmina O.V., Semukhin B.S., Elistratova A.W. Strengthening of foam glass materials // Adv. Mat. Res. 2013. 872. P. 79-83. 11. Казьмина О.В., Верещагин В.И., Семухин Б.С., Абияка А.Н. Низкотемпературный синтез
стеклогранулята из шихт на основе кремнеземсодержащих компонентов для получения
пеноматериалов // Стекло и керамика. 2009. № 10. С. 5–8. Таблица 2 Таблица 2 Основные свойства материала, полученного из фритты,
синтезированной при 900 °С Основные свойства материала, полученного из фритты,
синтезированной при 900 °С
Режим вспенивания Предел проч-
ности на сжа-
тие, МПа
Плотность, кг/м3
Коэффициент
теплопровод-
ности,
Вт/(м·К)
Водопог-
лощение,
%
Темпера-
тура, °С
Выдерж-
ка, мин
Средняя Насыпная
800
10
6,6
650
398
0,123
0,2
800
15
4,5
500
294
0,091
0,2
850
5
3,5
380
224
0,087
0,7
850
10
3,0
350
206
0,085
0,8
850
15
2,8
330
194
0,083
0,9
900
5
2,2
299
176
0,080
0,9
900
10
1,7
250
147
0,070
0,9 Основные свойства материала, полученного из фритты,
синтезированной при 900 °С При температуре вспенивания 800 °С гранулированный материал соот-
ветствует тяжелому керамзиту, в отличие от которого полученный пористый
материал имеет низкое водопоглощение (менее 1 %). При температуре вспе-
нивания 900 °С материал соответствует легкому керамзиту (менее 400 кг/м3),
но при этом имеет неравномерную пористую структуру, что снижает проч-
ность. Сравнительная характеристика образцов, полученных при 850 °С, ука-
зывает на то, что наиболее прочным является материал, полученный при вы-
держке 10 мин. В данном случае материал имеет большую, чем у традицион- Получение пористого теплоизоляционного материала 167 ного пеностекла, прочность, и его можно рекомендовать для использования
в качестве конструкционно-теплоизоляционного. Установлено, что при плот-
ности от 250 до 400 кг/м3 материал более прочный, чем керамзит, и имеет
меньшее водопоглощение. При относительно низкой плотности (до 250 кг/м3)
материал превосходит по прочности пеностекло. В результате проведенных исследований установлена возможность ути-
лизации хвостов обогащения медной руды и расширения сырьевой базы для
получения материалов теплоизоляционного назначения. Полученный матери-
ал обладает водопоглощением менее 1 %, что указывает на способность со-
хранять свои теплотехнические характеристики во времени, и имеет неогра-
ниченный срок службы. Гранулированный материал удовлетворяет основным
требованиям, предъявляемым к эффективным пористым заполнителям, и име-
ет расширенную область применения. Материал с плотностью до 300 кг/см3 рекомендуется в качестве тепло-
изоляционного для промышленного и строительного сектора. При плотности
выше 300 кг/см3 и прочности от 2,5 МПа назначение материала теплоизоля-
ционно-конструкционное. Технология получения пористого материала из от-
ходов обогащения медной руды по способу низкотемпературного синтеза
фритты является ресурсосберегающим и энергоэффективным решением. Сведения об авторах Казьмина Ольга Викторовна, докт. техн. наук, профессор, Национальный исследова-
тельский Томский политехнический университет, 630050, г. Томск, пр. Ленина, 30,
kazmina@tpu.ru Семке Анна Павловна, аспирант, Национальный исследовательский Томский поли-
технический университет, 630050, г. Томск, пр. Ленина, 30. Беляева Ирина Владимировна, магистрант, Национальный исследовательский Том-
ский политехнический университет, 630050, г. Томск, пр. Ленина, 30. Семухин Борис Семенович, докт. техн. наук, профессор, Томский государственный
архитектурно-строительный университет, 634003, г. Томск, пл. Соляная, 2, bsemu-
khin@mail.ru REFERENCES 1. Abd Rashid R.Z., Mohd Salleh H., Ani M.H., Yunus N.A., Akiyama T., Purwanto H. Reduction
of low grade iron ore pellet using palm kernel shell. Renew Energy. 2014. V. 63, Pp. 617–623. О.В. Казьмина, А.П. Семке, И.В. Беляева и др. 168 2. Adiansyah J.S., Rosano M., Vink S., Keir G. A framework for a sustainable approach to mine
tailings management: Disposal strategies. Journal of Cleaner Production. 2015. V. 108,
Pp. 1050–1062. p
3. Ali M.M., Agarwal S.K., Pahuja A. Potentials of copper slag utilisation in the manufacture of
ordinary Portland cement. Advances in Cement Research. 2013. V. 25. Pp. 208–216. 4. Liao Y.C., Huang C.Y. Effects of CaO addition on lightweight aggregates produced from water
reservoir sediment. Construction and Building Materials. 2011. V. 25. Pp. 2997–3002. 5. Mueller A., Schnell A., Ruebner K. The manufacture of lightweight aggregates from recycled
masonry rubble. Construction and Building Materials. 2015. V. 98. Pp. 376–387. 6. Volland S., Brötz J. Lightweight aggregates produced from sand sludge and zeolitic rocks. Construction and Building Materials. 2015. V. 85. Pp. 22–29. 7. Makowski C., Finkl C.W., Rusenko K. Suitability of Recycled Glass Cullet as Artificial Dune Fill
along Coastal Environments. Journal of Coastal Research. 2013. V. 29. No. 4. Pp. 772–782. 8. Chul-Tae Lee. Production of alumino-borosilicate foamed glass body from waste LCD glass. Journal of Industrial and Engineering Chemistry. 2013. V. 19. Рp. 1916–1925. 9. Kazmina O.V., Vereshchagin V.I. Fiziko-khimicheskoe modelirovanie sostava penosteklokris-
tallicheskikh materialov [Physicochemical modeling of composition of foam glass-crystal ma-
terials]. Fizika i khimiya stekla. 2015. V. 41. No. 1. Рp. 122–126. (rus) y
p
10. Kazmina O.V., Semukhin B.S., Elistratova A.W. Strengthening of foam glass materials. Ad-
vances in Cement Research. 2013. V. 872. Pp. 79–83. 11. Kazmina O.V., Vereshchagin V.I., Semukhin B.S., Abiyaka A.N. Nizkotemperaturnyi sintez
steklogranulyata iz shikht na osnove kremnezemsoderzhashchikh komponentov dlya polu-
cheniya penomaterialov [Low-temperature synthesis of granular glass from mixes based on sil-
ica-alumina-containing components for obtaining foam materials]. Steklo i keramika. 2009. V. 66. Pp. 341–344. (rus) Authors Details Olga V. Kazmina, DSc, Professor, National Research Tomsk Polytechnic University, 30,
Lenin Ave, 634028, Tomsk , Russia, kazmina@tpu.ru Olga V. Kazmina, DSc, Professor, National Research Tomsk Polytechnic University, 30,
Lenin Ave, 634028, Tomsk , Russia, kazmina@tpu.ru Anna P. Semke, Research Assistant, National Research Tomsk Polytechnic University, 30,
Lenin Ave, 634028, Tomsk, Russia. Anna P. Semke, Research Assistant, National Research Tomsk Polytechnic University, 30,
Lenin Ave, 634028, Tomsk, Russia. Irina V. Belyaeva, Graduate Student, National Research Tomsk Polytechnic University, 30,
Lenin Ave, 634028, Tomsk , Russia. Irina V. Belyaeva, Graduate Student, National Research Tomsk Polytechnic University, 30,
Lenin Ave, 634028, Tomsk , Russia. Boris S. Semukhin, DSc, Professor, Tomsk State University of Architecture and Building, 2,
Solyanaya Sq., 634003, Tomsk, Russia, semoukhin@yahoo.com Boris S. Semukhin, DSc, Professor, Tomsk State University of Architecture and Building, 2,
Solyanaya Sq., 634003, Tomsk, Russia, semoukhin@yahoo.com
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https://openalex.org/W2024504202
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380016/pdf/pone.0039379.pdf
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English
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Interaction of Caveolin-1 with Ku70 Inhibits Bax-Mediated Apoptosis
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PloS one
| 2,012
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cc-by
| 11,637
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Introduction Caveolae are 50 to 100 nm flask-shaped invaginations of the
plasma membrane enriched in cholesterol and glycosphingolipids. Caveolae can exist as individual invaginations or are found in
detached grape-like clusters and long tubular structures derived
from the fusion of single caveolae within the cytoplasm. Caveolae
were identified in the 1950’s in capillary endothelial cells and
epithelial cells from the gall bladder of the mouse [1,2]. Since then,
caveolae have been found in a wide variety of tissues and cell
types, including adipocytes, endothelial cells, striated and smooth
muscle cells, fibroblasts and epithelial cells [3]. Caveolae were
originally thought to function as macromolecular transport vesicles
[4]. From then on, their role has expanded to signal transduction,
cellular metabolism, cholesterol homeostasis, endocytosis and
tumor suppression [5–9]. Colorectal cancer is the fourth most commonly diagnosed
cancer and the second leading cause of cancer-related deaths in
the United States [16,17]. Apoptosis is an anti-tumorigenic event. In fact, tumor cells need to escape apoptosis in order to generate a
relevant tumor mass. Thus, apoptosis has a profound effect on the
malignant phenotype. In addition, most cytotoxic anticancer
agents promote apoptosis, suggesting that defective apoptotic
programs may contribute to treatment failure, which is commonly
seen in most human cancers. Studies have shown that caveolin-1 is
over-expressed in colon cancer tissues, as compared to normal
colonic mucosa, and colon cancer cells [18–20], suggesting that
caveolin-1 may have pro-tumorigenic properties in this type of
cancer. However,
whether
and
how
caveolin-1
modulates
apoptosis of colon cancer cells remains to be determined. Caveolin-1 is an essential protein component of caveolae. Caveolin-1 acts as a scaffolding protein that concentrates and
functionally regulates signaling molecules, including G-protein
alpha subunits, H-Ras, Nitric Oxide Synthase (NOS), Epidermal
Growth Factor Receptor (EGFR), Src-like Nonreceptor Tyrosine
Kinases (NRTK), Protein Kinase C (PKC), Protein Kinase A
(PKA), Mdm2, Thioredoxin Reductase 1 and Protein Phosphatase
type 2A [10–13]. The direct interaction between caveolin-1 and
signalling molecules occurs between the ‘‘caveolin scaffolding
domain’’ (CSD) of caveolin-1, which is represented by residues
82–101, and the caveolin binding domain (CBD: WXWXXXXW,
WXXXXWXXW, or WXWXXXXWXXW where W represents an
aromatic amino acid and X represents any amino acid) of
signaling molecules [14,15]. The direct interaction with caveolin-1 An important regulatory step in apoptosis is the activation of the
proapoptotic
factor
Bax,
which
translocates
to
the
outer
mitochondrial membrane and oligomerizes following its activa-
tion. Abstract Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling
molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis
and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding
protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82–101) to the caveolin-binding
domain (CBD) of Ku70 (amino acids 471–478). Cell culture data show that caveolin-1 binds Ku70 after treatment with
chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug-
induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of
apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we
found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 WRA),
limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic
protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death. e D, Galbiati F (2012) Interaction of Caveolin-1 with Ku70 Inhibits Bax-Mediated Apoptosis. PLoS ONE 7(6): e39379. doi:10.1371/ ation: Zou H, Volonte D, Galbiati F (2012) Interaction of Caveolin-1 with Ku70 Inhibits Bax-Mediated Apoptosis. PLoS ONE 7(6)
rnal.pone.0039379 Editor: Libing Song, Sun Yat-sen University Cancer Center, China Received June 16, 2011; Accepted May 24, 2012; Published June 20, 2012 Received June 16, 2011; Accepted May 24, 2012; Published June 20, 2012 pyright: 2012 Zou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, whic
e, distribution, and reproduction in any medium, provided the original author and source are credited. nding: This work was supported by grants from the National Institute on Aging (R01-AG022548 and R01-AG030636) (to F.G.). The funde
sign, data collection and analysis, decision to publish, or preparation of the manuscript. supported by grants from the National Institute on Aging (R01-AG022548 and R01-AG030636) (to F.G.). The funders had no role in study
nd analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by grants from the National Institute on Aging (R01-AG02254
design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abstract * E-mail: feg5@pitt.edu generally results in the sequestration of a given signaling molecule
within caveolar membranes and inhibition of its signaling activity. Huafei Zou, Daniela Volonte, Ferruccio Galbiati* epartment of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United Stat rmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America Interaction of Caveolin-1 with Ku70 Inhibits Bax-
Mediated Apoptosis Huafei Zou, Daniela Volonte, Ferruccio Galbiati* siRNA Treatment Knockdown of caveolin-1 protein expression was achieved by
transfection of HCT116 and HT29 cells with small interfering
RNA (siRNA) duplexes. The target sequence was as follows: 59-
aaccagaagggacacacag-39. Scrambled
siRNA
was
used
as
a
negative control. Transfection was performed using Lipofectamine
2000 according to the manufacture’s recommendations. Knockdown of caveolin-1 protein expression was achieved by
transfection of HCT116 and HT29 cells with small interfering
RNA (siRNA) duplexes. The target sequence was as follows: 59-
aaccagaagggacacacag-39. Scrambled
siRNA
was
used
as
a
negative control. Transfection was performed using Lipofectamine
2000 according to the manufacture’s recommendations. Clonogenic Assay HCT116 cells were transfected with either caveolin-1 siRNA or
scrambled siRNA as described above. After 24 hours, cells were
treated with different concentrations of either 5-FU (3.125 mg/ml,
6.25 mg/ml, 12.5 mg/ml and 25 mg/ml) or etoposide (200 mM,
400 mM and 600 mM) for 1 hour. Untreated cells were used as
control. 500 cells were then plated into 12-well plates and cultured
for 7 days. Cells were stained with crystal violet by incubating the
cells with 10% crystal violet in 70% ethanol for 2 minutes followed
by extensive washes with PBS. Quantification of crystal violet
staining from three independent experiments was performed by
counting the number of clones using Image J software analysis. Cloning
of
wild
type
(WT)
and
WRA
Ku70
constructs. The entire coding region of mouse Ku70 was
amplified by polymerase chain reaction amplification using a full-
length
cDNA
clone
(ID
3669715)
from
Open
Biosystems
(Huntsville, AL) and the following primers: forward 59-gaattcggt-
cagagtgggagtcctactac-39 and reverse 59-ctcgagtcagttcttctccaagtgtct-
39. The amplified DNA was sequenced to rule out the introduction
of unwanted mutations. The PCR fragment was digested to create
unique EcoRI and XhoI ends and subcloned into the pCMV-HA
expression vector (WT Ku70-HA). The WRA mutant of Ku70
(WRA Ku70-HA) was generated by replacement of the aromatic
residues within the caveolin binding motif of Ku70 (amino acids
471–478) with alanines by polymerase chain reaction amplifica-
tion
using
appropriate
internal
primers
(forward:
59- Cloning
of
wild
type
(WT)
and
WRA
Ku70
constructs. The entire coding region of mouse Ku70 was
amplified by polymerase chain reaction amplification using a full-
length
cDNA
clone
(ID
3669715)
from
Open
Biosystems
(Huntsville, AL) and the following primers: forward 59-gaattcggt-
cagagtgggagtcctactac-39 and reverse 59-ctcgagtcagttcttctccaagtgtct-
39. The amplified DNA was sequenced to rule out the introduction
of unwanted mutations. The PCR fragment was digested to create
unique EcoRI and XhoI ends and subcloned into the pCMV-HA
expression vector (WT Ku70-HA). The WRA mutant of Ku70
(WRA Ku70-HA) was generated by replacement of the aromatic
residues within the caveolin binding motif of Ku70 (amino acids
471–478) with alanines by polymerase chain reaction amplifica-
tion
using
appropriate
internal
primers
(forward:
59- Western Blotting Assay Protein samples were collected in boiling Laemmli buffer (0.2 M
Tris-HCl, pH 6.8, 2% SDS, 10% glycerol), vigorously resus-
pended and boiled for 5 min. Protein concentration was calculated
using the BCA protein assay kit (Thermo Fisher Scientific Inc.,
Rockford, IL). Equal amount of proteins were separated on 12.5%
Tris-HCl polyacrylamide gels after the addition of 0.01%
bromophenol blue and 20 mM DTT. Protein samples were then
transferred onto nitrocellulose membranes overnight at 4uC. Ponceau S staining was performed to confirm equal loading. Blots were incubated for 1 hr in TBST (10 mM Tris-HCl, pH 8.0,
150 mM NaCl, and 0.2% Tween 20) containing 2% dry milk and
1% bovine serum albumin. After three washes with TBST,
membranes were incubated overnight with the primary antibody
and for 1 hr with horseradish peroxidase-conjugated secondary
IgG. Bound antibodies were detected using a Super Signal
chemiluminescent solution (Pierce Chemical, Rockford, IL). In this study, we focused on the functional role of caveolin-1 in
chemotherapeutic-induced apoptosis of colon cancer cells and the
underlying molecular mechanism. We found that Ku70 is a novel
caveolin-1-binding protein and that binding of caveolin-1 to Ku70
inhibits chemotherapeutic drug-induced apoptosis of colon cancer
cells by limiting the release of Bax from Ku70. Materials Antibodies and their sources were as follows: anti-caveolin-1
(2297) and anti-Bax (6A7) IgGs were from BD Transduction
Laboratories (Lexington, KY); anti-caveolin-1 (H-97), anti-Ku70
(A-9) and anti-Bax (N-20) IgGs were from Santa Cruz Biotech-
nology (Santa Cruz, CA); anti-cleaved caspase-3 and anti-cleaved
PARP IgGs were from Cell Signaling Technology (Danvers, MA);
horseradish peroxidase-conjugated goat anti-mouse and anti-
rabbit secondary antibodies were from Pierce (Rockford, IL,
USA). McCoy’s 5a medium and fetal bovine serum were
purchased from Invitrogen (Carlsbad, CA). Other biochemicals
used were of the highest purity available and were obtained from
regular commercial sources. DAPI (4,6-Diamidino-2-Phenylindole) Staining
HCT116 and HT29 cells (untreated or treated with chemo-
therapeutic drugs) were harvested and incubated with staining
buffer (PBS containing 3.7% paraformaldehyde, 0.1% Triton,
10 mg/ml RNase A and 1 mg/ml DAPI) at room temperature for
1 hr. Nuclear morphology was examined under a Olympus Provis
fluorescent microscope. A total of 1,200 cells were scored from 4
independent viewing areas from three independent experiments
for each experimental point. DAPI (4,6-Diamidino-2-Phenylindole) Staining
HCT116 and HT29 cells (untreated or treated with chemo-
therapeutic drugs) were harvested and incubated with staining
buffer (PBS containing 3.7% paraformaldehyde, 0.1% Triton,
10 mg/ml RNase A and 1 mg/ml DAPI) at room temperature for
1 hr. Nuclear morphology was examined under a Olympus Provis
fluorescent microscope. A total of 1,200 cells were scored from 4
independent viewing areas from three independent experiments
for each experimental point. Introduction Bax promotes apoptosis by making the membrane permeable
to cytochrome c. Ku70 is a protein that was originally identified as
part of the Ku70/Ku80 complex that mediates the repair of DNA
double-strand breaks by non-homologous end joining (NHEJ)
[21]. However, Ku70 has also been shown to regulate apoptosis
through a Bax-dependent mechanism. Over-expression of Ku70
inhibits Bax-mediated apoptosis, whereas depletion of Ku70
enhances apoptosis [22]. In addition, Ku70 2/2 mice are
hypersensitive to ionizing radiation [23] and cells from these mice
are also hypersensitive to apoptosis induced by staurosporine [24]. PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e39379 1 Caveolin-1, a Novel Ku70-Interacting Protein Increased acetylation of Ku70 disrupts the Ku70-Bax interaction
and inhibits the antiapoptotic function of Ku70 [25]. Finally,
peptides based on the amino acid sequence of the Bax-binding
domain of Ku70 bind Bax and inhibit cell death [26,27]. Thus, by
interacting with Ku70, Bax is sequestered away from mitochon-
dria and is maintained in an inactive state. The molecular
mechanism underlying the sequestration of Bax by Ku70 and the
release of Bax from Ku70 after stimulation with an apoptotic
inducer remains to be fully identified. gttcaaaagctccgcgccacagccagatctgacagtgctgagaatccagtcctg-39;
re-
verse: 59-caggactggattctcagcactgtcagatctggctgtggcgcggagcttttgaac-
39) and subcloned EcoRI and XhoI into the pCMV-HA
expression vector. Transfection of WT Ku70-HA and WRA
Ku70-HA in HCT116 cells was performed using Lipofectamine
2000 according to the manufacture’s recommendations. Cell Culture and Drug Treatment Human colon carcinoma cells HT29, HCT116 Bax +/2
(HCT116) and HCT116 Bax 2/2 (HCT116 Bax null) (a
generous gift from Dr. Bert Vogelstein) were grown in McCoy’s
5a medium containing glutamine, antibiotics and 10% FBS. Cells
were incubated in growth medium supplemented with 50, 25,
12.5, 6.25, or 3.125 mg/ml of 5-FU for 12, 24 or 48 hours. HCT116 and HCT116 Bax null cells were also incubated in
growth medium supplemented with 800, 600, 400 or 200 mM of
etoposide for 24 hours. Human colon carcinoma cells HT29, HCT116 Bax +/2
(HCT116) and HCT116 Bax 2/2 (HCT116 Bax null) (a
generous gift from Dr. Bert Vogelstein) were grown in McCoy’s
5a medium containing glutamine, antibiotics and 10% FBS. Cells
were incubated in growth medium supplemented with 50, 25,
12.5, 6.25, or 3.125 mg/ml of 5-FU for 12, 24 or 48 hours. HCT116 and HCT116 Bax null cells were also incubated in
growth medium supplemented with 800, 600, 400 or 200 mM of
etoposide for 24 hours. Annexin
V
staining
and
flow
cytometry
analysis. HCT116 cells were transfected with caveolin-1 siRNA
and scrambled siRNA as described above. After 24 hours, cells
were treated with 25 mg/ml 5-FU for 48 hours. Untreated cells
were used as control. The Annexin-V-FITC Apoptosis Detection
Kit (BD Pharmingen, San Diego, CA) was used to detect apoptosis
by flow cytometry. Cells were harvested (including detached cells),
and processed according to the manufacturer’s instructions. Fluorescence-activated cell sorting analysis was carried out using
a FACScan flow cytometer (Becton Dickinson, San Diego, CA)
and CellQuest software. Annexin
V
staining
and
flow
cytometry
analysis. HCT116 cells were transfected with caveolin-1 siRNA
and scrambled siRNA as described above. After 24 hours, cells
were treated with 25 mg/ml 5-FU for 48 hours. Untreated cells
were used as control. The Annexin-V-FITC Apoptosis Detection
Kit (BD Pharmingen, San Diego, CA) was used to detect apoptosis
by flow cytometry. Cells were harvested (including detached cells),
and processed according to the manufacturer’s instructions. Fluorescence-activated cell sorting analysis was carried out using
a FACScan flow cytometer (Becton Dickinson, San Diego, CA)
and CellQuest software. PLoS ONE | www.plosone.org Co-Immunoprecipitation CellswerewashedtwicewithPBSandlysedfor45 minat4uCinan
IP lysis buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA,
1% Triton X-100, 60 mM octyl glucoside and protease inhibitors;
Bax immunoprecipitation was performed using 0.1 mM Hepes and
1%CHAPS).Subsequently,lysateswerecentrifugedat13,0006gfor
10 min at 4uC. The supernatant was pre-incubated with protein A
sepharose beads for 1 hr at 4uC. After centrifugation at 10,000 rpm
for 30 sec, the supernatant was collected and protein concentration
calculated using the BCA protein assay kit (Thermo Fisher Scientific
Inc.).Equalamountoftotalproteinswereincubatedovernightat4uC
with anti-caveolin-1 or anti-Bax antibodies and protein A sepharose
beads. The immunoprecipitates were washed three times with lysis
bufferandre-suspendedinLaemmlibuffer(0.2 MTris-HCl,pH 6.8,
2% SDS, 10% glycerol, 0.01% bromophenol blue, 20 mM DTT). Followingvigorousvortexingandboiling,sampleswereseparatedon
12.5% SDS-PAGE. Blots were then probed with appropriated
antibodies. Immunoprecipitations with protein A sepharose beads
were performed as control and did not produce any detectable band
by immunoblotting analysis usingtheappropriate antibody (data not
shown). RNA Isolation and RT-PCR Cells were collected and total RNA was isolated using the RNeasy
Mini kit from Qiagen (Valencia, CA). Equal amounts of RNA were
treated with RNase-free DNase, and subjected to reverse transcrip-
tion using the Advantage RT-for-PCR kit from Clontech (Mountain
View, CA), according to the manufacturer’s recommendations. PCR was then performed in the exponential linear zone of
amplification for each gene studied. The caveolin-1-specific primers
used were the following: forward 59-ctacaagcccaacaacaagg-39;
reverse 59- cagacagcaagcggtaaaa-39. A sequence corresponding to
GAPDH was also amplified as an internal control. Caveolin-1 Inhibits Chemotherapeutic Drug-induced
Apoptosis and Promotes Long-term Survival of Colon
Cancer Cells To directly address the role of caveolin-1 in chemotherapeutic
drug-induced apoptosis in colon cancer cells, we knocked-down
caveolin-1 protein expression by siRNA in HCT116 colon cancer
cells (Figure 1A). Scrambled and caveolin-1 siRNA-transfected
HCT116
cells
were
treated
with
different
concentrations
(3.125 mg/ml, 6.25 mg/ml, 12.5 mg/ml and 25 mg/ml) of the
chemotherapeutic drug 5-fluoracil (5-FU) for 48 hours (Figure 1B) Immunofluorescence Microscopy Cells grown on glass coverslips were washed three times with
PBS w/Ca++/Mg++ and fixed for 30 min at room temperature
with 2% paraformaldehyde in PBS w/Ca++/Mg++. Fixed cells
were rinsed with PBS w/Ca++/Mg++and permeabilized with 0.1%
Triton X-100, 0.2% bovine serum albumin for 10 min. Then cells
were treated with 25 mM NH4Cl in PBS w/Ca++/Mg++ for
10 min at room temperature to quench free aldehyde groups. Cells were treated with RNase (200 mg/ml) for 10 minutes and
with DAPI (1 mg/ml) for 20 minutes. Cells were rinsed with PBS
w/Ca++/Mg++ and incubated with the primary antibody (diluted
in PBS with 0.1% Triton X-100, 0.2% bovine serum albumin) for
2 h at room temperature. After three washes with PBS w/Ca++/
Mg++ (10 min each), cells were incubated with the secondary
antibody for 1 h at room temperature: lissamine rhodamine B
sulfonyl chloride-conjugated goat anti-rabbit antibody (5 mg/ml)
and fluorescein isothiocyanate-conjugated goat anti-mouse anti-
body (5 mg/ ml). Finally, cells were washed three times with PBS
w/Ca++/Mg++ (10 min each wash) and slides were mounted with
slow-Fade anti-fade reagent (Molecular Probes, Inc., Eugene, OR)
and observed using a Zeiss Confocal Microscope (LSM 5 Pascal). Cell Cycle Analysis by Fluorescence-activated Cell Sorter
(FACS) Cells were collected by trypsinization, pelleted, and washed with
PBS. Cells were resuspended and fixed overnight with 1 ml of ice- PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e39379 2 Caveolin-1, a Novel Ku70-Interacting Protein cold 70% ethanol at 4uC. On the day of analysis, cells were
centrifuged at 3000 rpm for 8 min and the supernatant was
carefully removed. Subsequently, cells were resuspended in 1.0 ml
of PI solution (PBS containing 0.1% glucose, 1.1 mg/ml RNAse,
and 0.05 mg/ml propidium iodide) and incubated at room
temperature for 30 min. Cell were then subjected to FACS
analysis with the use of a flow cytometer (LSR II, BD biosciences). For each analysis 10,000 gated events were collected to permit cell
cycle analysis. Data analysis was performed with the use of
CellQuest software. A representative cell cycle analysis from three
independent experiments is shown. for 15 min. Clarified lysates were subjected to centrifugation at
12,0006g at 4uC for 15 min. The mitochondrion-containing
pellets were resuspended in 2x SDS sample buffer. Mitochondrial
fractions were positive for the mitochondria marker cytochrome
oxidase subunit IV and negative for the cytoplasmic marker alpha-
tubulin (data not shown). Statistical Analysis Quantification studies were performed in triplicate and the
average 6 standard error of the mean (S.E.M.) is shown. Significance was calculated using the Student’s t-test. Quantifica-
tion of active Bax was obtained by scanning images of western
blotting analysis from three independent experiments using Image
J software. Immunoprecipitation and immunoblotting analysis
were performed three independent times and representative blots
are shown. Glutathione S-transferase (GST) Pull-down Assay y
GST-caveolin-1 (GST-Cav-1) constructs were as follows: GST
fused to full-length caveolin-1 (Cav-1 FL), GST fused to residues
82 to 101 of caveolin-1 (GST-Cav-1 (82–101)) and GST fused to
residues 1 to 101 of caveolin-1 (GST-Cav-1 (1–101)). GST-Cav-1
fusion protein constructs were transformed into Escherichia coli
(BL21 strain; Novagen, Inc.). After induction of expression
through addition of 0.5 mM isopropyl-b-D-galactoside (Sigma),
GST-Cav-1 proteins were affinity purified on glutathione-agarose
beads, using the detergent Sarcosyl for initial solubilization. GST-
Cav-1 and GST alone (bound to glutathione-agarose beads) were
washed 3 times with TNET buffer (50 mM Tris, pH 8.0, 150 mM
NaCl, 5 mM EDTA, 1% Triton X-100) containing protease
inhibitors. SDS-PAGE followed by Comassie staining was used to
determine the concentration of GST-Cav-1 per 100 ml of packed
bead volume. Pre-cleared cell lysates of HCT116 cells over-
expressing HA-Ku70 were diluted in buffer A (10 mM Tris,
pH 8.0, 0.1% Tween 20) and added to approximately 100 ml of
equalized bead volume for overnight incubation at 4uC. After
binding, the beads were extensively washed with phosphate-
buffered saline (6 times). Finally, the beads were resuspended in
2X sample buffer (0.4 M Tris-HCl, pH 6.8, 4% SDS, 20%
glycerol, 0.02% bromophenol blue) and subjected to SDS-PAGE. PLoS ONE | www.plosone.org Caveolin-1, a Novel Ku70-Interacting Protein Caveolin-1, a Novel Ku70-Interacting Protein Caveolin-1, a Novel Ku70-Interacting Protein and with the same concentration of 5-FU (25 mg/ml) for different
periods of time (12 h, 24 h and 48 h) (Figures S1A and S1B). Cells
were then stained with DAPI and apoptosis was quantified by
counting the number of cells showing nuclear condensation. We
show that 5-FU induced apoptosis in HCT116 cells transfected
with scrambled siRNA and that 5-FU-induced apoptosis was
significantly enhanced in HCT116 cells in which caveolin-1
protein expression was reduced by siRNA. These data were
confirmed by annexin V staining followed by flow cytometry
analysis (Figure 1C) and immunoblotting analysis using antibody
probes specific for cleaved caspase 3 and Poly (ADP-ribose)
polymerase (PARP) (Figure 1D). Studies have implicated reactive
oxygen species (ROS) in 5-FU-mediated apoptosis in colon cancer
cells [28]. To independently support our findings, we subjected
HCT116 cells transfected with either scrambled or caveolin-1
siRNA to oxidative stress and apoptosis was quantified by DAPI
staining. We found
that
knockdown of caveolin-1
protein
expression
potentiated
oxidative
stress-induced
apoptosis
in
HCT116 cells (data not shown). and with the same concentration of 5-FU (25 mg/ml) for different
periods of time (12 h, 24 h and 48 h) (Figures S1A and S1B). Cells
were then stained with DAPI and apoptosis was quantified by
counting the number of cells showing nuclear condensation. We
show that 5-FU induced apoptosis in HCT116 cells transfected
with scrambled siRNA and that 5-FU-induced apoptosis was
significantly enhanced in HCT116 cells in which caveolin-1
protein expression was reduced by siRNA. These data were
confirmed by annexin V staining followed by flow cytometry
analysis (Figure 1C) and immunoblotting analysis using antibody
probes specific for cleaved caspase 3 and Poly (ADP-ribose)
polymerase (PARP) (Figure 1D). Studies have implicated reactive
oxygen species (ROS) in 5-FU-mediated apoptosis in colon cancer
cells [28]. To independently support our findings, we subjected
HCT116 cells transfected with either scrambled or caveolin-1
siRNA to oxidative stress and apoptosis was quantified by DAPI
staining. We found
that
knockdown of caveolin-1
protein
expression
potentiated
oxidative
stress-induced
apoptosis
in
HCT116 cells (data not shown). Long-term clonogenic assays provide a more stringent mea-
surement of cell survival than short-term assays. Caveolin-1, a Novel Ku70-Interacting Protein To determine the
effect of reduced caveolin-1 expression on the long-term survival of
colon cancer cells, HCT116 cells were transfected with either
scrambled or caveolin-1 siRNA, treated with different concentra-
tions of 5-FU (3.125 mg/ml, 6.25 mg/ml, 12.5 mg/ml and 25 mg/
ml) and subjected to crystal violet staining 7 days after 5-FU
treatment. Figures 2A and 2B show that knockdown of caveolin-1
protein expression reduced the long-term survival of HCT116
cells, even at the lowest concentration of 5-FU (3.125 mg/ml). Interestingly, the number of clones, after crystal violet staining,
was reduced by the knockdown of caveolin-1 protein expression in
HCT116 cells even before drug treatment (Figure 2A). Since
caveolin-1 siRNA also reduced the number of cells in the S phase
of the cell cycle by 13%, without promoting apoptosis, before
treatment with 5-FU (Figure S2), caveolin-1 appears to have a
positive effect on cell growth in HCT116 cells in addition to its
anti-apoptotic function after stimulation with chemotherapeutic
drugs. This is consistent with data showing pro-tumorigenic
properties of caveolin-1 in colon cancer, as demonstrated by the Figure 1. Knockdown of caveolin-1 protein expression sensitizes HCT116 colon cancer cells to apoptosis. HCT116 colon cancer cells
were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. (A) Endogenous caveolin-1
expression was determined by immunoblotting analysis 24, 48 and 96 hours after transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-b-actin IgGs was done to show equal loading. (B–D) One day after transfection, HCT116 cells were treated with different Figure 1. Knockdown of caveolin-1 protein expression sensitizes HCT116 colon cancer cells to apoptosis. HCT116 colon cancer cells
were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. (A) Endogenous caveolin-1
expression was determined by immunoblotting analysis 24, 48 and 96 hours after transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-b-actin IgGs was done to show equal loading. (B–D) One day after transfection, HCT116 cells were treated with different
concentrations of 5-FU for 48 hours. Untreated cells were used as control. In (B), cells were stained with DAPI and the number of cells showing
nuclear condensation was quantified. Values represent mean 6 SEM; *P,0.001; #P,0.005. In (C), cells were subjected to Annexin V staining and flow
cytometry. Values represent mean. In (D), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3 and
PARP. Mitochondria Isolation Cells were washed once in PBS and resuspended in 600 mL of
mitochondrial isolation buffer containing 10 mM Hepes (pH 7.4),
250 mM sucrose, 1 mM EDTA and protease inhibitors. Cells
were broken in a Dounce homogenizer for 40 times. Nuclei and
unbroken cells were removed by centrifugation at 10006g at 4uC PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e39379 3 Caveolin-1, a Novel Ku70-Interacting Protein Immunoblotting with anti-b-actin IgGs was done to show equal loading. doi:10.1371/journal.pone.0039379.g001 Figure 1. Knockdown of caveolin-1 protein expression sensitizes HCT116 colon cancer cells to apoptosis. HCT116 colon cancer cells
were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. (A) Endogenous caveolin-1
expression was determined by immunoblotting analysis 24, 48 and 96 hours after transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-b-actin IgGs was done to show equal loading. (B–D) One day after transfection, HCT116 cells were treated with different
concentrations of 5-FU for 48 hours. Untreated cells were used as control. In (B), cells were stained with DAPI and the number of cells showing
nuclear condensation was quantified. Values represent mean 6 SEM; *P,0.001; #P,0.005. In (C), cells were subjected to Annexin V staining and flow
cytometry. Values represent mean. In (D), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3 and
PARP. Immunoblotting with anti-b-actin IgGs was done to show equal loading. doi:10.1371/journal.pone.0039379.g001 June 2012 | Volume 7 | Issue 6 | e39379 PLoS ONE | www.plosone.org 4 Caveolin-1, a Novel Ku70-Interacting Protein Figure 2. Caveolin-1 promotes long-term survival and inhibits apoptosis of colon cancer cells after treatment with
chemotherapeutic drugs. HCT116 (A, B, E and F) and HT29 (C and D) colon cancer cells were transfected with siRNA directed against
caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, cells were treated with different concentrations of 5-FU
for 48 h (A–D) or etoposide for 24 h (E and F). Untreated cells were used as control. In (A and B), cells were cultured for 7 days and stained with crystal
violet. A crystal violet staining that is representative of three independent experiments is shown in (A), quantification of the number of clones after
crystal violet staining is shown in (B). Values represent mean 6 SEM; *P,0.001; #P,0.005. In (C and E), cells were stained with DAPI and the number
of cells showing nuclear condensation was quantified. Values represent mean 6 SEM; *P,0.001; #P,0.005. In (D and F), cells were subjected to
immunoblotting analysis with antibody probes specific for cleaved caspase 3 and PARP. Immunoblotting with anti-b-actin IgGs was done to show
equal loading. doi:10.1371/journal.pone.0039379.g002 Figure 2. Caveolin-1 promotes long-term survival and inhibits apoptosis of colon cancer cells after treatment with
chemotherapeutic drugs. Caveolin-1, a Novel Ku70-Interacting Protein HCT116 (A, B, E and F) and HT29 (C and D) colon cancer cells were transfected with siRNA directed against
caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, cells were treated with different concentrations of 5-FU
for 48 h (A–D) or etoposide for 24 h (E and F). Untreated cells were used as control. In (A and B), cells were cultured for 7 days and stained with crystal
violet. A crystal violet staining that is representative of three independent experiments is shown in (A), quantification of the number of clones after
crystal violet staining is shown in (B). Values represent mean 6 SEM; *P,0.001; #P,0.005. In (C and E), cells were stained with DAPI and the number
of cells showing nuclear condensation was quantified. Values represent mean 6 SEM; *P,0.001; #P,0.005. In (D and F), cells were subjected to
immunoblotting analysis with antibody probes specific for cleaved caspase 3 and PARP. Immunoblotting with anti-b-actin IgGs was done to show
equal loading. doi:10 1371/journal pone 0039379 g002 over-expression of caveolin-1 in colon cancer tissues and colon
cancer cells [18–20]. PARP (Figure 2F). In addition, the long-term survival of HCT116
cells after treatment with etoposide was inhibited by knockdown of
caveolin-1 protein expression using siRNA (Figures S4A and S4B). To determine whether the caveolin-1-mediated inhibition of
apoptosis is not limited to the combination of HCT116 cells and 5-
FU, a different colon cancer cell line, HT29, was treated with
different concentrations of 5-FU after caveolin-1 protein expres-
sion was knockdown by siRNA (data not shown). Apoptosis was
then quantified by DAPI staining (Figures 2C, S3A and S3B) and
immunoblotting analysis to detect cleaved caspase 3 and PARP
(Figure 2D). We found enhanced 5-FU-induced apoptosis when
caveolin-1 protein expression was knockdown in HT29 cells. In
addition, scrambled and caveolin-1 siRNA-transfected HCT116
cells were treated with a different chemotherapeutic drug,
etoposide. We show enhanced etoposide-induced apoptosis in
HCT116 cells transfected with caveolin-1 siRNA, as compared to
scrambled siRNA-transfected cells, after DAPI staining (Figure 2E)
and immunoblotting analysis to detect activated caspase 3 and PLoS ONE | www.plosone.org The Interaction between Caveolin-1 and Ku70 is Induced
by Chemotherapeutic Drugs and Limits the Release of
Bax from Ku70 chemotherapeutic drugs, which induce apoptosis, promote the
interaction between caveolin-1 and Ku70 (Figures 4A and 4B). To
investigate the functional significance of the binding of caveolin-1 to
Ku70, we tested the hypothesis that binding of caveolin-1 to Ku70
inhibits apoptosis by limiting the release of Bax from Ku70. HCT116 cells were transfected with either scrambled or caveolin-1
siRNA and treated with either 5-FU or etoposide. Protein cell lysates
were then immunoprecipitated with an antibody probe specific for
Bax and immunoprecipitates subjected to immunoblotting analysis
with anti-Ku70 IgGs. Figure 5A shows that both 5-FU and etoposide
stimulated the release of Bax from Ku70 in scrambled siRNA-
transfected cells. Importantly, the dissociation of Bax from Ku70
was potentiated in cells lacking caveolin-1. Thus, the interaction of
caveolin-1 with Ku70 limits the release of Bax from Ku70 induced
by chemotherapeutic drugs. In support of the existence of a caveolin-
1/Ku70/Bax complex after treatment with chemotherapeutic
drugs, we show in Figure 4A that both 5-FU and etoposide
promoted the interaction between caveolin-1 and Bax, and in
Figure 5B that caveolin-1, Ku70 and Bax are all found by
immunoblotting analysis after immunoprecipitation of protein cell
lysates from 5-FU-treated HCT116 cells with antibody probes
specific for either caveolin-1 or Bax. The Interaction between Caveolin-1 and Ku70 is Induced
by Chemotherapeutic Drugs and Limits the Release of
Bax from Ku70 Once we demonstrated that Ku70 was a novel caveolin-1-
interacting protein, we then asked whether binding of caveolin-1 to
Ku70 was the molecular mechanism underlying the anti-apoptotic
properties of caveolin-1. HCT116 cells were treated with either 5-
FU or etoposide and the interaction between caveolin-1 and Ku70,
before and after treatment with chemotherapeutic drugs, was
evaluated by coimmunoprecipitation studies. We show in Figure 4A
that the interaction between caveolin-1 and Ku70 was marginal
under resting conditions but dramatically increased upon treatment
with either 5-FU or etoposide. Consistent with these data, Ku70 was
mainly localized in the nucleus and, to a lesser extent, diffuse in the
cytoplasm (see arrowheads in Figure 4B) in HCT116 cells under
resting conditions, away from caveolin-1, which was enriched at the
plasma membrane (Figure 4B). In contrast, although nuclear
localization was still evident, Ku70 was found to partially co-
localize with caveolin-1 at the plasma membrane and intracellular
caveolar membranes after 5-FU treatment (see arrows in Figure 4B). Ku70 is a Novel Caveolin-1-binding Protein Caveolin-1 is known to interact with signaling molecules
carrying a caveolin binding domain [14,15]. Ku70 has been
shown to regulate apoptosis [22–25]. Interestingly, Ku70 possesses
a putative caveolin binding domain between amino acids 471 and
478 (Figure 3A). To investigate the molecular mechanism
underlying the caveolin-1-mediated inhibition of apoptosis, we
asked whether Ku70 was a novel caveolin-1-interacting protein. To this end, we performed pulldown assays using a series of
caveolin-1 deletion mutants fused to GST (Figure 3B). Figures 3C
shows that Ku70 is a novel caveolin-1-binding protein and that the
caveolin scaffolding domain (CSD) of caveolin-1 (residues 82–101)
is sufficient for binding to Ku70. PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e39379 PLoS ONE | www.plosone.org 5 Caveolin-1, a Novel Ku70-Interacting Protein Figure 3. Ku70 binds to the scaffolding domain of caveolin-1. (A) The consensus caveolin binding domain (CBD) is shown together with the
caveolin binding domain of Ku70 (amino acids 471 to 478) and a mutant form of Ku70’s CBD in which aromatic residues were mutated to alanines. (B)
Ponceau S staining of GST alone and GST-caveolin-1 fusion proteins. (C) GST-caveolin-1 fusion protein pull-down assays were performed using cell
lysates from HCT116 cells transiently transfected with wild type Ku70-HA. A blot that is representative of two independent experiments is shown. doi:10.1371/journal.pone.0039379.g003 Figure 3. Ku70 binds to the scaffolding domain of caveolin-1. (A) The consensus caveolin binding domain (CBD) is shown together with the
caveolin binding domain of Ku70 (amino acids 471 to 478) and a mutant form of Ku70’s CBD in which aromatic residues were mutated to alanines. (B)
Ponceau S staining of GST alone and GST-caveolin-1 fusion proteins. (C) GST-caveolin-1 fusion protein pull-down assays were performed using cell
lysates from HCT116 cells transiently transfected with wild type Ku70-HA. A blot that is representative of two independent experiments is shown. doi:10.1371/journal.pone.0039379.g003 Figure 3. Ku70 binds to the scaffolding domain of caveolin-1. (A) The consensus caveolin binding domain (CBD) is shown together with the
caveolin binding domain of Ku70 (amino acids 471 to 478) and a mutant form of Ku70’s CBD in which aromatic residues were mutated to alanines. (B)
Ponceau S staining of GST alone and GST-caveolin-1 fusion proteins. (C) GST-caveolin-1 fusion protein pull-down assays were performed using cell
lysates from HCT116 cells transiently transfected with wild type Ku70-HA. The Interaction between Caveolin-1 and Ku70 is Induced
by Chemotherapeutic Drugs and Limits the Release of
Bax from Ku70 Interestingly, 5-FU, but not etoposide, also stimulated total caveolin-
1 protein expression in HCT116 cells (Figure 4A, lower panel). This
is consistent with the increased caveolin-1 mRNA level induced by
5-FU, but not etoposide, in HCT116 cells (Figure S5A), suggesting
that 5-FU also promotes caveolin-1 protein expression through a
transcriptional-dependent mechanism. Knockdown of Caveolin-1 Protein Expression Potentiates
the Chemotherapeutic Drug-induced Conformational
Change of Bax Ku70 is a Novel Caveolin-1-binding Protein A blot that is representative of two independent experiments is shown. doi:10.1371/journal.pone.0039379.g003 PLoS ONE | www.plosone.org Knockdown of Caveolin-1 Protein Expression Potentiates
the Chemotherapeutic Drug-induced Conformational
Change of Bax Consistent with these results, chemotherapeutic drug-
induced translocation of Bax to mitochondria was enhanced in
HCT116 cells in which caveolin-1 protein expression was reduced
by siRNA, as compared to HCT116 cells expressing scrambled
siRNA (Figure 5E). We conclude that binding of caveolin-1 to
Ku70 limits the release of Bax from Ku70 and the activation of
Bax induced by chemotherapeutic drugs. Knockdown of Caveolin-1 Protein Expression Potentiates
the Chemotherapeutic Drug-induced Conformational
Change of Bax Activation of Bax is associated with a conformational change
that leads to exposure of the N terminus and can be detected using
the anti-Bax antibody clone 6A7 [29,30]. We show in Figures 5C
and 5D that 6A7 antibody immunoprecipitated Bax only in
HCT116 cells treated with the apoptotic stimulus 5-FU but not in
control cells. Knockdown of caveolin-1 protein expression by
siRNA significantly potentiated binding of the 6A7 antibody to
Bax in cells treated with 5-FU (Figures 5C and 5D). However, Why does the chemotherapeutic drug-induced interaction
between caveolin-1 and Ku70 act in an anti-apoptotic manner? Ku70 has been shown to bind to Bax under resting conditions. Release of Bax from Ku70 and the translocation of Bax to
mitochondria are key steps in the signaling events that link an
apoptotic stimulus to cell death. The molecular mechanisms that
regulate these processes remain to be fully elucidated. Our data show
that caveolin-1 acts asan anti-apoptotic protein (Figures 1 and 2) and June 2012 | Volume 7 | Issue 6 | e39379 6 Caveolin-1, a Novel Ku70-Interacting Protein Figure 4. Chemotherapeutic drugs promote the Ku70-Cav-1 interaction. (A) HCT116 cells were treated with either etoposide or 5-FU for 24
hours. Untreated cells were used as control. Cell lysates were then immunoprecipitated with an antibody probe specific for caveolin-1 and
immunoprecipitates subjected to immunoblotting analysis with anti-Ku70, anti-Bax and anti-caveolin-1 IgGs. (B) HCT116 colon cancer cells were
transfected with HA-tagged wild type Ku70. One day after transfection, cells were treated with 5-FU for 24 hours. Untreated cells were used as
control. Cells were then subjected to immunofluorescence analysis using antibody probes specific for the HA tag (green) and caveolin-1 (red). Nuclei
were detected by DAPI staining (Blue). Yellow staining in the merged images shows co-localization between caveolin-1 and Ku70 only after 5-FU
treatment (see arrows). Representative images are shown. doi:10.1371/journal.pone.0039379.g004 Figure 4. Chemotherapeutic drugs promote the Ku70-Cav-1 interaction. (A) HCT116 cells were treated with either etoposide or 5-FU for 24
hours. Untreated cells were used as control. Cell lysates were then immunoprecipitated with an antibody probe specific for caveolin-1 and
immunoprecipitates subjected to immunoblotting analysis with anti-Ku70, anti-Bax and anti-caveolin-1 IgGs. (B) HCT116 colon cancer cells were
transfected with HA-tagged wild type Ku70. One day after transfection, cells were treated with 5-FU for 24 hours. Untreated cells were used as
control. Knockdown of Caveolin-1 Protein Expression Potentiates
the Chemotherapeutic Drug-induced Conformational
Change of Bax Cells were then subjected to immunofluorescence analysis using antibody probes specific for the HA tag (green) and caveolin-1 (red). Nuclei
were detected by DAPI staining (Blue). Yellow staining in the merged images shows co-localization between caveolin-1 and Ku70 only after 5-FU
treatment (see arrows). Representative images are shown. doi:10.1371/journal.pone.0039379.g004 To directly test this possibility, we took advantage of HCT116 Bax
(2/2) null isogenic cells [31], which do not express Bax (data not
shown). Caveolin-1 protein expression was knockdown by siRNA
in HCT116 and HCT116 Bax null cells (Figure 6A). Cells were
then treated with either 5-FU or etoposide and apoptosis was
quantified by DAPI staining. We demonstrate that knockdown of
caveolin-1 protein expression by siRNA potentiated 5-FU-induced
apoptosis in HCT116 cells by 81% but only by 46% in HCT116
Bax null cells (Figure 6B). Similarly, knockdown of caveolin-1
protein expression by siRNA enhanced etoposide-induced apop-
tosis in HCT116 cells by 84% but only by 10% in HCT116 Bax
null cells (Figure 6D). Consistent with these data, the enhanced
activation of caspase 3 by downregulation of caveolin-1 expression
following treatment with either 5-FU or etoposide observed in
HCT116 cells was dramatically inhibited in HCT116 Bax null
cells (Figures 6C and 6E, respectively). Thus, Bax expression plays
a central role in the potentiation of chemotherapeutic drug-
induced apoptosis following knockdown of caveolin-1 in HCT116
cells. The fact that we observed apoptosis after 5-FU treatment
even in HCT116 Bax null cells transfected with scrambled siRNA
(Figures 6B and 6D) suggests that apoptosis in these cells can also
be Bax-independent, confirming previously reported data showing
Bax-independent apoptosis in these cells [32,33]. Finally, the 46%
increase in apoptosis induced by 5-FU found in caveolin-1 siRNA- knockdown of caveolin-1 protein expression by itself in untreated
cells failed to promote the conformational change of Bax
(Figures 5C and 5D). Since our results show that chemothera-
peutic drugs stimulate the binding of caveolin-1 with Ku70 and
the interaction of Bax with Ku70 is known to prevent the
activation of Bax, our data suggest that caveolin-1 forms a
complex with Ku70 that inhibits the activation of Bax. The
dissociation of caveolin-1 from Ku70 is not sufficient to activate
Bax but potentiates Bax activation induced by chemotherapeutic
drugs. Enhancement of Apoptosis Induced by the Knockdown
of Caveolin-1 Protein Expression is Dependent upon the
Expression of Bax If knockdown of caveolin-1 protein expression potentiates
apoptosis in colon cancer cells by enhancing the release of Bax
from Ku70, one would not expect potentiation of apoptosis by
knockdown of caveolin-1 in HCT116 cells lacking Bax expression. PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e39379 June 2012 | Volume 7 | Issue 6 | e39379 7 Caveolin-1, a Novel Ku70-Interacting Protein Figure 5. Knockdown of caveolin-1 protein expression enhances the dissociation of Bax from Ku70 and the activation of Bax
induced by chemotherapeutic drugs. (A, C, D and E) HCT116 colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, HCT116 cells were treated with either 5-FU (A, C and D) or
etoposide (A and E) for 24 hours. In (B), untransfected cells were treated with 5-FU for 24 hours. In (A), cell lysates were immunoprecipitated with an
antibody probe specific for Bax and immunoprecipitates subjected to Western blotting analysis with anti-Ku70 and anti-Bax IgGs. In (B), cell lysates
were immunoprecipitated with either anti-caveolin-1 IgGs or anti-Bax IgGs. Immunoprecipitates were then subjected to immunoblotting analysis
with antibody probes specific for caveolin-1, Ku70 and Bax. In (C and D), cell lysates were immunoprecipitated with a Bax (6A7) antibody and
immunoprecipitates subjected to immunoblotting analysis with anti-Bax IgGs. A blot that is representative of three independent experiments is
shown in (C), quantification of Bax levels pulled down by the Bax (6A7) antibody is shown in (D). Values in (D) represent mean 6 SEM; *P,0.001. In
(E), mitochondrial fractions were isolated and expression of Bax in mitochondria was detected by immunoblotting analysis using an antibody probe
specific for Bax. doi:10.1371/journal.pone.0039379.g005 Figure 5. Knockdown of caveolin-1 protein expression enhances the dissociation of Bax from Ku70 and the activation of Bax
induced by chemotherapeutic drugs. (A, C, D and E) HCT116 colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, HCT116 cells were treated with either 5-FU (A, C and D) or
etoposide (A and E) for 24 hours. In (B), untransfected cells were treated with 5-FU for 24 hours. In (A), cell lysates were immunoprecipitated with an
antibody probe specific for Bax and immunoprecipitates subjected to Western blotting analysis with anti-Ku70 and anti-Bax IgGs. Enhancement of Apoptosis Induced by the Knockdown
of Caveolin-1 Protein Expression is Dependent upon the
Expression of Bax In (B), cell lysates
were immunoprecipitated with either anti-caveolin-1 IgGs or anti-Bax IgGs. Immunoprecipitates were then subjected to immunoblotting analysis
with antibody probes specific for caveolin-1, Ku70 and Bax. In (C and D), cell lysates were immunoprecipitated with a Bax (6A7) antibody and
immunoprecipitates subjected to immunoblotting analysis with anti-Bax IgGs. A blot that is representative of three independent experiments is
shown in (C), quantification of Bax levels pulled down by the Bax (6A7) antibody is shown in (D). Values in (D) represent mean 6 SEM; *P,0.001. In
(E), mitochondrial fractions were isolated and expression of Bax in mitochondria was detected by immunoblotting analysis using an antibody probe
specific for Bax. p
doi:10.1371/journal.pone.0039379.g005 expressing HCT116 Bax null cells, as compared to scrambled
siRNA-expressing HCT116 Bax null cells (Figure 6B), suggests
that, in addition to Bax-dependent apoptosis, caveolin-1 may also
inhibit, to a certain extent, Bax-independent cell death following
treatment with 5-FU. binding domain of Ku70 were mutated to alanines (WRA-Ku70). We show that the in vitro binding of WRA-Ku70 to caveolin-1 was
greatly inhibited, as compared to wild type Ku70 (Figures S5B and
S5C). We then overexpressed wild type Ku70 and WRA-Ku70 in
HCT116 cells. Cells were treated with 5-FU and the interaction of
caveolin-1 with Ku70 and Bax was examined by coimmunopre-
cipitation studies. We find that 5-FU promoted the interaction of
caveolin-1 with wild type Ku70 and Bax in cells overexpressing
wild type Ku70 (Figure 7A). In contrast, the interaction of
caveolin-1 with WRA-Ku70 and Bax was only marginally
stimulated by 5-FU treatment in cells overexpressing WRA-
Ku70 (Figure 7A). Consistent with our findings, WRA-Ku70,
similarly to wild type Ku70 (Figure 4B), did not co-localize with
caveolin-1 in HCT116 cells under resting conditions, being mainly
localized in the nucleus and, to a lesser extent, diffuse in the
cytoplasm (see arrowheads in Figure S6). However, in contrast to
wild type Ku70 (Figure 4B), 5-FU failed to promote the co- The Interaction of Ku70 with Caveolin-1 is Required for
the Ku70-dependent Protection against The Interaction of Ku70 with Caveolin-1 is Required for
the Ku70-dependent Protection against The Interaction of Ku70 with Caveolin-1 is Required for
the Ku70-dependent Protection against Chemotherapeutic Drug-induced Apoptosis Values
represent mean 6 SEM; *P,0.001. In (C and E), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3. Immunoblotting with anti-b-actin IgGs was done to show equal loading. doi:10.1371/journal.pone.0039379.g006 Figure 6. Knockdown of caveolin-1 protein expression fails to enhance apoptosis induced by chemotherapeutic drugs in the
absence of Bax expression. HCT116 and HCT116 Bax null cells were transfected with siRNA directed against caveolin-1. Transfection
with scrambled siRNA was used as control. In (A), expression of endogenous caveolin-1 was detected by immunoblotting analysis 48 hours after
transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-b-actin IgGs was done to show equal loading. In (B–E), 24
hours after transfection, cells were treated with either 5-FU (25 mg/ml) for 48 h (B and C) or etoposide (Etop.; 800 mM) for 24 h (D and E). Untreated
cells were used as control. In (B and D), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values
represent mean 6 SEM; *P,0.001. In (C and E), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3. Immunoblotting with anti-b-actin IgGs was done to show equal loading. doi:10.1371/journal.pone.0039379.g006 localization of WRA-Ku70 with caveolin-1 in HCT116 cells
(Figure S6). Interestingly, overexpression of wild type Ku70, but
not WRA-Ku70, promoted a ,40% decrease in apoptosis
induced by either 5-FU or etoposide (Figures 7B and 7C). In
support to this data, we show in Figure 7D that wild type Ku70, in
contrast to WRA-Ku70, prevented the activation of caspase 3 by
either 5-FU or etoposide and that overexpression of wild type
Ku70, but not WRA-Ku70, limited the Ku70/Bax dissociation
promoted by 5-FU in HCT116 cells (Figure 7E). These results
support our conclusion that the interaction of Ku70 with caveolin-
1 limits the chemotherapeutic drug-induced release of Bax from
Ku70 and therefore Bax-dependent apoptosis. whereas
reduced
apoptosis
may
lead
to
uncontrolled
cell
proliferation and cancer. Apoptosis is also a major cytotoxic effect
of chemotherapy. Thus, understanding, at the molecular level,
how apoptosis is regulated will not only bring insight into a
fundamental biological event but also contribute to the develop-
ment of more efficient therapeutic interventions for the treatment
of cancer. 5-fluorouracil is a chemotherapeutic drug that has been
the cornerstone of treatment for patients with colorectal cancer for
almost four decades. Chemotherapeutic Drug-induced Apoptosis 5-FU blocks the enzyme thymidylate synthase
and inhibits both RNA and DNA synthesis. In sensitive cells, 5-FU
induces apoptosis. Unfortunately, tumor responses to 5-FU is
limited by toxicity to tissues containing highly proliferative cell
populations [34]. In this report, we show that knockdown of
caveolin-1 protein expression in colon cancer cells enhances
apoptosis induced by 5-FU and etoposide. Mechanistically, our
data reveal a novel anti-apoptotic mechanism in which the binding
of caveolin-1 to Ku70 limits the dissociation of Bax from Ku70
and the activation of Bax induced by apoptotic stimuli. Consistent
with this conclusion, we show that the enhancement of 5-FU- PLoS ONE | www.plosone.org Chemotherapeutic Drug-induced Apoptosis Overexpression of Ku70 has been shown to protect against
apoptosis [22,25]. Based on our data showing that binding of
caveolin-1 to Ku70 protects the cells against apoptosis induced by
chemotherapeutic drugs, we would predict less protection against
apoptosis by a mutant form of Ku70 that cannot bind to caveolin-
1. Since we show in Figure 3 that caveolin-1 interacts with Ku70,
to directly test our hypothesis, we generated a mutant form of
Ku70 in which the critical aromatic residues of the caveolin PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e39379 8 Caveolin-1, a Novel Ku70-Interacting Protein Figure 6. Knockdown of caveolin-1 protein expression fails to enhance apoptosis induced by chemotherapeutic drugs in the
absence of Bax expression. HCT116 and HCT116 Bax null cells were transfected with siRNA directed against caveolin-1. Transfection
with scrambled siRNA was used as control. In (A), expression of endogenous caveolin-1 was detected by immunoblotting analysis 48 hours after
transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-b-actin IgGs was done to show equal loading. In (B–E), 24
hours after transfection, cells were treated with either 5-FU (25 mg/ml) for 48 h (B and C) or etoposide (Etop.; 800 mM) for 24 h (D and E). Untreated
cells were used as control. In (B and D), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values
represent mean 6 SEM; *P,0.001. In (C and E), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3. Immunoblotting with anti-b-actin IgGs was done to show equal loading. doi:10.1371/journal.pone.0039379.g006 Figure 6. Knockdown of caveolin-1 protein expression fails to enhance apoptosis induced by chemotherapeutic drugs in the
absence of Bax expression. HCT116 and HCT116 Bax null cells were transfected with siRNA directed against caveolin-1. Transfection
with scrambled siRNA was used as control. In (A), expression of endogenous caveolin-1 was detected by immunoblotting analysis 48 hours after
transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-b-actin IgGs was done to show equal loading. In (B–E), 24
hours after transfection, cells were treated with either 5-FU (25 mg/ml) for 48 h (B and C) or etoposide (Etop.; 800 mM) for 24 h (D and E). Untreated
cells were used as control. In (B and D), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. June 2012 | Volume 7 | Issue 6 | e39379 Discussion Apoptosis is the process of programmed cell death adopted by
multicellular organisms for proper development and maintenance
of adult individuals. Defective apoptosis has been linked to
pathological conditions. Excessive apoptosis may lead to atrophy June 2012 | Volume 7 | Issue 6 | e39379 June 2012 | Volume 7 | Issue 6 | e39379 9 Caveolin-1, a Novel Ku70-Interacting Protein Figure 7. WRA Ku70 fails to interact with caveolin-1, to protect against chemotherapeutic drug-induced apoptosis and to limit the
5-FU-induced dissociation of Bax from Ku70. HCT116 cells were transfected with either wild type Ku70-HA (WT Ku70) or WRA Ku70-HA (WRA
Ku70) and treated with different concentrations of either 5-FU (for 24 h in A and E; for 48 h in C and D) or etoposide (for 24 h in C and D). Untreated
cells were used as control. In (A), cell lysates were immunoprecipitated with an antibody probe specific for caveolin-1 and immunoprecipitates
subjected to immunoblotting analysis with anti-HA, anti-Bax and anti-caveolin-1 IgGs. In (B), total expression of WT Ku70-HA and WRA Ku70-HA was
detected by immunoblotting analysis with anti-HA IgGs before drug treatment. In (C), cells were stained with DAPI and the number of cells showing
nuclear condensation was quantified. Values represent mean 6 SEM; *P,0.001. In (D), cells were subjected to immunoblotting analysis with antibody
probes specific for cleaved caspase 3. Immunoblotting with anti-b-actin IgGs was done to show equal loading. In (E), cell lysates were
immunoprecipitated with and antibody probe specific for Bax and immunoprecipitates subjected to Western blotting analysis with anti-HA and anti-
Bax IgGs. doi:10.1371/journal.pone.0039379.g007 Figure 7. WRA Ku70 fails to interact with caveolin-1, to protect against chemotherapeutic drug-induced apoptosis and to limit the
5-FU-induced dissociation of Bax from Ku70. HCT116 cells were transfected with either wild type Ku70-HA (WT Ku70) or WRA Ku70-HA (WRA
Ku70) and treated with different concentrations of either 5-FU (for 24 h in A and E; for 48 h in C and D) or etoposide (for 24 h in C and D). Untreated
cells were used as control. In (A), cell lysates were immunoprecipitated with an antibody probe specific for caveolin-1 and immunoprecipitates
subjected to immunoblotting analysis with anti-HA, anti-Bax and anti-caveolin-1 IgGs. In (B), total expression of WT Ku70-HA and WRA Ku70-HA was
detected by immunoblotting analysis with anti-HA IgGs before drug treatment. Discussion In (C), cells were stained with DAPI and the number of cells showing
nuclear condensation was quantified. Values represent mean 6 SEM; *P,0.001. In (D), cells were subjected to immunoblotting analysis with antibody
probes specific for cleaved caspase 3. Immunoblotting with anti-b-actin IgGs was done to show equal loading. In (E), cell lysates were
immunoprecipitated with and antibody probe specific for Bax and immunoprecipitates subjected to Western blotting analysis with anti-HA and anti-
Bax IgGs. d i 10 1371/j
l
0039379 007 doi:10.1371/journal.pone.0039379.g007 doi:10.1371/journal.pone.0039379.g007 Caveolin-1 was recently shown to modulate the cellular
response to DNA damage by regulating both homologous
recombination (HR) and non-homologous end joining (NHEJ)
repair pathways [38]. Interestingly, Ku70 was originally discov-
ered as a molecule involved in the repair of DNA double-strand
breaks by non-homologous end joining (NHEJ). Since we
demonstrate in this report that Ku70 is a novel caveolin-1 binding
protein, our findings suggest that caveolin-1 may regulate DNA
repair through modulation of Ku70 functions which are indepen-
dent of apoptosis. How caveolin-1 regulates Ku70 in the context of
DNA repair mechanisms is currently under investigation. induced apoptosis after knockdown of caveolin-1 protein expres-
sion is dramatically prevented in the absence of Bax expression. Therefore, caveolin-1 acts as an anti-apoptotic molecule in colon
cancer cells by inhibiting Bax-dependent cell death. Overexpression of caveolin-1 was linked to drug-resistance of
cancer cells, including colon cancer cells [35,36]. Our findings
contribute to explain, at the molecular level, the role of caveolin-1
in drug resistance of cancer cells. Increased levels of caveolin-1
may promote resistance to chemotherapeutic drugs by bolstering
the inhibitory action of caveolin-1 on the Ku70-Bax complex. In
fact, in the presence of elevated caveolin-1 levels, the release of
Bax from Ku70 induced by chemotherapeutic drugs would be
further inhibited. This mechanism would also be consistent with a
proposed scenario in which caveolin-1 expression is lost in the
initial phases of cancer development but returns to high levels at
later stages when the tumor has acquired multi-drug resistance
properties (reviewed in [37]). Overexpression of Ku70 has been shown to protect against
apoptosis [22,25]. Our data contribute to explain this previous
finding at the molecular level. We confirmed that overexpression
of wild type Ku70 inhibits both 5-FU- and etoposide-induced
apoptosis of colon cancer cells. Acknowledgments WethankDr.BertVogelsteinforprovidingHCT116andHCT116Bax2/2
cells. Supporting Information One day after transfection, HCT116 cells
were collected, incubated with propidium iodide and subjected to
FACS analysis with the use of a fluorescence-activated cell sorter
(FACStar plus; Becton Dickinson). A representative cell cycle
analysis experiment is shown. Values represent mean from
independent experiments. Figure S2
Knockdown of caveolin-1 protein expression
reduces the number of cells in the S phase of the cell
cycle. HCT116 colon cancer cells were transfected with siRNA
directed against caveolin-1. Transfection with scrambled siRNA
was used as control. One day after transfection, HCT116 cells
were collected, incubated with propidium iodide and subjected to
FACS analysis with the use of a fluorescence-activated cell sorter
(FACStar plus; Becton Dickinson). A representative cell cycle
analysis experiment is shown. Values represent mean from
independent experiments. Figure S6
WRA Ku70 fails to co-localize with caveolin-1
after treatment with 5-FU. HCT116 colon cancer cells were
transfected
with
HA-tagged
WRA
Ku70. One
day
after
transfection, cells were treated with 5-FU for 24 hours. Untreated
cells were used as control. Cells were then subjected to
immunofluorescence analysis using antibody probes specific for
the HA tag (green) and caveolin-1 (red). Nuclei were detected by
DAPI staining (Blue). Representative images are shown. (TIF) Figure S3
Quantification of nuclear condensation after
knockdown of caveolin-1 protein expression in 5-FU-
treated HT29 colon cancer cells. HT29 colon cancer cells
were transfected with siRNA directed against caveolin-1. Trans-
fection with scrambled siRNA was used as control. One day after
transfection, cells were treated with 5-FU for different periods of
time. Untreated cells were used as control. Cells were then stained
with DAPI. Representative images are shown in (A). Arrows show
examples of cells with nuclear condensation. The number of cells Discussion Quantification of crystal violet staining after
etoposide treatment is shown in (B). Values represent mean 6
SEM; *P,0.001; #P,0.005. (TIF) Author Contributions Conceived and designed the experiments: HZ DV FG. Performed the
experiments: HZ DV. Analyzed the data: HZ DV FG. Contributed
reagents/materials/analysis tools: HZ DV. Wrote the paper: HZ FG. Conceived and designed the experiments: HZ DV FG. Performed the
experiments: HZ DV. Analyzed the data: HZ DV FG. Contributed
reagents/materials/analysis tools: HZ DV. Wrote the paper: HZ FG. 1. Palade GE (1953) The fine structure of blood capillaries. J Appl Physics 24:
1424. 10. Bartholomew JN, Volonte D, Galbiati F (2009) Caveolin-1 regulates the
antagonistic pleiotropic properties of cellular senescence through a novel
Mdm2/p53-mediated pathway. Cancer Res 69: 2878–2886. 2. Yamada E (1955) The fine structure of the gall bladder epithelium of the mouse.
J Biophys Biochem Cytol 1: 445–458. Supporting Information Figure S5
5-FU, but not etoposide, increases caveolin-1
mRNA levels in HCT116 cells. The binding of W RA
Ku70 to caveolin-1 is dramatically compromised. (A)
HCT116 cells were treated with either 5-FU or etoposide for 24
hours. Untreated cells were used as control. RNA was extracted
and RT-PCR was performed using primers specific for human
caveolin-1. Amplification of GAPDH was performed by RT-PCR
as an internal control. A representative blot is shown. (B) Ponceau
S staining of GST alone and the GST-caveolin-1 (82–101) fusion
protein. (C) GST-caveolin-1 fusion protein pull-down assays were
performed using cell lysates from HCT116 cells transiently
transfected with either wild type Ku70-HA or WRA Ku70-HA. A blot that is representative of two independent experiments is
shown. (TIF) Figure S5
5-FU, but not etoposide, increases caveolin-1
mRNA levels in HCT116 cells. The binding of W RA
Ku70 to caveolin-1 is dramatically compromised. (A)
HCT116 cells were treated with either 5-FU or etoposide for 24
hours. Untreated cells were used as control. RNA was extracted
and RT-PCR was performed using primers specific for human
caveolin-1. Amplification of GAPDH was performed by RT-PCR
as an internal control. A representative blot is shown. (B) Ponceau
S staining of GST alone and the GST-caveolin-1 (82–101) fusion
protein. (C) GST-caveolin-1 fusion protein pull-down assays were
performed using cell lysates from HCT116 cells transiently
transfected with either wild type Ku70-HA or WRA Ku70-HA. A blot that is representative of two independent experiments is
shown. (TIF) Figure S1
Quantification of nuclear condensation after
knockdown of caveolin-1 protein expression in 5-FU-
treated HCT116 colon cancer cells. HCT116 colon cancer
cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. One day
after transfection, cells were treated with 5-FU for different periods
of time. Untreated cells were used as control. Cells were then
stained with DAPI. Representative images are shown in (A). Arrows show examples of cells with nuclear condensation. The
number of cells showing nuclear condensation was quantified in
(B). Values represent mean 6 SEM; *P,0.001. Figure S2
Knockdown of caveolin-1 protein expression
reduces the number of cells in the S phase of the cell
cycle. HCT116 colon cancer cells were transfected with siRNA
directed against caveolin-1. Transfection with scrambled siRNA
was used as control. p
p
y
11. Parat MO (2009) The biology of caveolae: achievements and perspectives. Int
Rev Cell Mol Biol 273: 117–162. Discussion More importantly, we show that a
mutant form of Ku70 that cannot interact with caveolin-1 (WRA PL June 2012 | Volume 7 | Issue 6 | e39379 PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e39379 10 Caveolin-1, a Novel Ku70-Interacting Protein showing nuclear condensation was quantified in (B). Values
represent mean 6 SEM; *P,0.001. (TIF) Ku70) fails to inhibit chemotherapeutic drug-induced apoptosis. Thus, the interaction of Ku70 with caveolin-1 is critical for the
ability
of
Ku70
to
inhibit
apotosis. We
also
found
that
overexpression of WRA Ku70, in contrast to wild type Ku70,
fails to inhibit the Bax/Ku70 dissociation after stimulation with
chemotherapeutic drugs, which explains, at the molecular level,
why WRA Ku70 fails to protect against chemotherapeutic drug-
induced apoptosis. Thus, under cellular conditions where Ku70
fails to interact with caveolin-1 (i.e. cells in which caveolin-1
protein expression is knockdown by siRNA or cells expressing the
WRA mutant form of Ku70 that does not interact with caveolin-
1), the ability of Ku70 to limit the release of Bax is prevented. Together, our data indicate that disruption of the interaction
between caveolin-1 and Ku70 enhances apoptosis and propose the
caveolin-1/Ku70 complex as a novel potential therapeutic target
for the treatment of colon cancer. Figure S4
Knockdown of caveolin-1 protein expression
inhibits
long-term
survival
of
HCT116
cells
after
treatment with etoposide. HCT116 colon cancer cells were
transfected with siRNA directed against caveolin-1. Transfection
with scrambled siRNA was used as control. One day after
transfection, HCT116 cells were treated with different concentra-
tions of etoposide for 1 hour. Untreated cells were used as control. Cells were then cultured for 7 days and stained with crystal violet. A representative crystal violet staining after etoposide treatment is
shown in (A). Quantification of crystal violet staining after
etoposide treatment is shown in (B). Values represent mean 6
SEM; *P,0.001; #P,0.005. (TIF) Figure S4
Knockdown of caveolin-1 protein expression
inhibits
long-term
survival
of
HCT116
cells
after
treatment with etoposide. HCT116 colon cancer cells were
transfected with siRNA directed against caveolin-1. Transfection
with scrambled siRNA was used as control. One day after
transfection, HCT116 cells were treated with different concentra-
tions of etoposide for 1 hour. Untreated cells were used as control. Cells were then cultured for 7 days and stained with crystal violet. A representative crystal violet staining after etoposide treatment is
shown in (A). 6. Galbiati F, Razani B, Lisanti MP (2001) Caveolae and caveolin-3 in muscular
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oncogenic transformation and muscular dystrophy. J Cell Sci 113: 2103–2109. 3. Scherer PE, Lewis RY, Volonte D, Engelman JA, Galbiati F, et al. (1997) Cell-
type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize
and form a stable hetero-oligomeric complex in vivo. J Biol Chem 272: 29337–
29346. 4. Matveev S, Li X, Everson W, Smart EJ (2001) The role of caveolae and caveolin
in vesicle-dependent and vesicle-independent trafficking. Adv Drug Deliv Rev
49: 237–250. 4. Matveev S, Li X, Everson W, Smart EJ (2001) The role of caveolae and caveolin
in vesicle-dependent and vesicle-independent trafficking. Adv Drug Deliv Rev
49: 237–250. 5. Galbiati F, Razani B, Lisanti MP (2001) Emerging themes in lipid rafts and
caveolae. Cell 106: 403–411. PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e39379 June 2012 | Volume 7 | Issue 6 | e39379 11 Caveolin-1, a Novel Ku70-Interacting Protein PLoS ONE | www.plosone.org June 2012 | Volume 7 | Issue 6 | e39379 12
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OFD1 and Flotillins Are Integral Components of a Ciliary Signaling Protein Complex Organized by Polycystins in Renal Epithelia and Odontoblasts
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Abstract Mutation of the X-linked oral-facial-digital syndrome type 1 (OFD1) gene is embryonic lethal in males and results in
craniofacial malformations and adult onset polycystic kidney disease in females. While the OFD1 protein localizes to
centriolar satellites, centrosomes and basal bodies, its cellular function and how it relates to cystic kidney disease is largely
unknown. Here, we demonstrate that OFD1 is assembled into a protein complex that is localized to the primary cilium and
contains the epidermal growth factor receptor (EGFR) and domain organizing flotillin proteins. This protein complex, which
has similarity to a basolateral adhesion domain formed during cell polarization, also contains the polycystin proteins that
when mutant cause autosomal dominant polycystic kidney disease (ADPKD). Importantly, in human ADPKD cells where
mutant polycystin-1 fails to localize to cilia, there is a concomitant loss of localization of polycystin-2, OFD1, EGFR and
flotillin-1 to cilia. Together, these data suggest that polycystins are necessary for assembly of a novel flotillin-containing
ciliary signaling complex and provide a molecular rationale for the common renal pathologies caused by OFD1 and PKD
mutations. Citation: Jerman S, Ward HH, Lee R, Lopes CAM, Fry AM, et al. (2014) OFD1 and Flotillins Are Integral Components of a Ciliary Signaling Protein Complex
Organized by Polycystins in Renal Epithelia and Odontoblasts. PLoS ONE 9(9): e106330. doi:10.1371/journal.pone.0106330 ation: Jerman S, Ward HH, Lee R, Lopes CAM, Fry AM, et al. (2014) OFD1 and Flotillins Are Integral Components of a Ciliary Sign
ganized by Polycystins in Renal Epithelia and Odontoblasts. PLoS ONE 9(9): e106330. doi:10.1371/journal.pone.0106330 Editor: Wayne I. Lencer, Boston Children’s Hospital Harvard Medical School Pediatrics, United States of America Editor: Wayne I. Lencer, Boston Children’s Hospital Harvard Medical School Pediatrics, United States of America Received January 13, 2014; Accepted July 31, 2014; Published September 2, 2014 uary 13, 2014; Accepted July 31, 2014; Published September 2, 2 Copyright: 2014 Jerman 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. Stephanie Jerman1, Heather H. Ward2, Rebecca Lee1, Carla A. M. Lopes3, Andrew M. Fry3,
Mary MacDougall4, Angela Wandinger-Ness1* 1 Department of Pathology MSC08-4640 and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, United
States of America, 2 Department of Internal Medicine, Division of Nephrology MSC10-5550, University of New Mexico Health Sciences Center, Albuquerque, New Mexico,
United States of America, 3 Department of Biochemistry, University of Leicester, Leicester, United Kingdom, 4 Institute of Oral Health Research & Department of Oral and
Maxillofacial Surgery, School of Dentistry, University of Alabama, Birmingham, Alabama, United States of America Abstract Funding: These studies were supported in part by: grants from the National Institute of Dental and Craniofacial Research F31 DE023483-01 (SJ); a National
Institute of General Medical Sciences (NIGMS) Spatio Temporal Modeling Center Fellowship (STMC) P50 GM085273 (SJ, PI-Wilson, B.); The Wellcome Trust and
Kidney Research UK (AMF); the National Institute of Diabetes and Digestive and Kidney Diseases R01 DK050141 (AWN); NIGMS K12 GM088021 (AWN; HHW
scholar). The content is solely the responsibility of the authors and does not necessarily represent the official views from the National Institutes of Health. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors confirm that co-author Andrew Fry is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to
es and criteria. Competing Interests: The authors confirm that co-author Andrew Fry is a PLOS ONE Editorial Board member. This does not alter
PLOS ONE Editorial policies and criteria. Competing Interests: The authors confirm that co-author Andrew Fry is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to
PLOS ONE Editorial policies and criteria. * Email: wness@unm.edu September 2014 | Volume 9 | Issue 9 | e106330 Introduction proteins are mutant is thought to cause epithelial cell transdiffer-
entiation and contribute to renal cyst development [2]. While the
associations between defects in primary cilia, signaling and kidney
disease have been recognized for over a decade, it is only recently
that links between cilia, signaling, and tooth defects were revealed. Deletion of the Ofd1 gene (Ofd1D4-5/+-) in mice causes missing/
supernumerary teeth, enamel hypoplasia, and polycystic kidney
disease analogous to human oral-facial-digital syndrome type 1
[4]. The observed morphological defects in molars result from
altered differentiation and polarization of odontoblasts when Ofd1
is mutant [3,4,8]. Localization of OFD1 to the primary cilium of
tooth ectomesenchymal odontoblasts and renal epithelial cells is
therefore speculated to be crucial for proper cellular differentiation
of both cell types [3,4]. Oral-facial-digital syndrome type 1 (OFD1; OMIM #311200)
is an X-linked inherited disease characterized by the malformation
of the face, oral cavity, hands and feet caused by heterogeneous
mutations in the OFD1 gene also known as CXORF5. Systemic
manifestations of OFD1 mutations include polycystic kidneys that
resemble those caused by mutations in the PKD1 or PKD2 genes
associated with autosomal dominant polycystic kidney disease
(ADPKD) [1,2]. Due to the low rate of kidney transplantation,
many patients with both craniofacial disorders and cystic kidney
disease will eventually succumb to renal failure. Thus, there is an
urgent need to clarify how the OFD1 gene product might cross-
talk with the pathways regulated by the PKD1 and PKD2 genes to
result in a common disease phenotype. In related observations in the Tg737 mouse, ectopic teeth
(premolars normally evolutionarily silenced) arise from inactiva-
tion of IFT88/polaris in the embryonic jaw and the consequential
increase in sonic hedgehog signaling [9]. The Tg737 mouse was
first used to illuminate the central role of IFT88/Polaris in the
development of cystic kidneys [10]. The emerging molecular Many of the proteins associated with cystic kidney disorders,
including polycystin-1 (PC1) and polycystin-2 (PC2) that underlie
ADPKD, localize to and function in the primary cilium [3–7]. The
polycystins have pivotal roles in calcium dependent signaling to
multiple pathways and loss of signaling regulation when the September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 1 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain Figure 1. Key ciliary signaling proteins are expressed in RCTE and MO6-G3 cells. Introduction Renal cortical tubular epithelial (RCTE) cells and tooth
derived odontoblasts (MO6-G3) cells were lysed and probed with antibodies directed against indicated proteins. PC1 using NM002 pAb (A, B), PC2
using Santa Cruz pAb (C), OFD1 using Santa Cruz pAb [3] (D), EGFR using Santa Cruz pAb (E), ErbB2 using US Biological pAb (F), flotillin-1 using BD
Transduction mAb (H), and flotillin-2 using BD Transduction mAb (I) were found to be expressed in both cell types. Actin mAb from Millipore (G) was
used as a loading control for PC1, PC2, EGFR, ErbB2, and OFD1; a/b-tubulin pAb from Cell Signaling (J) was used as a loading control for flotillin-1 and
flotillin-2. Actin or a/b-tubulin was used to normalize results for quantification. Bar graph showing densitometric quantification of PC1 (a, b), PC2 (c),
OFD1 (d), EGFR (e), ErbB2 (f), flotillin-1 (g), flotillin-2 (h). Bar graph represents the mean 6 SD of four independent experiments. (*) p = 0.01 to 0.05,
(***) p,0.001. doi:10.1371/journal.pone.0106330.g001 Figure 1. Key ciliary signaling proteins are expressed in RCTE and MO6-G3 cells. Renal cortical tubular epithelial (RCTE) cells and tooth
derived odontoblasts (MO6-G3) cells were lysed and probed with antibodies directed against indicated proteins. PC1 using NM002 pAb (A, B), PC2
using Santa Cruz pAb (C), OFD1 using Santa Cruz pAb [3] (D), EGFR using Santa Cruz pAb (E), ErbB2 using US Biological pAb (F), flotillin-1 using BD
Transduction mAb (H), and flotillin-2 using BD Transduction mAb (I) were found to be expressed in both cell types. Actin mAb from Millipore (G) was
used as a loading control for PC1, PC2, EGFR, ErbB2, and OFD1; a/b-tubulin pAb from Cell Signaling (J) was used as a loading control for flotillin-1 and
flotillin-2. Actin or a/b-tubulin was used to normalize results for quantification. Bar graph showing densitometric quantification of PC1 (a, b), PC2 (c),
OFD1 (d), EGFR (e), ErbB2 (f), flotillin-1 (g), flotillin-2 (h). Bar graph represents the mean 6 SD of four independent experiments. (*) p = 0.01 to 0.05,
(***) p,0.001. doi:10.1371/journal.pone.0106330.g001 p
doi:10.1371/journal.pone.0106330.g001 molecular explanation for some of the observed commonalities in
the pathogenesis of multi-organ ciliopathies such as OFD1
syndrome and ADPKD. hierarchy requires OFD1 to recruit IFT88 as a prerequisite for
ciliogenesis and ciliary hedgehog signaling that also involves the
polycystins [11–13]. Introduction Collectively, these findings hint that OFD1
and the polycystins are likely part of the same or overlapping
protein assemblies that control similar ciliary signaling pathways in
both odontoblasts and renal epithelia. Results Expression of OFD1, polycystins, flotillins and ErbB
receptor family members in renal epithelia and
odontoblasts September 2014 | Volume 9 | Issue 9 | e106330 Expression of OFD1, polycystins, flotillins and ErbB
receptor family members in renal epithelia and
odontoblasts There are some striking similarities in the mechanism by which
odontoblasts and renal epithelial cells respond to external stimuli
with the primary cilium of odontoblasts believed to closely mimic
the sensory function of cilia in renal epithelial cells [14]; thus, the
primary cilium likely serves as the critical link between extracel-
lular mechanical stimuli and initiation of responding intracellular
signaling cascades in odontoblasts and renal epithelial cells. In
addition to altered Hedgehog signaling, aberrant expression and
signaling from the tyrosine kinase epidermal growth factor
receptor (EGFR) is implicated in both craniofacial disorders and
ADPKD [15–17], and in primary cilia of renal epithelia, EGFR
interacts with and regulates PC2 ion channel activity [7]. Collectively, these data demonstrate the need to characterize the
functional interactions and molecular assemblies of complexes
comprised of signaling receptors, domain-organizing proteins, and
ciliary proteins, which when mutant, cause similar disease
phenotypes. We therefore tested the hypothesis that OFD1 co-
assembles into protein complexes constituted of PC1 and PC2,
EGFR, and the flotillin lipid scaffolding proteins in the primary
cilium of renal epithelia and odontoblasts. Our findings provide a We suspected the membrane raft organizing flotillin proteins
[18,19] to be key players in the organization of ciliary signaling
complexes. The speculation is founded on our studies that first
identified a cholesterol-rich, flotillin-organized signaling domain –
constituted of polycystins, tyrosine kinases and phosphatases and
cholesterol – at the basolateral membrane of renal epithelia
[20,21]. Of interest is the recent finding that EGFR and
downstream signaling partners are scaffolded in cholesterol-rich
signaling domains by the flotillins [22]. Here, we tested if OFD1 is
co-assembled into protein complexes constituted of PC1 and PC2,
EGFR and the flotillin lipid scaffolding proteins in the primary
cilium of renal epithelia and odontoblasts. The expression of Erythroblastic Leukemia Viral Oncogene
(ErbB) receptor proteins and the flotillins in renal epithelia and
odontoblast cells is not well described. The expression of these and
cystic
kidney
disease
proteins
were
therefore
subjected
to
comparative immunoblot analyses on polarized renal cortical September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org September 2014 | Volume 9 | Issue 9 | e106330 2 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain Figure 2. Transiently transfected GFP-OFD1 and flotillin-2-GFP
localize to primary cilia of RCTE and MO6-G3 cells. Polarized
RCTE and MO6-G3 cells were transiently transfected with either GFP-
OFD1 or flotillin-2-GFP constructs. Colocalization of OFD1, polycystins, EGFR and flotillins to
the primary cilium of odontoblasts and renal epithelial
cells The
primary
cilium
has
a
distinct
protein
composition
compared to the apical surface from which it protrudes, and is
enriched in signaling proteins that also function at the basolateral
membrane [25,26]. The importance of the primary cilium as a
signaling command center has been suggested [7,27], yet little is
known about the organization of signaling proteins within primary
cilia. Figure 2. Transiently transfected GFP-OFD1 and flotillin-2-GFP
localize to primary cilia of RCTE and MO6-G3 cells. Polarized
RCTE and MO6-G3 cells were transiently transfected with either GFP-
OFD1 or flotillin-2-GFP constructs. Upon 16 hours post-transfection,
cells were fixed and acetylated a-tubulin (Sigma Aldrich mAb) was
labeled to identify cilia. GFP-OFD1 localized to primary cilia in RCTE cells
(A) and MO6-G3 cells (C). GFP-Flotillin-2 localized to primary cilia of
RCTE (B) and MO6-G3 (D) cells. Images captured using a Zeiss LSM510
confocal microscope (636 objective). Images are representative of at
least 3 independent experiments. Arrows denote cilia. Scale bar 10 mm. doi:10.1371/journal.pone.0106330.g002 Immunolocalization studies of endogenous and overexpressed
proteins were performed to specifically assess ciliary protein
localization patterns in odontoblasts and renal epithelial cells. Odontoblast cells and fully polarized RCTE cells are notoriously
difficult to transfect via conventional techniques. To circumvent
this problem, we found electroporation (iPorator) on filter surfaces
routinely yielded plasmid transfection efficiencies of 40–50% (see
Methods). Transiently expressed GFP-OFD1 (Figure 2A) and
flotillin-2-GFP (Figure 2B) localized to primary cilia in RCTE cells
with some flotillin-2 also on intracellular structures. Similarly,
GFP-OFD1 (Figure 2C) and flotillin-2-GFP (Figure 2D) localized
to primary cilia of odontoblast cells, though there were significant
pools of flotillin-2-GFP elsewhere in the plasma membrane and
intracellular structures. OFD1 is known to be associated with basal
bodies and, in some renal cells, to localize along the ciliary
axoneme [3]. Here we found that, particularly in odontoblasts,
GFP-OFD1 staining extended beyond the region expected to
contain just the basal bodies into the ciliary axoneme. we observed co-localization of transiently expressed Flotillin-2-
GFP and PC2 in primary cilia (Figure S1A) as well as Flotillin-2-
GFP and EGFR (Figure S1B), while endogenous EGFR and PC1
co-localized to primary cilia of RCTE cells (Figure S1C). The
ciliary localizations of OFD1, PC1, PC2 and EGFR are consistent
with previous reports showing individual proteins in different cell
types [3–7]. Expression of OFD1, polycystins, flotillins and ErbB
receptor family members in renal epithelia and
odontoblasts Upon 16 hours post-transfection,
cells were fixed and acetylated a-tubulin (Sigma Aldrich mAb) was
labeled to identify cilia. GFP-OFD1 localized to primary cilia in RCTE cells
(A) and MO6-G3 cells (C). GFP-Flotillin-2 localized to primary cilia of
RCTE (B) and MO6-G3 (D) cells. Images captured using a Zeiss LSM510
confocal microscope (636 objective). Images are representative of at
least 3 independent experiments. Arrows denote cilia. Scale bar 10 mm. doi:10.1371/journal.pone.0106330.g002 tubular epithelial (RCTE) cells and odontoblasts (MO6-G3). PC1
(Figure 1A, B), PC2 (Figure 1C) and OFD1 (Figure 1D), along
with the tyrosine kinase receptors EGFR (Figure 1E) and ErbB2
(Figure 1F) were robustly expressed in both human renal epithelial
cells and mouse odontoblasts in culture. The signaling domain
organizing proteins flotillin-1 (Figure 1H) and flotillin-2 (Figure 1I)
were similarly expressed in both cell types, although flotillin-1
exhibited a clear mobility shift in odontoblasts as compared to
renal epithelial cells. The native 460 kDa PC1 is known to be
cleaved in cells yielding several different C-terminal fragments that
are important for physiologic functions [6,23,24]. (N.B. Available
antibodies
to
PC1
recognize
different
epitopes
with
some
recognizing the full-length 460 kDa protein while other antibodies
preferentially recognize the 230–250 kDa or 150 kDa PC1 bands). Renal epithelia and odontoblasts expressed similar levels of both
the 460 kDa and the 150 kDa forms of PC1 (Figure 1a, b), along
with EGFR (Figure 1e), ErbB2 (Figure 1f ), flotillin-1 (Figure 1g),
and flotillin-2 (Figure 1h). Notably, protein expression levels of
PC2 were 3.4-fold higher (Figure 1c), while OFD1 levels were 3.8
fold lower in odontoblasts as compared to renal epithelia
(Figure 1d). These data show that key mechanosensory and
signaling proteins such as the polycystin proteins, OFD1, EGFR,
and the flotillins are common to both renal epithelia and
odontoblasts, though there are some variations in the quantitative
levels of the cation channel PC2 and OFD1. September 2014 | Volume 9 | Issue 9 | e106330 Colocalization of OFD1, polycystins, EGFR and flotillins to
the primary cilium of odontoblasts and renal epithelial
cells Endogenous OFD1, PC1, PC2, EGFR and flotillin-1 localize to primary cilia of RCTE and MO6-G3 cells. Polarized RCTE an
MO6-G3 cells were fixed and stained using the indicated antibodies. OFD1 from Novus Biologicals (A, F), PC1 using pAb NM002 (B, G), PC2 usin
AbCam pAb (C, H), EGFR using GeneTex pAb (D, I) and flotillin-1 using AbCam pAb (E, J) were found localized to 100% of primary cilia analyzed fo
both cell types. Acetylated a-tubulin (Sigma-Aldrich mAb) labeling identifies cilia. Zeiss LSM510 confocal microscope images (636objective). Arrow
denote the protein of interest within a cilium. Representative results from at least 5 independent experiments. Secondary antibody only controls we
negative (not shown). Scale bar 10 mm. doi:10.1371/journal.pone.0106330.g003 Figure 3. Endogenous OFD1, PC1, PC2, EGFR and flotillin-1 localize to primary cilia of RCTE and MO6-G3 cells. Polarized RCTE and
MO6-G3 cells were fixed and stained using the indicated antibodies. OFD1 from Novus Biologicals (A, F), PC1 using pAb NM002 (B, G), PC2 using
AbCam pAb (C, H), EGFR using GeneTex pAb (D, I) and flotillin-1 using AbCam pAb (E, J) were found localized to 100% of primary cilia analyzed for
both cell types. Acetylated a-tubulin (Sigma-Aldrich mAb) labeling identifies cilia. Zeiss LSM510 confocal microscope images (636objective). Arrows
denote the protein of interest within a cilium. Representative results from at least 5 independent experiments. Secondary antibody only controls were
negative (not shown). Scale bar 10 mm. doi:10.1371/journal.pone.0106330.g003 Polycystins and OFD1 form protein complexes with EGFR
and flotillins in odontoblasts and renal epithelial cells probed for EGFR revealing that EGFR was indeed in complex
with PC1 in both cell types (Figure 4D). The non-transmembrane,
ciliary protein OFD1 was also found to be part of the polycystin-
EGFR multimeric complex in renal epithelia and odontoblast cells
(Figure 4E), and flotillin-2 is in complex with PC1 in both cell
types (Figure 4F). Quantification of replicate experiments demon-
strated specific co-precipitation of PC2, EGFR, OFD1, and
flotillin-2 with PC1 (Figure 4a–f). In a converse experiment,
EGFR was immunoprecipitated from polarized renal epithelial
and odontoblast cell lysates and probed for PC1, PC2, OFD1, and
flotillin-2 (Figure 4G–K). As seen in the PC1 immunoprecipitates,
all of the proteins were co-enriched with EGFR (Figure 4g–k),
with a cleaved fragment of PC1 preferentially co-precipitated with
EGFR. Multiple OFD1 bands are present in RCTE and MO6-G3
cell lysates and a subset of these OFD1 bands are co-precipitated
with EGFR. Colocalization of OFD1, polycystins, EGFR and flotillins to
the primary cilium of odontoblasts and renal epithelial
cells However, we show for the first time the localization of
all 5 proteins, including the membrane organizing flotillins, to
primary cilia in a human kidney cell line. The findings that ciliary
protein distributions are similar in renal epithelia and odontoblasts
suggest that these proteins may be similarly organized in both cell
types. To support the microscopy data, polarized RCTE cells were
deciliated using dibucaine-HCl and immunoblot analyses per-
formed on the ciliary enriched fraction. The ciliary marker alpha/
beta-tubulin was expressed in abundance within the collected
fraction, whereas the nuclear marker lamin B was absent. Given
that the nuclear membrane is often tightly coupled to cilia via a
centrosomal anchoring mechanism, the data support the ciliary
enrichment in the absence of nuclear contamination of the cell
fraction. PC1, PC2, and EGFR were all co-enriched within the
ciliary fraction of RCTE cells providing further evidence for the
presence of signaling proteins within primary cilia (Figure S2). Endogenous protein localizations were evaluated by immuno-
fluorescence staining using antibodies specifically directed against
OFD1, PC1, PC2, EGFR, and flotillin-1. In RCTE cells OFD1,
PC1, PC2, and EGFR localized along the length of the primary
cilium (Figure 3A–D). The ciliary localization of flotillin-2-GFP
prompted us to look for the flotillin-2 scaffolding partner, flotillin-
1. As expected, endogenous flotillin-1 also localized to primary
cilia in RCTE cells (Figure 3E). Similarly, we observed endoge-
nous OFD1, PC1, PC2, EGFR, and flotillin-1 protein localization
to primary cilia in odontoblast cells (Figure 3F–J). In RCTE cells, September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 3 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain Figure 3. Endogenous OFD1, PC1, PC2, EGFR and flotillin-1 localize to primary cilia of RCTE and MO6-G3 cells. Polarized RCTE and
MO6-G3 cells were fixed and stained using the indicated antibodies. OFD1 from Novus Biologicals (A, F), PC1 using pAb NM002 (B, G), PC2 using
AbCam pAb (C, H), EGFR using GeneTex pAb (D, I) and flotillin-1 using AbCam pAb (E, J) were found localized to 100% of primary cilia analyzed for
both cell types. Acetylated a-tubulin (Sigma-Aldrich mAb) labeling identifies cilia. Zeiss LSM510 confocal microscope images (636objective). Arrows
denote the protein of interest within a cilium. Representative results from at least 5 independent experiments. Secondary antibody only controls were
negative (not shown). Scale bar 10 mm. doi:10.1371/journal.pone.0106330.g003 Figure 3. Colocalization of OFD1, polycystins, EGFR and flotillins to
the primary cilium of odontoblasts and renal epithelial
cells Interestingly, the flotillin-2 bands were notably
upshifted in both the PC1 and EGFR immunoprecipitates Polycystins and OFD1 form protein complexes with EGFR
and flotillins in odontoblasts and renal epithelial cells Normal rabbit IgG was used as a negative control. In a reciprocal
experiment EGFR was immunoprecipitated using Santa Cruz pAb from RCTE and MO6-G3 cell lysates. Immunoprecipitated proteins were separated
by SDS-PAGE and probed for EGFR using GeneTex pAb (G), PC1 using NM002 pAb (H), PC2 using Santa Cruz pAb (I), OFD1 using Novus Biologicals
pAb (J) and flotillin-2 using Cell Signaling Rabbit mAb (K). Bar graph showing a densitometric quantification of EGFR co-immunoprecipitation probed
for EGFR (g), PC1 (h), PC2 (i), OFD1 (j), and flotillin-2 (k). Normal rabbit IgG was used as a negative control. Lysate lane inputs were 5% of
immunoprecipitations. 25 mg of total protein was loaded into each well. Arrows indicate the quantified band of interest in each immunoblot panel. Bar represents the mean 6 SD of at least three independent experiments. (*) p = 0.01 to 0.05, (**) p = 0.001 to 0.01, (***) p,0.001. doi:10.1371/journal.pone.0106330.g004 Polycystins and OFD1 form protein complexes with EGFR
and flotillins in odontoblasts and renal epithelial cells PC1, PC2, EGFR, OFD1, and flotillin-2 are part of a multimeric protein complex in RCTE and MO6-G3 cells. RCTE and MO6-G3
cells were grown 5 days post-confluency to allow for polarization and ciliogenesis. PC1 was immunoprecipitated using NM002 pAb and precipitated
proteins were separated by SDS-PAGE and probed for PC1 using NM005 pAb (A, B), PC2 using AbCam pAb (C), EGFR using GeneTex pAb (D), OFD1
using Novus Biologicals pAb (E) and flotillin-2 using Cell Signaling Rabbit mAb (F). Bar graph showing a densitometric quantification of PC1 co-
immunoprecipitation for PC1 (a, b), PC2 (c), EGFR (d), OFD1 (e), and flotillin-2 (f). Normal rabbit IgG was used as a negative control. In a reciprocal
experiment EGFR was immunoprecipitated using Santa Cruz pAb from RCTE and MO6-G3 cell lysates. Immunoprecipitated proteins were separated
by SDS-PAGE and probed for EGFR using GeneTex pAb (G), PC1 using NM002 pAb (H), PC2 using Santa Cruz pAb (I), OFD1 using Novus Biologicals
pAb (J) and flotillin-2 using Cell Signaling Rabbit mAb (K). Bar graph showing a densitometric quantification of EGFR co-immunoprecipitation probed
for EGFR (g), PC1 (h), PC2 (i), OFD1 (j), and flotillin-2 (k). Normal rabbit IgG was used as a negative control. Lysate lane inputs were 5% of
immunoprecipitations. 25 mg of total protein was loaded into each well. Arrows indicate the quantified band of interest in each immunoblot panel. Bar represents the mean 6 SD of at least three independent experiments. (*) p = 0.01 to 0.05, (**) p = 0.001 to 0.01, (***) p,0.001. doi:10.1371/journal.pone.0106330.g004 OFD1, and flotillin-2 are part of a multimeric protein complex in RCTE and MO6-G3 cells. RCTE and MO6-G
fl
ll
f
l
i
i
d ili
i
PC
i
i i
d
i
NM002
Ab
d
i i
d Figure 4. PC1, PC2, EGFR, OFD1, and flotillin-2 are part of a multimeric protein complex in RCTE and MO6-G3 cells. RCTE and MO6-G3
cells were grown 5 days post-confluency to allow for polarization and ciliogenesis. PC1 was immunoprecipitated using NM002 pAb and precipitated
proteins were separated by SDS-PAGE and probed for PC1 using NM005 pAb (A, B), PC2 using AbCam pAb (C), EGFR using GeneTex pAb (D), OFD1
using Novus Biologicals pAb (E) and flotillin-2 using Cell Signaling Rabbit mAb (F). Bar graph showing a densitometric quantification of PC1 co-
immunoprecipitation for PC1 (a, b), PC2 (c), EGFR (d), OFD1 (e), and flotillin-2 (f). Polycystins and OFD1 form protein complexes with EGFR
and flotillins in odontoblasts and renal epithelial cells To determine whether the ciliary proteins of interest are part of
a multimeric complex we performed immunoprecipitation exper-
iments with specific antibodies against PC1 and EGFR and scored
for co-precipitation of the proteins of interest. RCTE and
odontoblast cells were grown to five days post-confluency to allow
for polarization and ciliogenesis. PC1 was immunoprecipitated
from renal epithelial and odontoblast cell lysate (Figure 4A) and
shown to be in complex with PC2 as expected (Figure 4C). The
native 460 kDa PC1 contains a region with potential metallopro-
tease recognition sequences yielding 230–250 kDa C-terminal
fragments [6,28], which were also co-precipitated (Figure 4B). PC1, along with PC2, becomes tyrosine phosphorylated poten-
tially through the actions of EGFR [7,21,29]. To test if EGFR is
part of the polycystin complex, PC1 immunoprecipitates were September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 4 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain Figure 4. PC1, PC2, EGFR, OFD1, and flotillin-2 are part of a multimeric protein complex in RCTE and MO6-G3 cells. RCTE and MO6-G3
cells were grown 5 days post-confluency to allow for polarization and ciliogenesis. PC1 was immunoprecipitated using NM002 pAb and precipitated
proteins were separated by SDS-PAGE and probed for PC1 using NM005 pAb (A, B), PC2 using AbCam pAb (C), EGFR using GeneTex pAb (D), OFD1
using Novus Biologicals pAb (E) and flotillin-2 using Cell Signaling Rabbit mAb (F). Bar graph showing a densitometric quantification of PC1 co-
immunoprecipitation for PC1 (a, b), PC2 (c), EGFR (d), OFD1 (e), and flotillin-2 (f). Normal rabbit IgG was used as a negative control. In a reciprocal
experiment EGFR was immunoprecipitated using Santa Cruz pAb from RCTE and MO6-G3 cell lysates. Immunoprecipitated proteins were separated
by SDS-PAGE and probed for EGFR using GeneTex pAb (G), PC1 using NM002 pAb (H), PC2 using Santa Cruz pAb (I), OFD1 using Novus Biologicals
pAb (J) and flotillin-2 using Cell Signaling Rabbit mAb (K). Bar graph showing a densitometric quantification of EGFR co-immunoprecipitation probed
for EGFR (g) PC1 (h) PC2 (i) OFD1 (j) and flotillin-2 (k) Normal rabbit IgG was used as a negative control Lysate lane inputs were 5% of re 4. PC1, PC2, EGFR, OFD1, and flotillin-2 are part of a multimeric protein complex in RCTE and MO6-G3
d
fl
ll
f
l
i
i
d ili
i
PC
i
i i
d
i
NM Figure 4. September 2014 | Volume 9 | Issue 9 | e106330 Discussion The data presented here identify the presence of a ciliary
signaling complex that is conserved between cells of kidney tubules
and the oral cavity. We show that this complex consists of the
polycystins (PC1 and PC2), the OFD1 ciliopathy disease gene
product, the EGFR receptor tyrosine kinase and the membrane
raft organizing flotillin proteins. Moreover, we demonstrated that
when one of these proteins is mutant it changes the assembly or
stability of the entire ciliary signaling complex in the affected cell
type, suggestive of co-assembly into a common domain. Indeed,
the decreased expression of OFD1 in primary cilia when PC1 is
mutant offers a molecular basis for some of the common
pathologies of OFD and ADPKD. Quantification of these proximity-induced fluorescent puncta
showed an enrichment in the cilia (62%) as compared to the cell
body (38%) of fully polarized RCTE cells (Figure 5D). The pattern
in odontoblasts was reversed with a greater fraction of puncta in
the cell body (90%), yet retaining a quantifiable fraction in cilia
(10%) (Figure 5D). Both cell types had similar numbers of ciliated
cells (RCTE 82%; MO6-G3 93%), although the average cilia
length was different (RCTE 6.10 mm; MO6-G3 0.49 mm). Both
RCTE and MO6-G3 had a similar percentage of primary cilia
containing puncta: RCTE 53.4%; MO6-G3 62.6% (Figure 5E). Taken in conjunction with previous data showing PC1 and PC2
interact in primary cilia [11] and PC2 forms a ciliary signaling
complex with EGFR [7], these data indicate that PC1 and PC2
likely form a three way complex with EGFR in cilia. There are two molecular explanations for the finding that
mutant PC1 results in reductions of OFD1 and other key signaling
proteins in primary cilia that immediately come to mind. First,
PC1 and OFD1 may have interdependent roles in the ciliary
transport and/or targeting of the proteins in the complex. Second,
PC1 may be essential for stabilizing the complex within the ciliary
membrane. Based on the fact that the levels of OFD1, PC2 and
flotillins were more significantly affected than EGFR, it is possible
that OFD1, PC2 and the flotillins are highly dependent on
functional PC1 for ciliary delivery and stabilization whereas
EGFR may arrive at the primary cilium independently of PC1 but
relies on functional PC1 for integration into ciliary signaling
protein assemblies. Mutant PC1 results in decreased ciliary localizations of
PC2, OFD1, EGFR and flotillin-1 Given the shared disease pathologies caused by mutant OFD1
and PKD genes, we tested if the expression of a mutant
component of our defined ciliary signaling complex would in turn
affect the formation and/or stability of these specialized complex-
es. In cells from patients with ADPKD, expression of mutant PC1
often results in its absence from primary cilia [31]. Immunolocal-
ization studies were therefore performed on cells from human
ADPKD patients with expression of mutant PC1 (Q4004X) [32]. In these cells, there was reduced expression not only of PC1, but
also of the associated PC2, EGFR, and flotillin-1 proteins along
the shaft of primary cilia (Figure 6A–H). Quantification of
multiple data sets verified statistically significant reductions (p,
0.0001 for PC1, PC2, and flotillin-2; p = 0.0105 for EGFR) in all
components of the complex (Figure 6I–L). A small residual pool of
EGFR was sometimes detectable at the ciliary base. OFD1
localization to primary cilia was also significantly decreased in
PKD cells as compared to RCTE cells (p,0.0001; data not
shown). As further confirmation, we also analyzed OFD1
localization
in
primary
human
normal
kidney
and
PKD
(46M06) cells. Whilst OFD1 localized to primary cilia of primary
normal proximal tubular epithelial cells (RPTEC) cells, it was
significantly decreased (P,0.0036) in primary cilia of 46M06 cells
(Figure 7B and Figure S3). p
p
OFD1 functions at the base of cilia to control ciliogenesis
through interactions with intraflagellar transport and possibly
vesicle docking machineries. The functional importance of OFD1
in recruitment of IFT components is reflected in that the loss of
either OFD1 or IFT88 results in similar phenotypes [4,8–10]. The
coiled-coil domains 2 through 4 of OFD1 interact with IFT88 and
recruit IFT particles to the distal appendages of centriolar
satellites, which are necessary for initiation of ciliogenesis [12]. New studies indicate autophagy is subsequently required for the
selective removal of OFD1 from centriolar satellites to allow
ciliogenesis to proceed [34]. Distal appendages are also implicated
in
the
trafficking
and
docking
of
ciliary
vesicles
carrying
membrane constituents [35–38]. If ciliary vesicle docking at the
distal appendages requires an interaction between PC1 and
OFD1, it is conceivable that the ciliary localizations of multiple
components of a preassembled complex would be similarly
affected. Discussion During cell division OFD1 localizes to
centrosomes, and later to basal bodies and the shaft of primary
cilia in polarized differentiated cells. Thus, OFD1 like the
polycystins may have multiple functions that are tied to different
locations and protein-protein interactions [2,3,33]. Polycystin-1 and EGFR form a complex within primary
cilia suggesting posttranslational modification within these complexes. These data indicate that key ciliary signaling proteins – PC1, PC2,
EGFR and OFD1 – may well reside in a complex with membrane
organizing flotillin proteins in renal and dental cell types. We utilized an antibody-based proximity ligation assay to
specifically evaluate if the protein complexes we identified by co-
immunoprecipitation were resident in cilia [30]. Using antibodies
directed against the extracellular domains of PC1 and EGFR, PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org September 2014 | Volume 9 | Issue 9 | e106330 5 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain these two proteins were found to interact specifically within the
primary cilium of odontoblasts (Figure 5A) and fully polarized
renal epithelial cells (Figure 5B and Movie S1). In both cell types
the fluorescent signal was notably concentrated in puncta,
suggestive of specialized domains wherein the proteins are
enriched. In the relatively short cilia of odontoblasts, a less
concentrated signal was also seen along the length of the shaft. Puncta associated with the cell body were largely intracellular with
a small fraction at or near the basolateral membrane (Figure 5A–B
and data not shown). Experiments performed without primary
antibody against PC1 yielded no fluorescent signal verifying the
specificity of the PC1-EGFR reaction (Figure 5C). epithelia and odontoblasts, these studies were complicated by the
fact that OFD1 is necessary for ciliogenesis and therefore siRNA
knockdown must be performed after cells are fully polarized and
have cilia. Under these conditions, a population of OFD1
remained resident in primary cilia even after 48 h of siRNA-
mediated knockdown precluding accurate interpretation. The
composite data demonstrate that the proteins are organized into a
functional complex that is disrupted when polycystin-1 is mutant
or absent. September 2014 | Volume 9 | Issue 9 | e106330 Mutant PC1 results in decreased ciliary localizations of
PC2, OFD1, EGFR and flotillin-1 The
C2
calcium-dependent
domain
containing
3
(C2cd3) protein is another essential regulator of primary cilium
biogenesis that localizes to distal appendages, though how C2cd3
is integrated with OFD1 regulated ciliogenesis remains to be
dissected [39]. Thus, the diminished OFD1 localization to cilia
when PC1 is mutant may adversely affect both ciliogenesis and Biochemical deciliation experiments were used to independently
verify that all components of the identified ciliary complex were
decreased in an interdependent manner when polycystin-1 is
mutant. Polarized
RCTE
and
PKD
(Q4004X)
cells
were
deciliated using Dibucaine-HCL and isolated ciliary fractions
analyzed by immunoblot. Quantification of OFD1, EGFR, and
flotillin-1 protein levels relative to a/b-tubulin levels confirmed
that all components were reduced in the ciliary fraction of the
PKD cells as compared to RCTE cells (Figure 8). These data
further substantiate the conclusion that the OFD1-EGFR-flotillin
complex is lost from cilia in cells with mutant PC1. While siRNA-
mediated ablation of OFD1 and PC1 were attempted in renal September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 6 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain gure 5. PC1 and EGFR interact in the primary cilium of MO6-G3 and RCTE cells. Polarized MO6-G3 (A) and RCTE (B) cells were gro
verslips 5 days post-confluency. Cells were incubated with antibodies against PC1 (Santa Cruz mAb) and EGFR (GeneTex pAb), followed by D
A Probes to identify points of PC1-EGFR interaction. Cilia identified by acetylated a-tubulin (Sigma-Aldrich mAb). Identical experiments perf
thout PC1 antibody are negative for fluorescent signal (C). Top panel shows XY plane, bottom panel shows XZ plane. Images from co
croscope (636 objective). Scale bar is 5 mm. Quantification of puncta in 3 representative images (11–29 cells/field) from 2 indepe
periments (D). Quantification of cilia containing PLA puncta (E). Puncta in cilia were counted based on colocalization with acetylated a-tu
atistical evaluation based on two-tailed t-test. p = 0.0194. i:10.1371/journal.pone.0106330.g005 Figure 5. PC1 and EGFR interact in the primary cilium of MO6-G3 and RCTE cells. Polarized MO6-G3 (A) and RCTE (B) cells were grown on
coverslips 5 days post-confluency. Cells were incubated with antibodies against PC1 (Santa Cruz mAb) and EGFR (GeneTex pAb), followed by Duolink
PLA Probes to identify points of PC1-EGFR interaction. Cilia identified by acetylated a-tubulin (Sigma-Aldrich mAb). Identical experiments performed
without PC1 antibody are negative for fluorescent signal (C). Top panel shows XY plane, bottom panel shows XZ plane. Mutant PC1 results in decreased ciliary localizations of
PC2, OFD1, EGFR and flotillin-1 Staining intensities for each protein
were quantified within the respective ciliary volume mask. Statistical evaluation based on two-tailed t-test. (*) p = 0.0105, (***) p,0.0001. doi:10.1371/journal.pone.0106330.g006 ciliary function based on the dual roles OFD1 has in regulating
intraflagellar transport components and membrane vesicles. important to distinguish the role(s) of polycystin-1 in complex
transport and/or stabilization. Our demonstration that the flotillins localize to primary cilia of
renal epithelia and odontoblasts and flotillin-2 interacts with the
polycystins, EGFR and OFD1 in both cell types is extremely
exciting for several reasons. The presence of the flotillins in cilia
supports previous speculation regarding commonalities between
basolateral membrane, immune synapse and cilia membrane
organization [6,42,43]. Flotillins are important in the formation of
ordered membrane domains in hematopoietic cells where they are
critical for cell polarization and lymphoid immune synapse
formation [18,44,45]. In polarized Madin-Darby canine kidney
cells, both flotillin-1 and -2 have a non-polarized distribution. Additionally, the flotillins were suggested to have a role in
polarization that is restricted to hematopoietic cells [44]. However,
as we showed in human renal epithelial cells flotillins bind
cholesterol and organize the polycystins, E-cadherin, tyrosine The fact that loss of ciliary PC1 led to decreased OFD1
localization to the ciliary shaft suggests that polycystins might also
serve to promote stable association of OFD1 with the ciliary
membrane. OFD1 contains five coiled coil domains [5]; and it is
possible that these domains have roles beyond enabling the distal
appendage localization of OFD1. For example, interactions with
coiled coil domains in the polycystins might mediate delivery to
and stable localization in the ciliary shaft [3]. Support for this
speculation is provided by previous reports showing PC1 and PC2
also contain CC domains that mediate their interaction, and are
important for their ciliary localization [40,41]. Further character-
izing the molecular basis for the interaction between membrane
proteins (such as polycystins) and soluble proteins (such as OFD1)
at the distal appendages and within the ciliary shaft will be Figure 7. OFD1 ciliary localization is altered in human primary cell lines. Human primary renal proximal tubule epithelial cells (RPTEC) and
PKD (44M06) cells were grown on glass coverslips 5 days post confluency to allow for ciliogenesis, fixed and stained using an antibody directed
against OFD1 (Santa Cruz pAb). OFD1 localizes to primary cilia in RPTEC cells (A) but is diminished in primary cilia PKD 46M06 cells (B). Mutant PC1 results in decreased ciliary localizations of
PC2, OFD1, EGFR and flotillin-1 Images from confocal
microscope (636 objective). Scale bar is 5 mm. Quantification of puncta in 3 representative images (11–29 cells/field) from 2 independent
experiments (D). Quantification of cilia containing PLA puncta (E). Puncta in cilia were counted based on colocalization with acetylated a-tubulin. Statistical evaluation based on two-tailed t-test. p = 0.0194. doi:10.1371/journal.pone.0106330.g005 September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 7 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain Figure 6. Key ciliary signaling proteins are significantly reduced in primary cilia of PKD cells. Human RCTE and PKD Q4004X cells (with
expression of mutant PC1) were grown on coverslips 5 days post-confluency to permit ciliogenesis. Cells were fixed and stained using antibodies
directed against indicated proteins: PC1 (NM002 pAb); PC2 (AbCam pAb); EGFR (GeneTex pAb); and flotillin-1 (AbCam pAb). PC1 (A), PC2 (C), EGFR (E)
and flotillin-1(G) are present in primary cilia of RCTE cells. PC1 (B), PC2 (D), EGFR (F) and flotillin-1 (H) were lacking in primary cilia of PKD Q4004X cells. Figure 6. Key ciliary signaling proteins are significantly reduced in primary cilia of PKD cells. Human RCTE and PKD Q4004X cells (with
expression of mutant PC1) were grown on coverslips 5 days post-confluency to permit ciliogenesis. Cells were fixed and stained using antibodies
directed against indicated proteins: PC1 (NM002 pAb); PC2 (AbCam pAb); EGFR (GeneTex pAb); and flotillin-1 (AbCam pAb). PC1 (A), PC2 (C), EGFR (E)
and flotillin-1(G) are present in primary cilia of RCTE cells. PC1 (B), PC2 (D), EGFR (F) and flotillin-1 (H) were lacking in primary cilia of PKD Q4004X cells. September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 8 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain Acetylated a-tubulin (Sigma-Aldrich mAb) staining identifies cilia. Arrows denote a small residual pool of EGFR detectable at the ciliary base of PKD
cells. Zeiss LSM510 confocal microscope images (636 objective). Representative results from at least 5 independent experiments. Comparative
images are from a single experiment and taken under identical settings. Arrows denote cilia. Scale bar 10 mm. Quantification shows individual ciliary
protein intensities normalized to the respective ciliary volume (I–L). Each protein was quantified in 30–100 cilia for each cell type. z-stack images were
imported into SlideBook and a volume mask for each cilium was created based on acetylated a-tubulin staining. Mutant PC1 results in decreased ciliary localizations of
PC2, OFD1, EGFR and flotillin-1 Acetylated a-
tubulin (Sigma-Aldrich mAb) staining identifies cilia. Zeiss LSM510 confocal microscope images (636 objective). Representative results from 3
independent experiments. Comparative images are from a single experiment and taken under identical settings. Arrows denote cilia. Scale bar 10 mm. Quantification shows OFD1 staining intensities normalized to ciliary volumes, performed as detailed in methods and Figure 6 (C). z-stack images were
imported into SlideBook and a mask for the cilia was created based on acetylated a-tubulin staining. Statistical evaluation based on two-tailed t-test. (**) p = 0.0036. doi:10.1371/journal.pone.0106330.g007 Figure 7. OFD1 ciliary localization is altered in human primary cell lines. Human primary renal proximal tubule epithelial cells (RPTEC) and
PKD (44M06) cells were grown on glass coverslips 5 days post confluency to allow for ciliogenesis, fixed and stained using an antibody directed
against OFD1 (Santa Cruz pAb). OFD1 localizes to primary cilia in RPTEC cells (A) but is diminished in primary cilia PKD 46M06 cells (B). Acetylated a-
tubulin (Sigma-Aldrich mAb) staining identifies cilia. Zeiss LSM510 confocal microscope images (636 objective). Representative results from 3
independent experiments. Comparative images are from a single experiment and taken under identical settings. Arrows denote cilia. Scale bar 10 mm. Quantification shows OFD1 staining intensities normalized to ciliary volumes, performed as detailed in methods and Figure 6 (C). z-stack images were
imported into SlideBook and a mask for the cilia was created based on acetylated a-tubulin staining. Statistical evaluation based on two-tailed t-test. (**) p = 0.0036. doi:10.1371/journal.pone.0106330.g007 September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 9 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain Figure 8. Expression of PC1, OFD1, EGFR, and Flotillin-1 is decreased in ciliary fractions of PKD cells compared to RCTE cells. RCTE
cells and PKD Q4004X cells were grown 6 days post-confluency to allow for cell polarization and ciliogenesis and treated with 1.5 mM Dibucaine-HCl
for 5 minutes to reduce cell loss and induce shedding of primary cilia. Cilia were collected by fractionation and the ciliary fraction was probed. PC1
(MN032 pAb), OFD1 (Novus Biologicals pAb), EGFR (GeneTex pAb), and flotillin-1 (BD Transduction mAb) were expressed in the ciliary fraction of
deciliated RCTE cells but not PKD cells. a/b-tubulin (Cell Signaling pAb) was used as a marker of primary cilia. The nuclear marker lamin B (Santa Cruz
pAb) was not present in the ciliary fraction (A). Quantification of 3 independent samples (B). Mutant PC1 results in decreased ciliary localizations of
PC2, OFD1, EGFR and flotillin-1 Mean values were found to be statistically different (p,
0.0001) using 1-way ANOVA. doi:10.1371/journal.pone.0106330.g008 Figure 8. Expression of PC1, OFD1, EGFR, and Flotillin-1 is decreased in ciliary fractions of PKD cells compared to RCTE cells. RCTE
cells and PKD Q4004X cells were grown 6 days post-confluency to allow for cell polarization and ciliogenesis and treated with 1.5 mM Dibucaine-HCl
for 5 minutes to reduce cell loss and induce shedding of primary cilia. Cilia were collected by fractionation and the ciliary fraction was probed. PC1
(MN032 pAb), OFD1 (Novus Biologicals pAb), EGFR (GeneTex pAb), and flotillin-1 (BD Transduction mAb) were expressed in the ciliary fraction of
deciliated RCTE cells but not PKD cells. a/b-tubulin (Cell Signaling pAb) was used as a marker of primary cilia. The nuclear marker lamin B (Santa Cruz
pAb) was not present in the ciliary fraction (A). Quantification of 3 independent samples (B). Mean values were found to be statistically different (p,
0.0001) using 1-way ANOVA. doi:10.1371/journal.pone.0106330.g008 kinases and phosphatases at the lateral membrane [20]. As shown
in this work, flotillins may also organize the polycystins, EGFR
and OFD1 in primary cilia into specialized domains. The fact that
ciliary EGFR levels were was less compromised than the other
components is interesting and suggests that the tyrosine kinase
may move in and out of cholesterol based raft domains organized
by flotillins and that this is likely to have functional importance in
regulating ciliary signaling. complex is suggestive of post-translational modifications that may
be important in their regulation of specific signaling events. kinases and phosphatases at the lateral membrane [20]. As shown
in this work, flotillins may also organize the polycystins, EGFR
and OFD1 in primary cilia into specialized domains. The fact that
ciliary EGFR levels were was less compromised than the other
components is interesting and suggests that the tyrosine kinase
may move in and out of cholesterol based raft domains organized
by flotillins and that this is likely to have functional importance in
regulating ciliary signaling. be important in their regulation of specific signaling events. In addition to EGFR, there are growing numbers of receptor
tyrosine kinases identified in primary cilia of epithelia, neuronal
cells and fibroblasts [52]. Of particular interest are the expression
of RON kinase in motile cilia of airway epithelia and the
documented cooperation between EGFR, RON and Met [53–55]. Mutant PC1 results in decreased ciliary localizations of
PC2, OFD1, EGFR and flotillin-1 Emerging data from studies in human embryonic kidney cells and
in breast, lung and colorectal cancer cells indicate that the RON
kinase interacts with EGFR and may contribute to direct
transcriptional regulation [56,57]. The connections between
RON, Met and EGFR are intriguing because both Met and
EGFR are pivotal in cystogenesis [58–60]. Counterbalancing the
activities
of receptor
tyrosine
kinases
are receptor
protein
phosphatases, with members of the LAR family shown to be
present in cilia of renal epithelia [21]. Additional regulation may
occur at the level of proteasomal degradation at the ciliary base
[57]. Therefore, the present studies lay the groundwork for how
receptor tyrosine kinases may be organized in cilia and warrant an
expanded characterization to determine how receptor protein
kinases and phosphatases along with other signaling proteins may
interact with or be co-assembled
with the
flotillin-EGFR-
polycystin-OFD1 protein complexes characterized here. Flotillins play a major role in the fidelity of cell signaling and
EGFR
function [46–48]. Knockdown of flotillin-2 disrupts
localization and phosphorylation of EGFR and activation of
downstream MAPK signaling components [22]. However, in
breast cancer cells with mutant PIK3CA, a well-known down-
stream target of EGFR signaling, knockdown of flotillin-1 causes
an
upregulation
of
EGFR
and
hyperactivation
of MAPK
signaling. These data indicate that the flotillins have dual roles
in enabling receptor tyrosine kinase activation and downstream
signaling, as well as in restraining signaling components such as
PIK3CA in an inactive state [49]. The importance of ciliary
organization of EGFR signaling is further reinforced by recent
studies showing that when cilia are ablated, EGFR mediated
activation of apical calcium channels is increased 64-fold [50]. Similarly, in inner medullary collecting duct cells (IMCD-3)
primary cilia were found to restrain interaction and cross-talk
between G-protein coupled receptors, which are also known to
cross-talk with receptor tyrosine kinases [51,52]. Hence, it appears
likely that the flotillins have an important role in the control of
ciliary signaling that extends beyond a purely scaffolding function. Notably, the upshift in molecular weight of flotillin proteins in the In sum, our findings of a protein complex containing the
polycystins, flotillins, EGFR and OFD1 in cilia of both renal and
dental cells provides evidence for the existence of ciliary signaling
microdomains. September 2014 | Volume 9 | Issue 9 | e106330 Cells and Reagents Dental pulp-derived odontoblasts were used as representative
neural crest derived cells from the oral cavity. The previously
characterized MO6-G3 immortalized mouse dental pulp-derived
odontoblast cell line was used as described [61]. MO6-G3 cells
were grown in alpha-MEM supplemented with 10% FCS, 100
units/ml penicillin and streptomycin, 50 mg/ml ascorbic acid at
33uC in a humidified atmosphere of 95% air and 5% CO2, with
media changes every two days. The previously characterized PKD
Q4004X PKD cells [32] were a generous gift from Dr. Robert
Bacallao (Indiana University School of Medicine, Indianapolis,
IN). Primary human cell line 46M06 was obtained from renal cysts
of an ADPKD kidney. Human cell lines from renal cortical
tubular epithelia (RCTE) were immortalized and cultured as
previously described (Nauli, 2006, Ward 2011). Renal proximal
tubule cells (RPTEC) were purchased from Lonza (Walkersville,
MD) and cultured using RCTE conditions. Cell culture reagents
were purchased from Invitrogen/Gibco (Carlsbad, CA). Primary
antibodies were purchased from the following vendors: mouse
mAb directed against acetylated a-tubulin (Sigma-Aldrich T7451,
St. Louis, MO); rabbit pAb directed against a/ß-tubulin (Cell
Signaling Technology #2148, Danvers, MA); mouse mAb anti-
actin, clone C4 (Millipore #MAB1501, Temecula, CA); goat pAb
directed against Lamin B (Santa Cruz sc-6217, Santa Cruz, CA);
rabbit pAb directed against PC1 NM005 raised by immunizing
rabbits with a distal carboxy-terminal fragment of polycystin-1
(amino acids 4070-4302) (Roitbak, 2004); rabbit anti-PC1 NM002
and NM032 raised in rabbits immunized with a peptide
corresponding to aa 3633–3645 of human PC1, and with an N-
terminal cysteine (CKRLHPDEDDTLVE; Suzanna Horvath,
California Institute of Technology, Pasadena, CA). The peptide
was conjugated to keyhole limpet hemocyanin using benzoqui-
none and was used to immunize rabbits NM002 and NM032
(Covance, Denver, PA). For affinity purification, the same peptide
was conjugated to Sulfalink gel (#20401; Pierce) according to the
manufacturer’s instructions (1 mg peptide/1 ml resin bed volume). Ten milliliters NM002 serum was incubated with 5 ml resin and
washed to remove unbound immunoglobulin and serum proteins. Bound immunoglobulin was eluted with 0.1 M glycine (pH 2.5),
immediately neutralized with Tris base, and used for immuno-
blotting and immunostaining experiments at a concentration of
10 mg/ml. Cells and Reagents (Ward, 2011); mouse mAb anti-polycystin-1 (Santa
Cruz Biotechnology sc-130554, Santa Cruz, CA); rabbit pAb anti-
polycystin-2 (AbCam ab78622, Cambridge, MA); rabbit pAb anti-
polycystin-2 (Santa Cruz Biotechnology sc25749, Santa Cruz,
CA); rabbit pAb anti-EGFR (GeneTex GTX121919, Irvine, CA);
rabbit pAb anti-EGFR (Santa Cruz Biotechnology sc-03, Santa
Cruz, CA); rabbit pAb anti-ErbB2 (US Biological E3451-27,
Swampscott, MA); rabbit mAb anti-ErbB2 (Epitomics #2064-1,
Burlingame, CA); rabbit pAb anti-OFD1 (Novus Biologicals
NBP1-89355, Littleton, CO); goat pAb anti-OFD1 (Santa Cruz
Biotechnology sc-168837, Santa Cruz, CA); rabbit pAb anti-
Flotillin-1 (AbCam ab-50671, Cambridge, MA); mouse mAb anti-
Flotillin-1 (BD Transduction Laboratories 610820, San Jose, CA);
rabbit mAb anti-Flotillin-2 (Cell Signaling Technology #3436,
Boston, MA); mouse mAb anti-Flotillin-2 (BD Transduction
Laboratories 610383, San Jose, CA); ECL Anti-Rabbit IgG-
HRP (GE Healthcare NA934V, Little Chalfont, Buckingham-
shire); ECL Anti Mouse IgG HRP (GE Healthcare NA931V Mutant PC1 results in decreased ciliary localizations of
PC2, OFD1, EGFR and flotillin-1 The requirement for the integrity of the complex
provides a plausible molecular rationale for the commonalities
between craniofacial disorders and cystic kidney disease and September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain informs strategies for the development of potential therapeutic
interventions that stabilize protein complexes. Little Chalfont, Buckinghamshire); donkey anti-goat IgG-HRP
(Santa Cruz sc-2020, Santa Cruz, CA); Rabbit TrueBlot ULTRA:
Anti-Rabbit Ig HRP (eBioscience 18–8816, San Diego, CA);
Mouse TrueBlot ULTRA: Anti-Mouse Ig HRP (eBioscience 18–
8817, San Diego, CA); mouse IgG2a, k mouse isotype control
(Sigma-Aldrich M5409, Saint Louis, MO) normal rabbit IgG
isotype control (Santa Cruz Biotechnology sc2027, Santa Cruz,
CA). The following fluorophore-conjugated secondary antibodies
(Alexa Fluor dyes) were used for immunofluorescence assays:
Alexa Fluor 488 Donkey Anti-Mouse IgG (Invitrogen A-21202,
Grand Island, NY); Alexa Fluor 488 Donkey Anti-Rabbit IgG
(Invitrogen A-21206, Grand Island, NY); Alexa Fluor 555 Donkey
Anti-Mouse IgG (Invitrogen A-31570, Grand Island, NY); Alexa
Fluor 488 Donkey Anti-Goat IgG (Invitrogen A-11055, Grand
Island, NY); Alexa Fluor 555 Donkey Anti-Rabbit IgG (Invitrogen
A-31572, Grand Island, NY); Alexa Fluor 647 Donkey Anti-
Mouse IgG (Invitrogen A-31571, Grand Island, NY); Alexa Fluor
647 Donkey Anti-Rabbit IgG (Invitrogen A-31573, Grand Island,
NY); Alexa Fluor 647 Donkey Anti-Goat IgG (Invitrogen A-
21082, Grand Island, NY). September 2014 | Volume 9 | Issue 9 | e106330 Immunofluorescence Staining For ciliary immunolocalization experiments, cells were grown
on glass coverslips or 0.4 mm filter supports (Falcon-BD 353090,
Franklin Lakes, NJ) for 4–6 days post-confluence and fixed with
3% paraformaldehyde (PFA) and processed for immunostaining as
previously described [6]. Briefly, cells were permeabilized using
0.1% Triton-X 100 in 0.2% cold fish gelatin (blocking agent), and
primary and secondary antibody incubations were performed in a
humidified chamber at 37uC. Cells were labeled with antibodies
directed against OFD1, EGFR, ErbB2, PC1, PC2, or flotillin-1. Cells were co-labeled with anti-acetylated a-tubulin or anti-a/b-
tubulin to identified primary cilia. Controls for specificity and
auto-fluorescence included staining with secondary antibodies
alone. Ciliary colocalization of proteins with acetylated a-tubulin
was defined as exhibiting colocalization of the two signals across at
least two pixels within the cilium as imaged in the confocal Z-stack
data. Confocal immunofluorescence images were collected using a
Zeiss LSM510 or Zeiss LSM 510-META laser-scanning confocal
microscope (Carl Zeiss, Thornwood, NY) with 40x, 1.3 numerical
aperture (NA) or 63x, 1.4 NA oil immersion objectives. LSM 510
Image Acquisition software (Carl Zeiss) was used to acquire
images. Confocal Z-stacks were processed with the Zeiss Image
Browser or Voxx2 (provided freely for noncommercial use by the
Indiana Center for Biological Microscopy, Indianapolis, IN, www. nephrology.iupui.edu/imaging/voxx/index.htm), and assembled
using Adobe Photoshop and Illustrator (Adobe, San Jose, CA). Quantification was performed by importing z-stack images (all
taken at identical settings) into SlideBook software (Intelligent
Imaging) and creating a ciliary mask based on acetylated a-tubulin
staining. Ciliary marker (OFD1, PC1, PC2, flotillin-1 and EGFR)
staining intensities within the ciliary mask were quantified and
normalized based on ciliary volume. At least 30–100 cilia were
quantified from multiple experiments. Movies were generated using the movie maker feature in Voxx2
software, and converted to .avi and .mpg file formats using
QuickTime Player (Apple, Cupertino, CA) and TMPGEnc
(Pegasys, Tokyo, Japan), respectively. OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain frame at the N-terminus to pEGFP-N1 vector [3] was provided by
Dr. Andrew Fry. Cells were plated at confluency on filter supports
and cultured as described above 4–6 d post-confluency to ensure
ciliogenesis. Transfections were performed by electroporation
(iPoration-Primax Biosceinces, Menio Park, CA) according to the
manufacturer’s instructions. The iPorator (Primax Biosciences,
Inc.) yields 50–60% transfection efficiency in both cell types
(Figure S4). Flotillin-2-GFP and GFP-OFD1 transfection efficien-
cy of ,40% was consistently observed. Cells were fixed with 3%
PFA 16 h post-transfection, and viewed directly or processed for
co-immunostaining. frame at the N-terminus to pEGFP-N1 vector [3] was provided by
Dr. Andrew Fry. Cells were plated at confluency on filter supports
and cultured as described above 4–6 d post-confluency to ensure
ciliogenesis. Transfections were performed by electroporation
(iPoration-Primax Biosceinces, Menio Park, CA) according to the
manufacturer’s instructions. The iPorator (Primax Biosciences,
Inc.) yields 50–60% transfection efficiency in both cell types
(Figure S4). Flotillin-2-GFP and GFP-OFD1 transfection efficien-
cy of ,40% was consistently observed. Cells were fixed with 3%
PFA 16 h post-transfection, and viewed directly or processed for
co-immunostaining. Proximity Ligation Assay (PLA) PCR-based visualization was performed using Duolink II
Rabbit/Mouse Red Kit (Olink Bioscience #92101) following
manufacturer’s instructions. Primary antibodies used were: rabbit
anti-EGFR (GeneTex, Irvine, CA) and anti-polycystin-1 (Santa
Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitations Cells were grown 3–5 days post-confluency to ensure cilial
formation. Cells were washed in PBS and lysed on ice in lysis
buffer (0.5% NP40, 10 mM Tris-HCL pH 7.4, 150 mM NaCl,
5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 60 mM n-Octyl-b-
D-glucopyranosid (AppliChem A1010, Saint Luios, MO), supple-
mented
with
protease
inhibitor
cocktail
(Calbiochem/EMD
Chemicals, Gibbstown, NJ). Lysates were precleared by centrifu-
gation at 10,000 rpm in an Eppendorf microcentrifuge for
10 minutes and pretreated with protein G Sepharose or protein
A Sepharose (Amersham Biosciences/GE Healthcare, Piscataway,
NJ); 500 mg of total protein was used for immunoprecipitations. 500 mg cell lysate/40 mg protein A Sepharose or protein G
Sepharose at 4uC for 3 hours on a rotator. Precleared lystate
supernatant was transferred to a clean microfuge tube and
incubated with 1–3 mg of antibody directed against specified
protein at 4uC for 2 hours with gentle rotation. Antibody
complexes were recovered by incubation with Protein G or
Protein A Sepharose beads at 4uC overnight. The immunopre-
cipitates were washed three times in lysis buffer and supplemented
with SDS-PAGE loading buffer, heated for 5 minutes at 94uC,
and resolved by SDS gel electrophoresis. Figure S2
Deciliation of RCTE cells. Polarized renal
epithelial cells were treated with 5 mM Dibucaine-HCl for
15 minutes causing the cells to shed their primary cilia. Cilia
were collected by fractionation and the ciliary fraction was probed. PC1 (NM032 pAb), PC2 (Santa Cruz pAb), and EGFR (Santa
Cruz pAb) were expressed in the ciliary fraction of deciliated
RCTE cells. The nuclear marker lamin B (Santa Cruz pAb) was
not present in the ciliary fraction. Representative results from 4
independent experiments. a/b-tubulin was used as a marker of
primary cilia. (TIF) Supporting Information Figure S1
Two Protein Co-Localization in Primary Cilia
of RCTE. Flotillin-2 was transiently transfected into polarized
RCTE and cells and then fixed and stained using the AbCam pAb
against PC2 (A) or the GeneTex pAb against EGFR (B), Polarized
RCTE cells were fixed and stained using the GeneTex pAb
against EGFR and NM002 pAb against PC1 (C). Acetylated a-
tubulin
(Sigma-Aldrich
mAb)
labeling
identifies
cilia. Zeiss
LSM510 confocal microscope images (636 objective). Arrows
denote the protein of interest within a cilium. Representative
results from at least 3 independent experiments. Secondary
antibody only controls were negative (not shown). Scale bar
10 mm. (TIF) Statistical Analysis For biochemical assays, means and standard errors were used to
summarize the data from each experiment. For immunofluores-
cence experiments a minimum of 30–100 cells/condition were
counted. All experiments were repeated at least three times. For
immunoprecipitations comparisons across groups were made using
One-way analysis of variance (ANOVA) to determine whether the
group
means
were
significantly
different. When
significant
differences in group means are found, post-hoc multiple compar-
ison tests (e.g., Tukey’s test) were used to determine which groups
are significantly different from one another. For protein expression
analyses and immunofluorescence quantifications differences were
analyzed by unpaired, two-tailed Student’s t test. GraphPad Prism
version 5.0 was used to perform statistical analyses. Deciliation Assay Figure S3
OFD1 Reduced in Primary Cilia of PKD
46M06 Cells. Polarized 46M06 PKD cells were fixed and
stained
using
an
antibody
directed
against
OFD1
(Novus
Biologicals pAb). Acetylated a-tubulin (Sigma-Aldrich mAb)
labeling identifies cilia. 636 objective with no zoom (A), 636
zoomed in image of panel A cilia marked with arrows (B), 636
zoomed in images of separate fields of view (C–D). Zeiss LSM510
confocal microscope images. Representative results from at least 3
independent experiments. Secondary antibody only controls were
negative (not shown). Scale bar 10 mm. (TIF) y
Renal epithelial cells were deciliated using 1.5 mM dibucaine-
HCL for 5 minutes or 5 mM dibucaine-HCL in culture media for
5 minutes or 15 minutes, both of which rapidly and selectively
caused the intact shedding of primary cilia from the cell surface as
was observed previously for IMCD3 cells [62,63]. Dibucaine
treatment conditions were optimized by testing different concen-
trations and treatment times with the goal of promoting cilial
release without cell detachment. A stock solution of dibucaine HCl
(25 mM) was diluted to 1.5 mM or 5 mM in Tyrodes balanced
salt solution. RCTE and MO6-G3 cells were grown on 60 mM
collagen-1 coated cell culture dishes for 5–6 days post-confluency
to allow for ciliogenesis. Media was aspirated and cells were
washed three times in PBS+. Dibucaine was then added for 5–
15 minutes with agitation causing deciliation. Tyrodes/dibucaine-
HCl solution containing the cilia were then collected and
centrifuged at 850 gmax for 10 minutes to collect cell bodies. The supernatant containing the ciliary fraction was collected and
cilia
were
harvested
by
centrifugation
at
28,000 gmax
for
30 minutes. Supernatant was removed and pellet containing
ciliary fraction was resuspended using 30 ml of lysis buffer and
run on a SDS-PAGE gel. Figure S4
iPoration yields high transfection efficiencies
for plasmid expression vectors and siRNA in post-
confluent, polarized MO6-G3 and RCTE cells. MO6-G3
odontoblasts are an inherently more difficult cell line to transfect. This problem is compounded further by the necessity to transfect
these cells after they are 100% confluent and have a completely
polarized phenotype with very tight cell-cell contacts. With
iPoration transfection efficiencies were 60–70% for FITC-labeled
siRNA and 40–60% for flotillin-2-GFP in both MO6-G3 and
RCTE cell types. As a comparison, traditional lipofection using Figure S4
iPoration yields high transfection efficiencies
for plasmid expression vectors and siRNA in post-
confluent, polarized MO6-G3 and RCTE cells. Transient transfections with flotillin-2-GFP or GFP-OFD1 Flotillin-2–EGFP (where EGFP stands for enhanced green
fluorescent protein) encoding full-length rat reggie-1 fused in
frame at the C-terminus to EGFP was provided by Dr. Ritva
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PC1 and EGFR interact within primary cilia
of RCTE cells. Polarized RCTE cells were grown on coverslips 5
days post-confluency. Cells were incubated with antibodies against
PC1 (Santa Cruz mAb) and EGFR (GeneTex pAb), followed by
Duolink PLA Probes to identify points of PC1-EGFR interaction
(red). Cilia identified by acetylated a-tubulin (green). Movie
generated using Voxx2 (provided freely for noncommercial use by
the Indiana Center for Biological Microscopy, Indianapolis, IN,
www.nephrology.iupui.edu/imaging/voxx/index.htm). Scale bar
is 5 mm. Author Contributions Conceived and designed the experiments: SJ HHW AWN. Performed the
experiments: SJ. Analyzed the data: SJ HHW AWN. Contributed
reagents/materials/analysis tools: HHW RL CAML. Wrote the paper:
SJ HHW AMF MM AWN. Conceived and designed the experiments: SJ HHW AWN. Performed the
experiments: SJ. Analyzed the data: SJ HHW AWN. Contributed
reagents/materials/analysis tools: HHW RL CAML. Wrote the paper:
SJ HHW AMF MM AWN. Deciliation Assay MO6-G3
odontoblasts are an inherently more difficult cell line to transfect. This problem is compounded further by the necessity to transfect
these cells after they are 100% confluent and have a completely
polarized phenotype with very tight cell-cell contacts. With
iPoration transfection efficiencies were 60–70% for FITC-labeled
siRNA and 40–60% for flotillin-2-GFP in both MO6-G3 and
RCTE cell types. As a comparison, traditional lipofection using September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 12 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain Lipofectamine 2000 (Life Technologies) resulted in transfection
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Water usage under the concession model in the law of Republika Srpska
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Zbornik radova Pravnog fakulteta u Nišu
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cc-by-sa
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Босиљка Чубриловић Стаменић,*
Виши асистент,
Правни факултет, Универзитет у Бањој Луци
Република Српска Виши асистент,
Правни факултет, Универзитет у Бањој Луци
Република Српска UDK: 628.1:341.223.2](497.6)
UDK: 556.1:005]:34(497.6)
Рад примљен: 03.06.2023. Рад прихваћен: 30.08.2023. * bosiljka.cubrilovic-stamenic@pf.unibl.org Кључне ријечи: концесија, воде, BOT модел, „државна имовина“, јавна
својина. оригинални научни рад
DOI: 10.5937/zrpfn1-44616 оригинални научни рад
DOI: 10.5937/zrpfn1-44616 оригинални научни рад
DOI: 10.5937/zrpfn1-44616 1 Закон о о концесијама (ЗоК), Сл. гласник РС, 59/13, 16/18, 70/20 и 111/21. ВОДЕ КАО ПРЕДМЕТ КОНЦЕСИЈЕ У
ПРАВУ РЕПУБЛИКЕ СРПСКЕ Апстракт: Интересовање за тему коришћења вода по систему
концесија у домаћем законодавству, аутору је побудила Одлука
Уставног суда Босне и Херцеговине у предмету бр. У-16/20. Анализа
наведене одлуке отворила је шири простор за промишљање, те је
предочени чланак настао као плод истраживања ове тематике. Рад је методолошки заснован на нормативној анализи Закона о
концесијама, те Закона о стварним правима, који има функцију
кровног законодавног акта, када се дискутује о стварноправним
институтима у Републици Српској. Иако се у првом дијелу рада
бавимо посебним правним уређењем појединих врста ствари и
дефинисањем појмова воде, водних добара и водних објеката, као
предмета права својине, у фокусу рада су ипак релевантна питања
везана за воде, као концесионо добро. Предмет концесије може бити
право обављања широког опсега привредних дјелатности, а када су у
питању воде, најчешће се ради о коришћењу јавног водног добра (воде
за технолошки процес у обављању привредних дјелатности, воде
и водног земљишта за коришћење хидроакумулација, купалишта,
рибњака, водног земљишта за реализацију неке од привредних
дјелатности, као и вађење материјала из водотока). Поред наведеног,
енергетски потенцијал воде може да се користи и када је објекат
концесије изградња и коришћење енергетских постројења, као једног
од најважнијих економских аспеката експлоатације вода. У последњем
дијелу рада обрађена је пракса Уставног суда, превасходно у вези са
институтом концесија, али и једним ширим питањем које се све више
потенцира у домаћој јудикатури, а то је концепт „државна имовина“. Кључне ријечи: концесија, воде, BOT модел, „државна имовина“, јавна
својина. * bosiljka.cubrilovic-stamenic@pf.unibl.org 215 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 2 Одлука Уставног суда БиХ бр. У-9/19, Службени гласник, 16/20.
3 Закон о стварним правима (ЗСП), Сл. гласник РС, 124/2008, 3/2009 − испр. 58/09,
95/11, 60/15, 18/16 − одлука УС, 107/19, 1/21 − одлука УС и 119/21 − одлука УС.
4 Закон о водама (ЗВ), Сл. гласник РС, 50/06, 92/09, 121/12 и 74/17; Закон о рударству,
Сл. гласник РС, 62/18; Закон о ловству, Сл. гласник РС, 60/09 и 50/13 и др. 1. Увод На концу,
анализирана је пракса Уставног суда Босне и Херцеговине (Уставни суд),
демистификована конструкција „државна имовина“, као и њено довођење
у везу са надлежностима за додјелу концесија, те је извршена детаљнија
разрада одлуке донијете у предмету бр. У-16/202. модел пројектног финансирања инфраструктурних пројеката. На концу,
анализирана је пракса Уставног суда Босне и Херцеговине (Уставни суд),
демистификована конструкција „државна имовина“, као и њено довођење
у везу са надлежностима за додјелу концесија, те је извршена детаљнија
разрада одлуке донијете у предмету бр. У-16/202. 1. Увод У Републици Српској (РС) област концесија уређена је Законом о
концесијама1 који је, уз извијесне измјене и допуне, на снази од 2013. године. Законом је поближе регулисана материја која се односи на предмет
и надлежност за додјелу концесија, политику додјеле концесија, начин и
поступак додјеле концесија, елементе уговора о концесији и престанак
уговора о концесији, права и обавезе концесионара и концендента и
друга питања од значаја за остваривање концесија, с тим да ћемо се у
овом раду бавити искључиво концесијама у контексту искоришћавања
вода, као природног ресурса. Концесија је право обављања привредних
дјелатности коришћењем јавних добара, природних богатстава и других
добара од општег интереса, као и право на обављање дјелатности од
општег интереса, a то право се уз плаћање концесионе накнаде уступа
концесионару на одређено вријеме, под условима прописаним ЗоК. Успостављање концесионог система у националном праву имало је за циљ
креирање подстицајаног правног оквира за привлачење страних и домаћих
инвестиција, уз истовремено инсистирање на одговорном управљању
природним богатствима и општим добрима. Република Српска располаже
значајним капацитетима и природним ресурсима, који уз правилну
употребу умногоме могу да допринесу развоју и јачању домаће привреде. На овом мјесту укратко ћемо изложити систематику рада који је подијељен
у три цјелине. У првом дијелу рада аутор настоји да прецизира поједине
врсте ствари, сходно њиховом посебном правном регулисању. Дефинисани
су институти опште добро, јавно добро и добро од општег интереса, онако
како је то нормирано позитивним законодавством РС. За дискусију о
концесијама круцијално је схватање разлика између ових појмова, јер
поједине врсте ствари због својих особина или правне регулативе нису
подобне да буду објекат права својине и других стварних права. Такође,
аутор прави дистинкцију између појмова воде, водна добра и водни објекти,
као потенцијалних објеката права својине, односно предмета концесије. У централном дијелу рада, обрађена је тема коришћења вода по моделу
концесија у праву РС. Воде су опште добро које је под посебном заштитом
државе (ентитета), те се користе под условима и на начин које обезбјеђује
њихову рационалну употребу. Према домаћем позитивном законодавству,
воде се могу користити у режиму концесија и у режиму јавно-приватног
партнерства, с тим да је овај први модел нешто више заступљен у
пракси. Овдје смо се бавили и појмом, предметом и различитим врстама
концесија, са посебним освртом на BOT модел који је најчешће коришћени 216 Б. Чубриловић Стаменић | стр. 215-236 модел пројектног финансирања инфраструктурних пројеката. 2. Претходно разјашњење − посебно правно
уређење појединих врста ствари Ствари су материјални дијелови природе, који могу бити у својини
физичког или правног лица (чл. 5, ст. 1 Закона о стварним правима3). Међутим, постоје ствари које због својих особина или правног регулисања
нису подобне да буду објекат права својине и других стварних права. Закон
о стварним правима све ствари нормира на општи и посебан начин. Општи режим стварноправног уређења обухвата универзална правила
која се примењују на све врсте ствари над којима физичко или правно лице
може да има право својине. С друге стране, посебно уређење стварних права
представља изузетак од овог општег режима и односи се искључиво на
одређене врсте ствари које су од ширег друштвеног значаја, попут: општих
добара, јавних добара и добара од општег интереса. Између ових добара
никако не можемо ставити знак једнакости, иако су, неријетко, границе
врло флуидне. Потенцијални проблем код разграничења наведених
појмова представља чињеница да ЗСП, али и други посебни закони4, не
дефинишу концепт општег добра, јавног добра и добра од општег интереса,
већ су поједине врсте ствари наведене exempli causa. Сходно томе, из праксе
Уставног суда произилазе различита, често неадекватна, тумачења ових
појмова, која за крајњи резултат имају погрешну примјену правних правила
за посебне врсте ствари. Нису објекат права својине и других стварних права они дијелови природе
који по својим особинама не могу бити у власти физичког или правног лица
појединачно, него су на употреби свима под једнаким условима (чл. 7, ст. 1
ЗСП). Дакле, општа добра (res communes omnium) као што су ваздух, вода
у ријекама, изворима, језерима и мору, нису уопште ствари, односно нису
подобна да буду објекат својине, јер не испуњавају физички услов (не могу
бити у људској власти), али ни правни услов (res extra commercium). Општа 217 217 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 добра су „...само једна подврста добара од општег интереса...“ (Бабић, Медић,
Хашић, Повлакић, Велић, 2011: 145). Општа добра не могу бити ни у својини
државе, њима само управља држава или неки субјект јавне власти, али
не као власник, већ као империјум (Gavella, 2011: 137). Иако општа добра
не могу бити у власти физичког или правног лица, сви субјекти права
их могу употребљавати под једнаким условима. На општим добрима се
може успоставити и одређени стварноправни однос издвајањем појединих
сегмената општег добра, након чега они постају предмет својине и других
стварних права (чл. 7, ст. 3 ЗСП). Тако нпр. 5 Види чл. 8, ст. 1 ЗСП, у вези са чл. 59 Устава Републике Српске (Устав РС), Сл.
гласник РС, 21/1992 − пречишћен текст, 28/1994 − Амандмани XXVI-XLIII, 8/1996 −
Амандмани XLIV-LI, 13/1996 − Амандман LII, 15/1996 − испр., 16/1996 − Амандман
LIII, 21/1996 − Амандмани XLIV-LI, 21/2002 − Амандмани LIV-LXV, 26/2002 − испр.,
30/2002 − испр., 31/2002 − Амандмани XCIII-XCVIII, 69/2002 − Амандмани XCIX-CIII,
31/2003 − Амандмани CIV и CV, 98/2003 − Амандмани CVI-CXII, 115/2005 − Амандман
CXIV, 117/2005 − Амандмани CXV-CXXI и 48/2011 − Амандман CXXII и Сл. гласник БиХ,
73/2019 − одлука УС БиХ. 2. Претходно разјашњење − посебно правно
уређење појединих врста ствари слободни дијелови природе, као
што је вода у ријеци, начелно не могу бити у људској власти, све док се вода
из ријеке не издвоји у посебан чанак (посуду), када прелази у својину лица
које ју је издвојило. Такође, ЗоК регулисана је могућност да концендент
(Влада РС, у име РС или скупштина јединице локалне самоуправе, у
име јединице локалне самоуправе) на одређени временски период и уз
плаћање концесионе накнаде уступи концесионару (привредно друштво)
на употребу и коришћење неко опште добрo. Иако се налазе у несвојинском
правном режиму, општа добра се уписују као својина РС, а такав упис има
деклaративан карактер, јер својство општег добра ствари стичу на основу
саме своје природе, без доношења посебног акта о проглашењу општег
добра (Бабић, Јотановић, 2020: 265). Јавна добра су ствари које су по закону намијењене да служе свима под
једнаким условима, као што су: јавни путеви, улице, тргови и друго (чл. 7, ст
2 ЗСП). Јавна добра (res publicae) су у истом правном режиму као и општа
добра, јер нису подобна да буду објекти стварноправних односа, с тим
да јавна добра не испуњавају правни услов. Дакле, по својим природним
карактеристикама јавна добра би могла бити предмет права својине, али
им је правни поредак одузео својство објекта, а право својине и друга
стварна права не могу егзистирати без објекта (предмета). Управо у овом
сегменту можемо да идентификујемо дистинкцију између општих и јавних
добара – оно што по својој природи не може бити објекат стварноправног
односа је опште добро, а оно што је законом намијењено за општу употребу
свих представља јавно добро (Бабић et. al. 2011: 146). Добра од општег интереса, за разлику од општих и јавних добара, имају
другачији правни статус. Она су genus proximum, јер поред општих,
обухватају и низ других добара, природних ресурса, ствари које су
подвргнуте посебном правном режиму. Добра од општег интереса, а која
нису општа добра, јесу ствари у грађанскоправном смислу (грађевинска
земљишта, пољопривредна земљишта, шуме и шумска земљишта,
заштићени дијелови природе, биљни и животињски свијет, ствари од 218 Б. Чубриловић Стаменић | стр. 215-236 културног, историјског и еколошког значаја5), те могу бити објекат
стварних права. Да би одређена ствар била добро од општег интереса,
као таква треба да буде проглашена посебним законом (чл. 2, ст 2 ЗСП). 2. Претходно разјашњење − посебно правно
уређење појединих врста ствари Закон о стварним правима није мјеродаван за одређивање да ли је нека
ствар добро од општег интереса, његова улога је да створи правни оквир
за режим који се примјењује на стварима које су примјеном lex specialis
проглашене добрима од општег интереса.6 6 Више о овим питањима (Gavella, 2012: 1445−1490; Јотановић, 2015: 187−207). 7 Сви појмови су дефинисани Основним одредбама закона, чл. 4, ст. 1, т. 2−5 ЗВ. 6 Више о овим питањима (Gavella, 2012: 1445−1490; Јотановић, 2015: 187−207).
7 Сви појмови су дефинисани Основним одредбама закона, чл. 4, ст. 1, т. 2−5 ЗВ. 3. Воде, водна добра и водни објекти као предмет права својине Воде представљају природно богатство које се користи под условима и
на начин који обезбјеђује њихову рационалну употребу. У националном
законодавству, воде су дефинисане као опште добро које је под посебном
заштитом РС (чл. 8 ЗВ, у вези са чл. 7, ст. 1 ЗСП). За разумијевање правног
статуса вода, неопходно је да укажемо на разлике између појмова воде,
водно добро и водни објекти. У смислу ЗВ, „вода“ означава сву воду у
унутрашњости Републике Српске, која је стајаћа или текућа по површини
земље и свака подземна вода ближе земљи дуж базне линије границе
територије (чл. 4, ст 1, т. 1 ЗВ). Дакле, под концептом „вода“ подводимо
сва водна тијела у унутрашњости РС: површинске воде, подземне воде,
ријеке и језера.7 За разлику од воде, „водно добро“ подразумијева водно
земљиште, воде и водне објекте, који због свог нарочитог значаја јесу добра
од општег интереса и под посебном су заштитом (чл. 9, ст 1 ЗВ). Дакле,
водно добро представља јединство водног земљишта, воде и објеката
који су у чврстој вези са земљиштем. На основу претходно наведеног,
прилично је јасна тачка раздвајања појмова вода и водно добро, као и
њихов потпуно другачији правни статус. За разлику од вода у морима,
ријекама, језерима, изворима које су опште добро, те стога не могу бити
објекат права својине, водно добро, као добро од општег интереса, може
бити предмет стварних права, па самим тим и у својини титулара права. Потенцијални проблем може да представља колизија у регулацији правног 219 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 режима вода (у ријекама, изворима, језерима, морима), јер је у ЗСП8 вода
нормирана као опште добро, док је у ЗВ, вода, као саставни дио концепта
водно добро, регулисана као добро од општег интерса (упор. чл. 7, ст. 1
ЗСП и чл. 9, ст. 1 ЗВ). Друга категорија за уређење права и обавеза субјеката права јесте „јавно
водно добро“, које чине сви сегменти водног добра, као што су: земљишне
честице, водни или хидротехнички објекти, ријеке и потоци са дефинисаним
морфолошким, геолошким и хидролошким садржајем, који су до дана ступања
на снагу овог закона били уписани у земљишним књигама, односно катастру
непокретности као јавно водно добро од општег интереса или су дефинисани
у другим документима јавног карактера, односно који су на основу закона
постали државна својина (чл. 10, ст. 1 ЗВ). Нормирано на овакав начин,
јавно водно добро је добро од општег интереса у својини РС. 8 Исто и у чл. 8, ст. 1 ЗВ.
9 О управљању водама, водним добрима и водним објектима (Радић, 2021: 64−66). 8 Исто и у чл. 8, ст. 1 ЗВ. 10 „Сви људи, без обзира на степен развоја друштва, те социјалне и економске услове
у којима живе, имају право на приступ пијаћој води у квантитету и квалитету који
одговара њиховим основним потребама.“ (прев. аут). (II Community Water Supply).
Report of the United Nations Water Conference. Преузето 30. 3. 2022. https://www.ielrc.
org/content/e7701.pdf. 3. Воде, водна добра и водни објекти као предмет права својине Овлашћења
управљања јавним водним добром врше органи управе, јавна установа
или надлежни субјекти са јавним овлашћењима, у оном обиму и на начин
како им је законом или другим актима надлежног органа пренесено у
надлежност (чл. 10, ст. 3 ЗВ). Припадност јавном водном добру утврђује
се на основу уписа у јавне евиденције. Јавно водно добро не може бити
предмет промета, али трећа лица могу бити закупци или концесионари
(чл. 11, ст. 1 и 3 ЗВ). И последња релевантна група јесу „водни објекти“. Водни објекти и системи,
заједно са припадајућом опремом који чине техничку, односно технолошку
цјелину, служе за уређење водотока и других вода, заштиту од штетног
дјеловања вода, захватања вода ради њиховог намјенског коришћења,
за праћење стања вода и за заштиту вода од загађивања и одржавање
акватичних екосистема (чл. 14 ЗВ). С обзиром на то која је њихова намјена,
водни објекти могу бити: заштитни, објекти за заштиту од ерозија и
бујица, водни објекти за одводњавање и водни објекти за коришћење
и искоришћавање вода. Својински статус ових објеката првенствено
зависи од њихове намјене, па су тако заштитни водни објекти у својини
РС, а остали само уколико то буде утврђено посебном одлуком надлежног
органа управе (чл. 16, ст. 1−2 ЗВ). Изузетак од овог правила односи се на
објекте за заштиту од ерозија и бујица, водне објекте за одводњавање,
водне објекте за коришћење и искоришћавање вода, који могу бити и
у својини јединице локалне самоуправе, односно у својини физичких и
правних лица, који имају својину над земљиштем на коме је изграђен
водни објекат (чл. 16, ст. 2−3 ЗВ).9 220 Б. Чубриловић Стаменић | стр. 215-236 Након што смо разријешили разлике између појмова воде, водно добро и
водни објекти, у наставку рада биће говора о коришћењу (употреби) вода
по моделу концесија у домаћем праву. 11 Закон о јавно-приватном партнерству у Републици Српској, Сл. гласник РС, 59/09,
63/11 и 68/20. 12 Види нпр. чл. 11, ст. 3, чл. 115, чл. 138 ЗВ и др. 4. Коришћење вода у режиму концесија Цијенећи значај вода као општег добра, позитивним законодавством
прецизиран је појам коришћења вода, као поступак захватања,
црпљења и употребе површинских и подземних вода за различите
намјене (снадбијевање водом за пиће, санитарне и технолошке потребе,
наводњавање и друго), односно искориштавање водних снага за
производњу електричне енергије и друге погонске потребе, за узгој рибе,
за пловидбу као и за спорт, купање, рекреацију и друге сличне намјене. Приступ води је основно људско право, те је важно апострофирати да је
свакоме дозвољена тзв. општа употреба вода, односно сваком лицу је
дозвољено да користи воде обичним начином који не захтијева посебне
направе и не искључује друге од једнаког коришћења.10 У супротном,
неопходна је сагласност или дозвола надлежног органа (чл. 50, ст. 1 и чл. 51, ст. 1 ЗВ). Воде се могу користити у режиму концесија и у режиму јавно-приватног
партнерства,11 с тим да ћемо на овом мјесту анализирати само правне
аспекте коришћења вода по моделу концесија. Правни оквир за регулисање
ове материје чини чл. 59, ст. 2 Устава РС, који прописује да се законом
уређује заштита, коришћење, унапређивање и управљање добрима од
општег интереса, као и плаћање накнаде за коришћење добара од општег
интереса и градског грађевинског земљишта, затим ЗоК (чл. 6, ст. 1, т. б) који
нормира предмет концесија, као и релевантне одредбе ЗВ12. Дакле, за свако
коришћење вода које прелази опсег опште употребе, односно слободног
коришћења воде, титулар мора добити посебну дозволу у виду концесије. Један од разлога за то је и чињеница да усљед прекомјерног или коришћења
вода без надзора, може доћи до озбиљних посљедица по животну средину. Посматрано са историјског аспекта, под појмом концесије подразумијевала
се одређена привилегија, али је израз временом попримио прецизнију 12 Види нпр. чл. 11, ст. 3, чл. 115, чл. 138 ЗВ и др. 221 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 конотацију, па данас овај институт представља класично уступање
одређеног права физичком или правном лицу, од стране државе која иступа
са позиције суверенитета (iure imperii). Иако концесија није нов правни
институт, о њеној правној природи егзистирају различите теорије. Постоје
становишта да је концесиони посао административне природе, тј. да се даје
у форми управног акта, те да се не ради о приватно-правном, већ о јавно-
правном односу између државе као носиоца јавне власти и привредног
друштва. 14 Комисија за концесије РС води регистар уговора о концесијама који представља
јединствену евиденцију уговора о концесијама додијељеним на територији РС. Уговор
о концесији се обавезно уписује у регистар уговора о концесијама. Регистар уговора о
концесијама садржи податке о називу концесионара, називу концендента, предмету
концесије, висини концесионе накнаде, датуму закључења концесионог уговора и
периоду трајања концесије. (чл. 35 ЗоК). 4. Коришћење вода у режиму концесија Друго схватање представља теорија о уговорној природи
концесије, са образложењем да је правни основ концесије уговор између
концедента и концесионара, док је по трећем мишљењу концесиони посао,
правни посао мјешовите природе (Ђурђев, 2004: 400−401). Иако држава
може преносити одређена управна права на приватни сектор (привредна
друштва) кроз концесије, она и даље задржава свој основни суверенитет
над тим ресурсима. Дакле, узевши у обзир кумулативне чињенице да је
држава једна од уговорних страна у уговору о концесији, а да је предмет
уговора обављање привредне дјелатности од јавног интереса, у овом раду
приклонили смо се првој теорији, према којој држава као концесионар
иступа са позиције суверенитета. Концесија (lat. concession − уступање, допуштење, давање повластице) је
право обављања привредних дјелатности коришћењем јавних добара,
природних богатстава и других добара од општег интереса, као и право
на обављање дјелатности од општег интереса, a то право се уступа
концесионару на одређено вријеме, уз плаћање концесионе накнаде
(чл. 2 ЗоК). Дакле, ради се о праву које титулару омогућује да користи
одређене ресурсе или да врши дјелатности од општег интереса уз плаћање
одговарајуће новчане накнаде, при чему концесионар сноси ризик везан
за економску експлоатацију предмета концесије. Концесионар је дужан да
конценденту плаћа концесиону накнаду у износу и на начин утврђен ЗоК
и уговором о концесији. Концесиона накнада је обавезан елемент уговора
о концесији, новчаног је карактера, а састоји се из: 1. накнаде за уступљено
право, која се плаћа једнократно при закључивању уговора о концесији
и 2. концесионе накнаде за коришћење. Зависно од предмета концесије,
концесиона накнада за коришћење изражава се процентуално у односу на
годишњи приход остварен од обављања концесионе делатности, или по
јединици мјере. Концесиона накнада за уступљено право приход је буџета
РС, односно буџета јединице локалне самоуправе, зависно од надлежности
за додјелу концесије (чл. 29 ЗоК). Правни основ за регулисање међусобних права и обавеза представља
уговор о концесији закључен у писаној форми између концендента, с 222 Б. Чубриловић Стаменић | стр. 215-236 једне стране, и концесионара као изабраног најповољнијег понуђача,13 с
друге стране. Уговор о концесији се закључује на рок који не може бити
дужи од 50 година (у пракси је већина уговора додијељена на 30 година,
са могућношћу да се у преговарачком поступку продужи рок трајања
уговора). Рок концесионог уговора одређује се у зависности од предмета
концесије, времена потребног за поврат уложеног капитала и остваривања
планиране добити на основу концесионе дјелатности. 14 једне стране, и концесионара као изабраног најповољнијег понуђача,13 с
друге стране. 13 Понуђач је свако домаће или страно правно или физичко лице, конзорцијум два
или више уговором повезаних правних лица, који је доставио понуду у поступку
додјеле концесије (чл. 5, ст. 1, т. г. ЗоК). 15 Свеобухватан предмет концесија у праву РС регулисан је чл. 6 ЗоК. 16 Документ о политици додјеле концесија садржи опис привредних и других области
у којима се могу додјељивати концесије, циљеве и приоритете, врсте БОТ и других
модела за додјелу концесије, елементе за израду студије, као и мјере и активности
које се предузимају ради остваривања дугорочних циљева у области концесија,
утврђених стратешким и другим планским документима. Види више: Документ о
политици додјеле концесија. Преузето 13. 2. 2023. https://koncesije-rs.org/wp-content/
uploads/2019/06/Politika_dodjela-Cir.pdf. 4. Коришћење вода у режиму концесија 2 ЗоК нормира да је дозвољено уговарање
и других модела за реализацију концесија, у складу са Документом о
политици додјеле концесија16 и предметом концесија. Под утицајем UNI-
CITRAL правила све ове модалитете можемо подвести под генерични појам
„приватно финансирање инфраструктурних пројеката“ (енг. Privately Finan-
ced Infrastructure Projekts – PFIP). У основи, под њим се подразумевају сви
облици улагања у јавне инфраструктурне објекте, при чему финансирање и
ризик пословног неуспјеха преузима привредно друштво. Оно то остварује
по основу добијене концесије од стране државне или локалне власти за
изградњу и управљање инфраструктурним објектом (Дивљак, 2006: 277). Дакле, BOT модел финансирања омогућава држави (у конкретном случају
РС, као ентитету) да без ангажовања финансијских средстава постане
титулар значајних инфраструктурних објеката, с тим да у уговореном року
нема право да их експлоатише, јер то чини концесионар који кроз остварену
добит враћа уложени капитал. С друге стране, овај модел пројектног
финансирања инфраструктуре омогућује и приватном сектору да се
укључи у финансирање, изградњу, санацију, проширење, функционисање
и одржавање пројеката, које уобичајено финансира и којима управља
РС, те сходно томе можемо рећи да су на овај начин свој интерес нашли
сви учесници. По истицању рока предвиђеног уговором о концесији и по
повраћају инвестиције, врши се трансфер свих постројења у оквиру BOT
пројекта према РС, с тим да та постројења треба да буду у добром стању и
неоптерећена. У многим земљама концесије за коришћење воде користе
се у сврху побољшања квалитета воде, унапређења инфраструктуре, као
и за финансирање нових пројеката. Међутим, да би се избјегли негативни
аспекти (нпр. успостављање монопола на тржишту, повећање цијена воде
за потрошаче, те ограничавања права локалних заједница на коришћење
воде), неопходно је осигурати законитост и транспарентност у поступку
додјеле концесија. имај у својини – управљај − пренеси (енг. Build – Own – Operate - Transfer)],
МОТ посао [модернизуј – управљај − пренеси (енг. Moderate – Operate − Tran-
sfer)], RОТ посао [реновирај – управљај − пренеси (енг. Refurbish − Operate
− Transfer)] и др. Тако и чл. 7, ст. 2 ЗоК нормира да је дозвољено уговарање
и других модела за реализацију концесија, у складу са Документом о
политици додјеле концесија16 и предметом концесија. Под утицајем UNI-
CITRAL правила све ове модалитете можемо подвести под генерични појам
„приватно финансирање инфраструктурних пројеката“ (енг. Privately Finan-
ced Infrastructure Projekts – PFIP). У основи, под њим се подразумевају сви
облици улагања у јавне инфраструктурне објекте, при чему финансирање и
ризик пословног неуспјеха преузима привредно друштво. 4. Коришћење вода у режиму концесија Уговор о концесији се закључује на рок који не може бити
дужи од 50 година (у пракси је већина уговора додијељена на 30 година,
са могућношћу да се у преговарачком поступку продужи рок трајања
уговора). Рок концесионог уговора одређује се у зависности од предмета
концесије, времена потребног за поврат уложеног капитала и остваривања
планиране добити на основу концесионе дјелатности. 14 Предмет концесије може бити право обављања широког опсега привредних
дјелатности, с тим да се најчешће ради о областима које државни
органи из одређених разлога не могу или неће да врше самостално, те
их преносе на друго лице уз одговарајућу новчану накнаду.15 На тај се
начин оставарују јавни интереси али и обезбјеђује функционисање јавних
служби и привредни развој. Када су у питању воде, предмет концесије
може бити коришћење јавног водног добра [воде за технолошки процес у
обављању привредних дјелатности, воде и водног земљишта за коришћење
хидроакумулација, купалишта, рибњака, водног земљишта за реализацију
неке од привредних дјелатности или за реализацију других предмета
концесије, као и вађење материјала из водотока (шљунка, пијеска, камена)
према програму уређења водотока (чл. 6, ст 1, т. б. ЗоК)]. Поред овога,
енергетски потенцијал воде може да се користи и када је објекат концесије
изградња и коришћење енергетских постројења, као једног од најважнијих
економских аспеката експлоатације вода. Концесије којима се предвиђа изградња или реконструкција објеката,
постројења или погона све више се реализују по BOT моделу [изгради –
користи − предај (енг. Build − Operate − Transfer)], што подразумијева изградњу
или реконструкцију и финансирање комплетног објекта, постројења или
погона, његово коришћење и предају у својину конценденту у уговореном
року. BOT модел је најчешће коришћени модел пројектног финансирања
инфраструктуре, с тим да су се у пословној пракси обликовали и други
подоблици пројектног финансирања као што су: BOOT посао [изгради − 13 Понуђач је свако домаће или страно правно или физичко лице, конзорцијум два
или више уговором повезаних правних лица, који је доставио понуду у поступку
додјеле концесије (чл. 5, ст. 1, т. г. ЗоК). 223 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 имај у својини – управљај − пренеси (енг. Build – Own – Operate - Transfer)],
МОТ посао [модернизуј – управљај − пренеси (енг. Moderate – Operate − Tran-
sfer)], RОТ посао [реновирај – управљај − пренеси (енг. Refurbish − Operate
− Transfer)] и др. Тако и чл. 7, ст. 17 У БиХ је у претходном периоду изграђено 108 малих хидроелектрана, од чега је 65
у ФБиХ, 43 у РС и у припреми је изградња додатних 338 МХЕ. Види: Zabrana izgradnje
malih hidroelektrana na Zapadnom Balkanu − Uporedna analiza moratorijuma i zabrane
izgradnje malih hidroelektrana u Bosni i Hercegovini, Crnoj Gori i Srbiji. Преузето 7. 2. 2023.
https://www.reri.org.rs/wp-content/uploads/2022/01/Uporedna-analiza-moratorijuma-i-
zabrane-izgradnje-malih-hidroelektrana-u-Bosni-i-Hercegovini-Crnoj-Gori-i-Srbiji.pdf, 4. 18 Рјешење о продужењу прелиминарног права на подстицај за производњу
електричне енергије – МХЕ „Самар“. Преузето 15. 2. 2023. https://reers.ba/wp-content/
uploads/2021/11/Rjesenje-o-produzenju-preliminarnog-prava-na-podsticaj-za-proizvodnju-
elektricne-energije-MHE-Samar-cirilica-1.pdf. Накнадно је Регулаторна комисија за
енергетику РС у априлу 2022. године одбила захтјев за продужење прелиминарног права
на подстицај, као неоснован. Рјешење о одбијању захтјева за одобрење прелиминарног
права на подстицај – МХЕ „Самар“. Преузето 15. 2. 2023. https://reers.ba/wp-content/
uploads/2022/04/Rjesenje-o-odbijanju-zahtjeva-za-odobrenje-preliminarnog-prava-na-
podsticaj-MHE-Samar-cirilica.pdf. 4. Коришћење вода у режиму концесија преусмјеравање подстицаја − Декларација
сугерише да би Народна скупштина РС требало да у новој регулативи о
обновљивим изворима енергије минимизира давање јавних подстицаја
специфично за МХЕ. Уместо тога, фокус би требало да буде на подстицању
производње електричне енергије кроз друге, еколошки прихватљивије,
обновљиве изворе енергије. Међутим, иако представља корак напред,
Декларација нема обавезујућу правну моћ, што значи да њено спровођење
зависи од политичке воље и притиска јавности. То је постало јасно на
примјеру МХЕ „Самар“ гдје је, упркос постојању Декларације, Влада РС дала
зелено свјетло за њену изградњу. Ситуација се додатно закомпликовала
када је Окружни суд у Бањој Луци, по тужби једног удружења који се
бави промоцијом и очувањем здраве животне средине, интервенисао
и поништио еколошку дозволу за ову МХЕ, али радови на изградњи су
настављени, уз додатну подршку Регулаторне комисије за енергетику.18
Након доношења одлуке од стране Окружног суда у Бањој Луци, надлежно
министарство је уложило ванредни правни лијек, односно захтјев за на изградњу и експлоатацију малих хидроелектрана (МХЕ) због све веће
забринутости о њиховим негативним ефектима на животну средину. 17 У
фебруару 2021. године, Република Српска је донијела кључан документ
у овом контексту − Декларацију о заштити ријека у Републици Српској
(Декларација). Ова Декларација није само симболичан документ, већ
поставља конкретне смјернице и обавезе: 1. замрзавање додјеле нових
концесија − Влада РС је обавезна да обустави све нове захтјеве за додјелу
концесија за МХЕ док се не изврши темељна анализа свих претходно
потписаних уговора о концесијама из периода 2005−2020. године. (остављен
рок од шест мјесеци) и 2. преусмјеравање подстицаја − Декларација
сугерише да би Народна скупштина РС требало да у новој регулативи о
обновљивим изворима енергије минимизира давање јавних подстицаја
специфично за МХЕ. Уместо тога, фокус би требало да буде на подстицању
производње електричне енергије кроз друге, еколошки прихватљивије,
обновљиве изворе енергије. Међутим, иако представља корак напред,
Декларација нема обавезујућу правну моћ, што значи да њено спровођење
зависи од политичке воље и притиска јавности. То је постало јасно на
примјеру МХЕ „Самар“ гдје је, упркос постојању Декларације, Влада РС дала
зелено свјетло за њену изградњу. 4. Коришћење вода у режиму концесија Оно то остварује
по основу добијене концесије од стране државне или локалне власти за
изградњу и управљање инфраструктурним објектом (Дивљак, 2006: 277). Дакле, BOT модел финансирања омогућава држави (у конкретном случају
РС, као ентитету) да без ангажовања финансијских средстава постане
титулар значајних инфраструктурних објеката, с тим да у уговореном року
нема право да их експлоатише, јер то чини концесионар који кроз остварену
добит враћа уложени капитал. С друге стране, овај модел пројектног
финансирања инфраструктуре омогућује и приватном сектору да се
укључи у финансирање, изградњу, санацију, проширење, функционисање
и одржавање пројеката, које уобичајено финансира и којима управља
РС, те сходно томе можемо рећи да су на овај начин свој интерес нашли
сви учесници. По истицању рока предвиђеног уговором о концесији и по
повраћају инвестиције, врши се трансфер свих постројења у оквиру BOT
пројекта према РС, с тим да та постројења треба да буду у добром стању и
неоптерећена. У многим земљама концесије за коришћење воде користе
се у сврху побољшања квалитета воде, унапређења инфраструктуре, као
и за финансирање нових пројеката. Међутим, да би се избјегли негативни
аспекти (нпр. успостављање монопола на тржишту, повећање цијена воде
за потрошаче, те ограничавања права локалних заједница на коришћење
воде), неопходно је осигурати законитост и транспарентност у поступку
додјеле концесија. На концу овог дијела рада, направићемо кратак осврт на тему о којој се
много говори у јавности. Ради се о потенцијалном увођењу мораторијума 16 Документ о политици додјеле концесија садржи опис привредних и других области
у којима се могу додјељивати концесије, циљеве и приоритете, врсте БОТ и других
модела за додјелу концесије, елементе за израду студије, као и мјере и активности
које се предузимају ради остваривања дугорочних циљева у области концесија,
утврђених стратешким и другим планским документима. Види више: Документ о
политици додјеле концесија. Преузето 13. 2. 2023. https://koncesije-rs.org/wp-content/
uploads/2019/06/Politika_dodjela-Cir.pdf. 224 Б. Чубриловић Стаменић | стр. 215-236 на изградњу и експлоатацију малих хидроелектрана (МХЕ) због све веће
забринутости о њиховим негативним ефектима на животну средину. 17 У
фебруару 2021. године, Република Српска је донијела кључан документ
у овом контексту − Декларацију о заштити ријека у Републици Српској
(Декларација). Ова Декларација није само симболичан документ, већ
поставља конкретне смјернице и обавезе: 1. замрзавање додјеле нових
концесија − Влада РС је обавезна да обустави све нове захтјеве за додјелу
концесија за МХЕ док се не изврши темељна анализа свих претходно
потписаних уговора о концесијама из периода 2005−2020. године. (остављен
рок од шест мјесеци) и 2. 4. Коришћење вода у режиму концесија Ситуација се додатно закомпликовала
када је Окружни суд у Бањој Луци, по тужби једног удружења који се
бави промоцијом и очувањем здраве животне средине, интервенисао
и поништио еколошку дозволу за ову МХЕ, али радови на изградњи су
настављени, уз додатну подршку Регулаторне комисије за енергетику.18
Након доношења одлуке од стране Окружног суда у Бањој Луци, надлежно
министарство је уложило ванредни правни лијек, односно захтјев за 225 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 преиспитивање судске одлуке, па је овај случај тренутно пред Врховним
судом РС и још увијек се чека његов правни исход.19 Осим овог конкретног случаја, постоји још један законски аспект
који је изазвао контроверзе. Актуелни закон дозвољава преношење и
продају уговора о концесијама, као и промјене у власничкој структури
концесионара. Ове одредбе пружају прилику за спекулације, где се
концесије често прибављају с примарном намјером продаје, а не стварне
изградње и експлоатације ресурса. Ово може довести до ситуације где се
примарни циљеви, попут заштите животне средине, занемарују у корист
финансијске добити. Да би се наведено избјегло, неопходно је преиспитати
и евентуално ревидирати постојеће законске одредбе. Сматрамо да
ограничење преноса својинских права није добро рјешење, али би свакако
требало да се предуприједи понашање које омогућава изигравање слова
закона. 21 Поред ЗСП који се примјењује на територији РС, види и Закон о стварним правима,
Службене новине ФБиХ, 66/13, 100/13 и 32/19 − одлука УС, као и Закон о власништву и
другим стварним правима Брчко Дистрикта Босне и Херцеговине, Сл. гласник Брчко
Дистрикта БиХ, 11/01, 8/03, 40/04, 19/07, 26/21 и 44/22. 22 Одлука бр. У-1/11, Службени гласник БиХ, 73/12. Иначе, овај предмет се тицао
уставне надлежности РС за регулисање правне материје која је предмет Закона
о статусу државне имовине која се налази на територији Републике Српске и под
забраном је располагања, Службени гласник РС, 135/10, наметнут од стране Високог
представника за БиХ. 5. Пракса Уставног суда по питању „државне имовине“ Надлежности институција БиХ дефинисане су чл. III, т. 1 Уставa БиХ20 по
принципу позитивне енумерације. Истим чланом, у т. 3 предвиђено је
да ентитетима припадају све владине функције и овлашћења која нису
Уставом БиХ изричито повјерена институцијама БиХ. То значи да је у
јурисдикцији ентитета и Брчко Дистрикт БиХ, све оно што explicite није
у надлежности институција на државном нивоу. Важно је нагласити да
на нивоу БиХ не постоји пропис који регулише стварноправне односе, јер
су закони из овог домена у искључивој надлежности ентитета и Брчко
Дистрикта БиХ.21 Ово је потврђено и у Одлуци Уставног суда бр. У-1/1122,
19 Више о економској оправданости концесионих накнада за МХЕ, види: Analiza
ekonomske opravdanosti koncesionih naknada i podsticaja za male hidroelektrane na teritoriji
Bosne i Hercegovine. Преузето 16. 2. 2023. https://czzs.org/wp-content/uploads/2018/11/
Analiza-MHE1.pdf. 20 Устав БиХ је саставни дио Општег оквирног споразума за мир у БиХ (Дејтонски
мировни споразум). Преузето 23. 2. 2023. https://www.paragraf.ba/propisi/bih/ustav-
bosne-i-hercegovine.html. 21 Поред ЗСП који се примјењује на територији РС, види и Закон о стварним правима,
Службене новине ФБиХ, 66/13, 100/13 и 32/19 − одлука УС, као и Закон о власништву и
другим стварним правима Брчко Дистрикта Босне и Херцеговине, Сл. гласник Брчко
Дистрикта БиХ, 11/01, 8/03, 40/04, 19/07, 26/21 и 44/22. 22 Одлука бр. У-1/11, Службени гласник БиХ, 73/12. Иначе, овај предмет се тицао
уставне надлежности РС за регулисање правне материје која је предмет Закона
о статусу државне имовине која се налази на територији Републике Српске и под
забраном је располагања, Службени гласник РС, 135/10, наметнут од стране Високог
представника за БиХ. Надлежности институција БиХ дефинисане су чл. III, т. 1 Уставa БиХ20 по
принципу позитивне енумерације. Истим чланом, у т. 3 предвиђено је
да ентитетима припадају све владине функције и овлашћења која нису
Уставом БиХ изричито повјерена институцијама БиХ. То значи да је у
јурисдикцији ентитета и Брчко Дистрикт БиХ, све оно што explicite није
у надлежности институција на државном нивоу. Важно је нагласити да
на нивоу БиХ не постоји пропис који регулише стварноправне односе, јер
су закони из овог домена у искључивој надлежности ентитета и Брчко
Дистрикта БиХ.21 Ово је потврђено и у Одлуци Уставног суда бр. У-1/1122,
19 Више о економској оправданости концесионих накнада за МХЕ, види: Analiza
ekonomske opravdanosti koncesionih naknada i podsticaja za male hidroelektrane na teritoriji
Bosne i Hercegovine. Преузето 16. 2. 2023. https://czzs.org/wp-content/uploads/2018/11/
Analiza-MHE1.pdf. 20 У
Б Х ј
О
Б Х (Д ј Надлежности институција БиХ дефинисане су чл. III, т. 20 Устав БиХ је саставни дио Општег оквирног споразума за мир у БиХ (Дејтонски
мировни споразум). Преузето 23. 2. 2023. https://www.paragraf.ba/propisi/bih/ustav-
bosne-i-hercegovine.html. 5. Пракса Уставног суда по питању „државне имовине“ 1 Уставa БиХ20 по
принципу позитивне енумерације. Истим чланом, у т. 3 предвиђено је
да ентитетима припадају све владине функције и овлашћења која нису
Уставом БиХ изричито повјерена институцијама БиХ. То значи да је у
јурисдикцији ентитета и Брчко Дистрикт БиХ, све оно што explicite није
у надлежности институција на државном нивоу. Важно је нагласити да
на нивоу БиХ не постоји пропис који регулише стварноправне односе, јер
су закони из овог домена у искључивој надлежности ентитета и Брчко
Дистрикта БиХ.21 Ово је потврђено и у Одлуци Уставног суда бр. У-1/1122, 20 Устав БиХ је саставни дио Општег оквирног споразума за мир у БиХ (Дејтонски
мировни споразум). Преузето 23. 2. 2023. https://www.paragraf.ba/propisi/bih/ustav-
bosne-i-hercegovine.html. 22 Одлука бр. У-1/11, Службени гласник БиХ, 73/12. Иначе, овај предмет се тицао
уставне надлежности РС за регулисање правне материје која је предмет Закона
о статусу државне имовине која се налази на територији Републике Српске и под
забраном је располагања, Службени гласник РС, 135/10, наметнут од стране Високог
представника за БиХ. 226 Б. Чубриловић Стаменић | стр. 215-236 у којој je навeдено сљедеће: „Уставни суд се слаже с мишљењем Народне
скупштине РС да Устав БиХ не садржи експлицитну одредбу која утврђује
надлежност БиХ да регулише питање државне имовине која припада БиХ...“. Расправљајући о расподјели надлежности између појединих нивоа власти
у БиХ, Уставни суд је донио низ контроверзних одлука, а на овом мјесту
ћемо апстраховати само неке од њих којима се задире у својинскоправне
односе, па сам тим и у питање концесија.23 У наведеним одлукама Уставни
суд, најблаже речено, узурпира основне постулате стварноправног уређења
РС и БиХ, те прави низ кардиналних грешака које су резултат погрешног
тумачења релевантних прописа. Тако је овај суд, ван оквира својих
ингеренција, установио нову правну категорију „државна имовина“, као
посебан облик својине. У предмету бр. У-1/11, у т. 62 образложења одлуке
наводи се: „Државна имовина, иако је то облик власништва који је по својој
структури сличан грађанскоправном приватном власништву, представља
посебан правни концепт, те, из тог разлога, ужива посебан статус. Државна
имовина је карактеристична по јавноправној природи односа субјеката и
кориштења те имовине, као и њезиног титулара. Она обухвата, с једне
стране, покретне и непокретне ствари које су у рукама јавне власти и које
јој служе ради вршења те власти, с друге стране, она може обухватити
ʼјавно доброʼ (морска вода и морско дно, ријечна вода и ријечна корита, језера,
планине и друга природна богатства, јавна саобраћајна мрежа, саобраћајна
инфраструктура итд.). 23 Одлука бр. У-9/19; Одлука бр. У-16/20, Службени гласник БиХ, 49/21. 5. Пракса Уставног суда по питању „државне имовине“ Оно, по својој природи, приоритетно служи свим
људима у држави.“ у којој je навeдено сљедеће: „Уставни суд се слаже с мишљењем Народне
скупштине РС да Устав БиХ не садржи експлицитну одредбу која утврђује
надлежност БиХ да регулише питање државне имовине која припада БиХ...“. у којој je навeдено сљедеће: „Уставни суд се слаже с мишљењем Народне
скупштине РС да Устав БиХ не садржи експлицитну одредбу која утврђује
надлежност БиХ да регулише питање државне имовине која припада БиХ...“. Премиса од које полази Уставни суд да је „државна имовина“ облик
својине који је само сличан грађанскоправној приватној својини, те да
преставља посебан правни концепт, напросто не стоји. Прву теоријску
нелогичност у овој констатацији представља чињеница да суд ставља знак
једнакости између појмова „имовина“ и „својина“. Иако је имовина (patri-
monium) вишезначан израз који се лаички често поистовјећује са својином,
а нерјетко и са појмом ствари, концепт имовине је далеко шири и не може
се свести само на право својине. Како наводи проф. Водинелић, имовина
„...представља неки шири или ужи скуп, неку групацију права и обавеза одн. имовинских права или права и обавеза у оквиру свеукупности права и обавеза“
(Vodinelić, 2012: 272). За разлику од имовине, право својине је једно од
неколико стварних права, из кога, истина, произлази највиша правна
и фактичка власт на ствари (држање, коришћење, односно употреба и
располагање), али имовина је ипак genus proximum, институт који је по
свом садржају шири од појма својине. 23 Одлука бр. У-9/19; Одлука бр. У-16/20, Службени гласник БиХ, 49/21. 227 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 Надаље, говорити о „државној имовини“ као посебном правном концепту,
различитом од грађанскоправне приватне својине, резон је противан
закону који регулише материју стварних права (ЗСП), а који право својине
нормира по принципу једноврсности. Право својине се не може дијелити
по овлашћењима између различитих субјеката, као што је то било у
феудализму, јер, само је једна врста права својине, без обзира о којем се
носиоцу права ради (чл. 3, ст. 2 ЗСП). Закон о стварним правима предвиђа
и то да РС, јединица локалне самоуправе, јавно предузеће, јавна установа
и друге јавне службе које су носиоци права својине јавног права имају
као власници у правним односима исти положај као и други власници,
ако посебним законом није другачије одређено (чл. 22, ст. 1 ЗСП). 5. Пракса Уставног суда по питању „државне имовине“ Начелно
говорећи, за садржај права својине је сасвим ирелевантно ко је носилац
права, јер је само једна врста права својине и било који титулар (субјекат
јавног или приватног права) има иста овлашћења. У горенаведеној одлуци, Уставни суд погрешно дефинише и појам јавног
добра, под који подводи морску воду и морско дно, ријечну воду и ријечна
корита, језера, планине и друга природна богатства, јавну саобраћајну
мрежу, саобраћајну инфраструктуру итд. Евидентно је да суд под исти
правни режим ставља општа и јавна добра, не правећи суштинску разлику
између њих. Узевши у обзир појашњења из првог дијела овог рада, општа
добра не могу бити предмет права својине, па је нејасно на основу чега
Уставни суд тврди да је вода у морама, ријекама, језерима својина државе,
односно „државна имовина“. Сву нелогичност судске праксе поткријепићемо са још два става које је заузео
Уставни суд: 1) рјешавајући спор у вези са одлукама о концијама у погледу
концесионог добра и надлежности за њихово доношење, суд у предмету
У-16/20, у т. 31 образложења одлуке, изричито наводи да не постоји закон
који је донесен на нивоу БиХ којим би било регулисано питање статуса
„државне имовине“ („...у вези са статусом државне имовине, у којима није
донесен закон на нивоу Босне и Херцеговине којим би било регулисано питање
статуса државне имовине...“) и 2) у предмету У-1/11, т. 63 образложења суд
констатује: „Мада Устав Босне и Херцеговине дијели надлежности између
државе и ентитета, он не садржи нити једну одредбу која се односи на
државну имовину“. На основу цитираних сентенци, јасно проистиче да је
институт „државна имовина“ неуставан и незаконит, да не постоји нити
један позитивни пропис који дефинише појам и/или прецизира садржај
појма „државна имовина“, да је то измишљена конструкција чији је креатор
сам Уставни суд, а не уставотворац нити законодавац, те да не може
бити дио позитивног законодавства БиХ (ентитета и Брчко Дистрикта
БиХ). Начелно говорећи, у европско-континенталном правном систему 228 Б. Чубриловић Стаменић | стр. 215-236 судска пракса представља примјену а не стварање правних норми, те
стога није формалан извор права (судови суде на основу Устава и закона,
чл. 121, ст. 2 Устава РС). Иако је примјетна тенденција да и у европско-
континенталном правном систему судови све више имају креативистичку
улогу, ипак наглашавамо да је Уставни суд у овом случају изашао ван
креативистичких оквира и ставио се у функцију законотворца, што је
свакако неприхватљиво са правног аспекта. 24 Одлука о утврђивању услова за додјелу концесије путем преговарачког поступка за
изградњу и коришћење ХЕ „Бук Бијела“ на ријеци Дрини, општина Фоча, бр. 04/1-012-2-
472/16 од 4. марта 2016, Сл. гласник РС, 20/16, Рјешење о додјели концесије за изградњу
и коришћење ХЕ „Бук Бијела“ на ријеци Дрини, општина Фоча, бр. 04/1-012-2-1099/16
од 20. маја 2016, Сл. гласник РС, 42/16 и Уговор о концесији број 05.05/012-274-16/16 од
3. јуна 2016; Одлука о утврђивању услова за додјелу концесије путем преговарачког
поступка за изградњу и коришћење ХЕ „Фоча“ на ријеци Дрини бр. 04/1-012-2-81/19
од 10. јануара 2019, Сл. гласник РС, 4/19, Рјешење о додјели концесије за изградњу и
коришћење ХЕ „Фоча“ на ријеци Дрини бр. 04/1-0122-221/19 од 24. јануара 2019, Сл.
гласник РС, 9/19 и Уговор о концесији бр. 05.05/360-2-24/19 од 19. фебруара 2019;
Одлука о утврђивању услова за додјелу концесије путем преговарачког поступка
за изградњу и коришћење ХЕ „Паунци“ на ријеци Дрини број 04/1-012-2-80/19 од 10.
јануара 2019, Сл. гласник РС, 4/19, Рјешење о додјели концесије за изградњу и коришћење ХЕ „Паунци“ на ријеци Дрини
бр. 04/1-012-2-214/19 од 24. јануара 2019, Сл. гласник РС, 9/19 и Уговор о концесији бр.
05.05/360-2-22/19 од 19. фебруара 2019. (даље: оспорени акти). 25 Види нпр. одлуку у предмету У-9/19. 6. Концесије у пракси Уставног суда Реферишући се на закључке изведене у претходном дијелу рада [не
постоји закон који је донесен на нивоу БиХ којим би било регулисано
питање статуса „државне имовине“, као и то да су воде општа добра (а не
јавна добра, по тврдњама Уставног суда)], детаљније ћемо се позабавити
питањем концесија у пракси Уставног суда на примјеру предмета У-16/20. Наводни спор у вези надлежности између БиХ и РС за доношење одлука
о концесијама у погледу концесионог добра, настао је поводом Одлуке о
утврђивању услова за додјелу концесије путем преговарачког поступка за
изградњу и коришћење три ХЕ: „Бук Бијела“, „Фоча“, те „Паунци“, на ријеци
Дрини, општина Фоча.24 Подносиоци захтјева (24 члана Представничког
дома Парламентарне скупштине БиХ), навели су да се спор односи на
питање уставноправне обавезе ентитетских власти да поштују Устав
БиХ и подјелу надлежности између државе и ентитета, те су се позвали
на одлуке Уставног суда25 у којима је заузет став да ентитети немају
право управљања и располагања државном имовином, а што укључује 229 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 и успостављање концесије на државној имовини. Даље наводе „како се
види из спорних одлука, рјешења и уговора, власти ентитета РС додјељују
концесију за коришћење водних потенцијала ријеке Дрине без обзира на то
што ти потенцијали, према досадашњим ставовима Уставног суда БиХ,
чине државну имовину... Уставни суд БиХ је утврдио и да право располагања
државном имовином, па и овдје спорним водним потенцијалима ријеке
Дрине, све до доношења закона о статусу државне имовине на нивоу БиХ
има искључиво држава БиХ“. Дакле, у конкретном случају подносиоци
захтјева тврде да постоји спор између БиХ и РС који је настао доношењем
оспорених аката од стране РС, за који РС наводно није надлежана јер се
ради о додјели концесија на концесионом добру које је „државна имовина“,
те је сходно томе надлежна власт на нивоу БиХ, а не на нивоу ентитета. Подносиоци захтјева су предложили да Уставни суд усвоји њихов захтјев,
утврди да су оспорени акти неуставни, да их стави ван снаге, као и да
обавеже Владу РС да поништити све супсидијарне одлуке и активности
које су засноване на оспореним актима. Такође, предложено је да Уставни
суд донесе привремену мјеру којом би се обуставила примјена оспорених
одлука. Примјећујемо да је у овом захтјеву спорно неколико хипотеза. 26 Закон о концесијама БиХ (ЗоК БиХ), Сл. гласник БиХ, 32/02 и 56/04. Друго, Уставни суд није надлежан да одлучује о спору о концесијама јер је,
како је то и у одговору на захтјев наведено, „...додјела концесија иницијално 6. Концесије у пракси Уставног суда Као прво,
Закон о концесијама БиХ26 не нормира ко је овлаштени субјект за покретање
спора из надлежности Комисије за концесије, као ни начин покретања
истог, што потврђује и суд у т. 32 образложења одлуке када наводи да
„Уставни суд примјећује да Закон о концесијама Босне и Херцеговине не
садржи посебне одредбе којима би се утврдио начин и овлашћени субјекат за
покретање спора из надлежности Комисије за концесије Босне и Херцеговине у
својству Заједничке комисије за концесије. Дакле, постоји правна празнина коју
Уставни суд мора, сходно томе, попунити“. Дакле, иако ЗоК БиХ не нормира
ко је овлаштени субјект за покретање спора из надлежности Комисије за
концесије БиХ (у својству Заједничке комисије за концесије), нити начин
покретања истог, Уставни суд се упушта у расправљање овог питања,
попуњавајући правне празнине у позитивним актима. У надлежности
Уставног суда је да тумачи законодавна рјешења и идентификује правне
празнине, али његова примарна функција није да „попуњава“ те празнине
у смислу креирања нове норме. Сматрамо да би на овом мјесту било
примјереније да је кроз своје одлуке Уставни суд указао на потребу за
таквим промјенама. Право и дужност креирања и мијењања закона лежи
искључиво на законодавној власти. Друго, Уставни суд није надлежан да одлучује о спору о концесијама јер је,
како је то и у одговору на захтјев наведено, „...додјела концесија иницијално
26 Закон о концесијама БиХ (ЗоК БиХ), Сл. гласник БиХ, 32/02 и 56/04. Друго, Уставни суд није надлежан да одлучује о спору о концесијама јер је,
како је то и у одговору на захтјев наведено, „...додјела концесија иницијално 230 Б. Чубриловић Стаменић | стр. 215-236 и примарно уређена законима, а секундарно и деривативно подзаконским
актима. Законодавна надлежност у регулисању концесија подијељена је
између Босне и Херцеговине и ентитета. (…) У складу с тим, концесије су
законски уређене Законом о концесијама Босне и Херцеговине, Законом о
концесијама Републике Српске, Законом о концесијама Федерације БиХ,
законима о концесијама кантона у Федерацији БиХ“. Законом о концесијама
БиХ, уређен је начин рјешавања спора који потенцијално настане између
БиХ и/или РС приликом додјеле концесије на концесионим добрима (чл. 4,
ст. 3). Дакле, спор између БиХ и РС своди на питање да ли је БиХ искључиво
надлежна за додјелу концесија на свим природним ресурсима у БиХ која се
могу ставити у функцију концесионог добра. 6. Концесије у пракси Уставног суда Сматрамо да овакав став нема
упориште ни у Уставу БиХ, а ни у законима којима је регулисана област
концесија, јер концесионо добро у конкретном случају није „државна
имовина“, у смислу одлука Уставног суда (т. 12 образложења одлуке). И треће, питање надлежности за регулисање статуса „државне имовине“
не може се прелијевати на питања надлежности за додјелу концесија. Ове двије области имају различите правне основе и не би требало да се
преплићу. Надлежност за додјелу концесија регулисана је законима на
државном и ентитетском нивоу, а у складу са подјелом надлежности која
произлази из Устава БиХ. С друге стране, не постоји законски оквир који
регулише питање „државне имовине“, већ само неконзистентно тумачење
и пракса Уставног суда, која није извор права. У закључку Дјелимичне одлуке о допустивости и меритуму, Уставни суд
је оставио одговарајући рок за рјешавање спора од најдуже три мјесеца од
дана достављања одлуке Комисији за концесије БиХ, у својству Заједничке
комисије за концесије. Увидом у Рјешење од 3. децембра 2021. и Рјешење од
19. јануара 2023, јасно произилази да Комисија за концесије БиХ, у својству
Заједничке комисије за концесије још увијек није извршила Дјелимичну
одлуку Уставног суда БиХ бр. У-16/20 од 16. јула 2021, те ово питање и даље
стоји отворено пред правосудним органима. накнаду, али и уз стриктне услове који обезбеђују одрживост и дугорочну
заштиту водених ресурса. На глобалном нивоу, концесије представљају средство које доприноси
различитим циљевима: од унапређења квалитета воде и заштите
екосистема, до развоја инфраструктуре и стимулисања инвестиција у
водопривредне пројекте. Оне такође играју важну улогу у мобилисању
приватног капитала за финансирање великих пројеката који би били
финансијски неприступачни за јавни сектор сам по себи. Да би се избјегли
негативни аспекти коришћена овог модела пројектног финансирања
инфраструктурних пројеката, неопходно је осигурати законитост и
транспарентност у поступку додјеле концесија. Једнако важно је и да
се правосудни органи приликом доношења одлука крећу у оквирима
надлежности које су успостављене, не само између три гране власти већ
и између појединих нивоа власти у БиХ. Из наведених примјера видјели
смо да Уставни суд у појединим одлукама негира основне постулате
стварноправног уређења РС и БиХ. Уставни суд је допустио себи да преузме
улогу законодавца: као прво, нормирајући институт „државна имовина“
који не постоји у позитивним прописима РС и БиХ и друго, третирајући
„државну имовину“ као посебан облик права својине, која има специјалан
правни статус. Надаље, Уставни суд под исти правни режим ставља општа
и јавна добра, не правећи суштинску разлику између њих. Како општа
добра не могу бити предмет права својине, нејасно нам је на основу којих
правних чињеница Уставни суд тврди да је вода у морима, ријекама,
језерима својина државе, односно „државна имовина“. Проблематично је
и то што су одлуке у вези регулисање статуса „државне имовине“ директно
утицале на питања надлежности за додјелу концесија. Такве одлуке могу
да имају значајне правне и метаправне консеквенце на функционисање
цјелокупног система у РС и БиХ. 7. Закључак Воде представљају природно богатство које има непроцењив значај за
опстанак и развој друштва. Под посебном заштитом РС, ови ресурси су
не само еколошки важни, већ такође и економски, културно и социјално. Закон о концесијама у РС детаљно дефинише услове и оквире у којима
приватни и јавни сектор могу користити ове ресурсе. Кроз додјелу
концесија, дозвољено је коришћење и управљање водама уз одговарајућу 231 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 накнаду, али и уз стриктне услове који обезбеђују одрживост и дугорочну
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концесијама Републике Српске. Зборник радова Правног факултета у Нишу. 69(LIV). 295−311. 232 Б. Чубриловић Стаменић | стр. 215-236 Vassileva, A. Ignjatijević S. (2020). BOT (build-operate transfer) projects as a
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đanskog prava. Beograd: Pravni fakultet Univerziteta Union. Дивљак, Д. (2006). BOT модел пројектног финансирања инфраструктуре. Зборник радова Правног факултета у Новом Саду. 3. 275−288. Ђурђев, Д. (2004). Концесије. Зборник радова Правног факултета у Новом
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28/1994 − Амандмани XXVI-XLIII, 8/1996 − Амандмани XLIV-LI, 13/1996
− Амандман LII, 15/1996 − испр., 16/1996 − Амандман LIII, 21/1996 −
Амандмани XLIV-LI, 21/2002 − Амандмани LIV-LXV, 26/2002 − испр., 30/2002
− испр., 31/2002 − Амандмани XCIII-XCVIII, 69/2002 − Амандмани XCIX-CIII,
31/2003 − Амандмани CIV и CV, 98/2003 − Амандмани CVI-CXII, 115/2005
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CXXII и Сл. гласник БиХ, бр. 73/2019 − одлука УС БиХ. Закон о концесијама, Сл. гласник РС, бр. 59/13, 16/18, 70/20 и 111/21. Закон о концесијама БиХ, Сл. гласник БиХ, 32/02 и 56/04. Закон о стварним правима, Сл. гласник РС, бр. Литература / References 124/2008, 3/2009 − испр.,
58/2009, 95/2011, 60/2015, 18/2016 − одлука УС, 107/2019, 1/2021 − одлука
УС и 119/2021 − одлука УС. Закон о водама, Сл. гласник РС, 50/06, 92/09, 121/12 и 74/17. Закон о рударству, Сл. гласник РС, 62/18. Закон о рударству, Сл. гласник РС, 62/18. Закон о ловству, Сл. гласник РС, 60/09 и 50/13. Закон о јавно-приватном партнерству у Републици Српској, Сл. гласник
РС, 59/09, 63/11 и 68/20. Закон о стварним правима, Службене новине ФБиХ, 66/13, 100/13 и 32/19
− одлука УС. 233 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 Закон о власништву и другим стварним правима Брчко Дистрикта Босне
и Херцеговине, Сл. гласник Брчко Дистрикта БиХ, 11/01, 8/03, 40/04, 19/07,
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bosne-i-hercegovine.html. Устав БиХ. Преузето 23. 2. 2023. https://www.paragraf.ba/propisi/bih/ustav-
bosne-i-hercegovine.html. Analiza ekonomske opravdanosti koncesionih naknada i podsticaja za male
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електричне енергије − МХЕ „Самар“. Преузето 15. 2. 2023. https://reers.ba/
wp-content/uploads/2021/11/Rjesenje-o-produzenju-preliminarnog-prava-na-
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liza moratorijuma i zabrane izgradnje malih hidroelektrana u Bosni i Hercegovi-
ni, Crnoj Gori i Srbiji. Преузето 7. 2. 2023. https://www.reri.org.rs/wp-content/
uploads/2022/01/Uporedna-analiza-moratorijuma-i-zabrane-izgradnje-malih-
hidroelektrana-u-Bosni-i-Hercegovini-Crnoj-Gori-i-Srbiji.pdf. Report of the United Nations Water Conference. Преузето 30. 3. 2022. https://
www.ielrc.org/content/e7701.pdf. Report of the United Nations Water Conference. Преузето 30. 3. 2022. https://
www.ielrc.org/content/e7701.pdf. Одлука Уставног суда БиХ, бр. У-1/11, Службени гласник БиХ, 73/12. Одлука Уставног суда БиХ, бр. У-9/19, Службени гласник БиХ, 16/20. Одлука Уставног суда БиХ, бр. У-16/20, Службени гласник БиХ, 49/21. Одлука о утврђивању услова за додјелу концесије путем преговарачког
поступка за изградњу и коришћење ХЕ „Бук Бијела“ на ријеци Дрини,
општина Фоча, бр. 04/1-012-2-472/16 од 4. марта 2016, Сл. гласник РС, 20/16. Одлука о утврђивању услова за додјелу концесије путем преговарачког
поступка за изградњу и коришћење ХЕ „Фоча“ на ријеци Дрини бр. 04/1-
012-2-81/19 од 10. јануара 2019, Сл. гласник РС, 4/19. Рјешење о додјели концесије за изградњу и коришћење ХЕ „Бук Бијела“
на ријеци Дрини, општина Фоча, бр. 04/1-012-2-1099/16 од 20. маја 2016,
Сл. гласник РС, 42/16. Рјешење о додјели концесије за изградњу и коришћење ХЕ „Фоча“ на ријеци
Дрини бр. 04/1-0122-221/19 од 24. јануара 2019, Сл. гласник РС, 9/19. Рјешење о додјели концесије за изградњу и коришћење ХЕ „Паунци“ на
ријеци Дрини бр. 04/1-012-2-214/19 од 24. јануара 2019, Сл. гласник РС, 9/19. Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 Bosiljka Čubrilović Stamenić,
Senior Teaching Assistant,
Faculty of Law, University of Banja Luka
Republika Srpska Keywords: concession, water, Build-Operate-Transfer (BOT) model, “state proper-
ty”, public property. Литература / References Одлука о утврђивању услова за додјелу концесије путем преговарачког
поступка за изградњу и коришћење ХЕ „Паунци“ на ријеци Дрини број
04/1-012-2-80/19 од 10. јануара 2019, Сл. гласник РС, 4/19. 234 Б. Чубриловић Стаменић | стр. 215-236 Рјешење о додјели концесије за изградњу и коришћење ХЕ „Бук Бијела“
на ријеци Дрини, општина Фоча, бр. 04/1-012-2-1099/16 од 20. маја 2016,
Сл. гласник РС, 42/16. Рјешење о додјели концесије за изградњу и коришћење ХЕ „Бук Бијела“
на ријеци Дрини, општина Фоча, бр. 04/1-012-2-1099/16 од 20. маја 2016,
Сл. гласник РС, 42/16. Рјешење о додјели концесије за изградњу и коришћење ХЕ „Фоча“ на ријеци
Дрини бр. 04/1-0122-221/19 од 24. јануара 2019, Сл. гласник РС, 9/19. Рјешење о додјели концесије за изградњу и коришћење ХЕ „Фоча“ на ријеци
Дрини бр. 04/1-0122-221/19 од 24. јануара 2019, Сл. гласник РС, 9/19. Рјешење о додјели концесије за изградњу и коришћење ХЕ „Паунци“ на
ријеци Дрини бр. 04/1-012-2-214/19 од 24. јануара 2019, Сл. гласник РС, 9/19. Рјешење о додјели концесије за изградњу и коришћење ХЕ „Паунци“ на
ријеци Дрини бр. 04/1-012-2-214/19 од 24. јануара 2019, Сл. гласник РС, 9/19. Уговор о концесији бр. 05.05/012-274-16/16 од 3. јуна 2016. Уговор о концесији бр. 05.05/360-2-24/19 од 19. фебруара 2019. Уговор о концесији бр. 05.05/360-2-22/19 од 19. фебруара 2019. 235 235 Зборник радова Правног факултета у Нишу | Број 98 | Година LXII | 2023 Summary Summary The author’s interest in water usage under the concession model envisaged in the
domestic legislation of Republika Srpska was sparked by the Decision of the Con-
stitutional Court of Bosnia and Herzegovina in Case No. U-16/20. The analysis of
this decision has paved the way for broader reflection, leading to further research
which resulted in the creation of this article. Methodologically, the paper is rooted
in the normative analysis of the Concessions Act and the Real Rights Act, the latter
being the primary legislative act when discussing real property institutions in
Republika Srpska. While the initial segment of this article addresses the specific
legal regulation of certain kinds of properties and delineates the concepts of water,
water assets, and water entities as subjects of property rights, the main focus is
on pertinent issues related to water as the subject matter of concession. The scope
of concession can encompass a vast array of economic activities. In the context of
water, it typically pertains to the utilization of public water assets (e.g., water for
technological processes in economic activities, water and waterfronts for hydro
reservoirs, swimming pools, ponds, waterfronts for conducting certain economic
tasks, and extraction of materials from waterways). Furthermore, the energy po-
tential of water can be tapped when the objective of concession is the construction
and operation of energy plants, which stand as one of the paramount economic
facets of water utilization. The concluding section delves into the Constitutional
Court’s practices regarding the concession institution, as well as the notion of “state
property” as an increasingly emphasized topic in domestic jurisprudence. 236
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A Review Study on the Effect of Nanomaterials and Local Materials on Soil Geotechnical Properties
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E3S Web of Conferences 427, 01010 (2023)
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https://doi.org/10.1051/e3sconf/202342701010
A Review Study on the Effect of Nanomaterials and Local Materials on
Soil Geotechnical Properties
Eman Adnan1, a, Maki J. Al Waily1,b, and Zahraa F. Jawad1,c *
1Construction
and Building Department, Al Furat Al Awsat Technical University, Najaf, Iraq.
aeman.adnan.tcm.18@student.atu.edu.iql, bmaki_jafar@atu.edu.iq
and cdr_zahraajawad@atu.edu.iq
*Corresponding author
Abstract. Considerable strides have been made in nanotechnology in recent years, with many
nanotechnology-based achievements in the geotechnical-engineering. It seems certain that nanoparticles
would be widely utilized to develop the geotechnical qualities for soils in modern applications. This study covers
prior research on using nanomaterials in soil, their characteristics, and their influence on soil engineering. It
seems certain that nanoparticles will be widely utilized to develop soil geotechnical properties in the near future.
The application of current technologies to improve soil resistance requirements has resulted in the stability of
many soil types with formation issues. This study analyzes the use of some nanomaterials and lime as
additives or stabilization materials to improve soil resistance properties by geotechnical researchers.
Researchers have demonstrated that adding local materials such as lime enhances the properties of soil. Still,
a remarkable and interesting percentage of improvement was observed when adding nanoparticles to the soil
by improving its engineering qualities as well as those of some other components, such as lowering stability
and compressibility, raising density, and raising shear resistance.
Keywords: Soil Stabilization, Clayey Soil, Nano Silica, Nano Technology, Geotechnical, Resistance Metrics.
1. INTRODUCTION
The high compressibility and low shear strength of structures constructed on soft clay soils are just two of
the issues they encounter. The price of raw materials and the cost of the machinery needed to complete the
job make conventional installation methods pricey [1]. Of all the building resources, the soil is one of the most
plentiful ;when building embankments, highways, railways, and other engineering projects that call for a
significant volume of soil, where soils are the most frequently employed material. In most countries, clay soils
cover a sizable portion of the world's surface [2-4]. To enhance the undesired geotechnical qualities of soils
gypseous, soil stabilization is required [5]. The layers of soil used as a foundation for buildings, structures, and
substrates to be utilized as a substrate, the soil must be able to resist loading conditions within the range of
permitted deformations and transfer them to the ground. Strong and deformable characteristics are important.
The nanoscale is a feasible size for soil stability when additives are used. It is a technique used in engineering
to increase the strength and properties of soil. Nanotechnology has recently spread widely in many scientific
disciplines [6].
Because nanomaterials actively interact with other particles, even very small amounts can significantly
change the soil's physicochemical behavior and technical qualities [7,8]. By increasing the particular surface
area and favoring quantum effects [9]. In order to stabilize problematic soils with complex and erratic behavior,
like gypsum soils, which are mostly found in semi-dry and dry parts of the world and are prone to collapse
when wet, a variety of additions can be utilized. Nanomaterials were employed to examine their impact on the
geotechnical qualities of the soil to lessen the likelihood of soil collapse [10,11]. It was discovered that soil
behavior was altered at the nanoscale. With particle sizes from 1 to 100 nm, they offered a new kind of soil
particles, dubbed nano-cells. This study presents the discussion and evaluation of the effect of using
conventional materials (lime) and nanomaterials on the physical properties of soft soil by making an analysis
based on the results of geotechnical researchers' investigations.
2. STUDY METHOD
2.1 Statistical Frameworks
The method for choosing search terms and databases was followed to systematically identify sources for
review. A comprehensive analysis differs from a standard assessment in that it follows a sufficiently clear
method to allow other investigators to duplicate it, notably by giving an audit record of pertinent actions and
choices. To choose and assess the works, we employed the search window evaluation procedure and
recommended premium quality analysis techniques.
2.2 Criteria for Inclusion
Understanding at least one of our studies is required for a source to be regarded as credible and pertinent
to our review. A research project with a peer review standard training file for systematic and works reviews
must also be accessible in English. Peer-reviewed sources have been the only ones used in the literature
because they are highly relevant. The ultimate query phrases were Geotechnical engineering, soft clay,
resistance soil, and nanomaterial.
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/).
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a) EBSCO: Education Research Complete
b) Google Scholar
c) ACM Digital Library
d) Research gate
3. RESULTS AND DISCUSSION
The marine mud was taken from the Cochin District's Vipin District for the study. To contrast the effect of
silica nanoparticles on the mechanical properties of mud, investigations were conducted into how lime affected
the characteristics of marine mud. The geometric features investigated were Atterberg’s limit, Maximum dry
unit weight, appropriate moisture level, and unrestricted compressive resistance. Increasing proportions
decreased the indicator of plasticity [12], as shown in Figure 1.
Figure 1: Changes in plastic limit with time of curing in lime-added clay[12].
A 3%, 4%, and 5% soil lime ratio has been applied. In the study, nanosilica comprised 0.5%, 0.8%, and 1%
of the total material. For the periods of treatment 3, 7, 14, and 28 days, Atterberg boundary variability and
unconfined compressive strength variations were assessed. In lime-treated soils, a higher lime content
decreases the dry density. The soil treated with lime has a lower maximum dry density to boost the lime
concentration. The electrolyte concentration rises, and the diffuse double layer falls at greater lime
concentrations. The pressure curve is depicted in Figure 2.
Figure 2: Compaction curve of lime-modified clay [9].
For the nano silica-modified mud, the forte has increased three times. Lime-stabilized clay's ideal lime and
nano-silica contents were 4% and 0.8%, respectively. The plasticity properties were considerably altered by
comparing lime-modified clay to nano-silica additions (Figure 3) .
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Figure 3: Changes in unconfined compressive strength UCS of clay with lime and curing time [9].
Figure 4 illustrates how the unconfined compressive strength has improved. The plasticity index decreases
by 34, 38, and 41% compared to natural clay with 0.5, 0.8, and 1% of nanosilica, respectively.
Figure 4: Changes in plasticity index with drying time in clay treated with nano-silica [12].
One may conclude that for all nanoscale silica concentrations, an immediate drop in the plasticity index
can be seen because of the declining liquid limit and rising plastic limit, Figure 5.
Figure 5: Dry density and Water Content with Various Nano-Silica Content [12].
Figure 5 depicts how clay's water content and dry density are connected to the various nano-silica content
ratios. It becomes denser when soil treated with nano-silica includes more microscopic silica particles. As a
result, the unconfined compressive strength of soil is modified with nano-silica. The UCS increases as the
amount of nano-silica in the soil increases. The graph indicates that increasing the amount of silica
nanoparticles by 0.5, 0.8, and 1% increases the strength. The UCS values of 0.8% and 1% are comparable.
According to the slope of the UCS curves, more silica nanoparticles may result in higher resistance levels [12].
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Figure 6: Changes in UCC of clay amended with nano-silica with curing time [12].
Extensive soil was dosed with up to 12% LSD in 3% increments. The test results demonstrated that the
maximum dry density, hydraulic conductivity, swelling pressure, and liquid limit dropped consistently. In
contrast, the plastic limit, shrinkage limit, and ideal moisture content increased to 12% LSD addition. However,
when LSD was added at 9%, the UCS, wet CBR, cohesiveness, and internal friction angle increased to their
greatest values. The optimum dose of LSD for stabilizing expanding soils was discovered to be 9% [13].
Various amounts of limestone were applied to high-expansion clay. The findings suggested that adding
limestone powder could improve the geotechnical qualities of the soil. It is also recommended to include as
much limestone as possible in the expanded soil [14].
Limestone was added to the clay soil to improve it. The chosen soil, a fine clay from southeast Spain, had
its dry weight increased by 5, 10, 15, 20, and 25% by dry limestone dust. X-ray fluorescence and diffraction
are two typical geotechnical tests. Enhancing the geotechnical properties of the mixture Soils were assessed
using the one-dimensional uniformity test, free bulging index, unconfined compressive strength, and change
in Atterberg limits. Scanning electron microscopy was used to investigate the microstructure of mixed soils.
Overall, the results demonstrated decreased soil deformation and improved soil toughness in limestone [15].
The role of lime and nano-silica particles in soil enhancement was examined. According to the data, the UCC
force produced by the nano-mixed soil was 589 kN/m2, 1.2 times larger than that produced by non-nano
particles in soil compared to soil without nanoparticles [16]. Kalhor et al. [17] studied the evaluation of the
performance of clay soil that has been treated with nano-silica as an addition. The goal Tests on the Atterberg
boundary, typical pressure and permeability, and ungrouped (UU) were carried out in order to achieve this.
Using 1, 2, 3, and 4 wt% of NS mixed with fine-grained soil. Also utilized to view was scanning electron
microscopy (SEM). Specimens treated with NS and those not treated have different textures. X-ray diffraction
(XRF) fluorescence test was performed on a soil sample to assess the proportion of components present.
When NS is introduced to soil, the results show that it decreases. The variation of the plasticity index, plastic
limit, and liquid limit are shown in Figure 7.
Figure 7: Impact of nano silica content on plastic+ liquid limit and plasticity index [17].
The permeability coefficient of the NS-treated samples was discovered to be lower than that of the control
sample. Additionally, by performing 3-axis UU Tests on a material treated with 2% NS under confined pressure,
an additional 60–68% maximum strength was attained. Additionally, internal friction and cohesion angle In
comparison to the sample that wasn't treated with NS, the sample treated had an increase [17], as shown in
Figure 8(a,b,c).
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Figure 8: Stress-strain relation with different pressures of a) 30, b) 60, and c) 90 kPa [17].
Clay was tested for reaction to five to a hundred nanometer-sized silica nanoparticles, consolidation tests,
three-axis, and compressive strength. The results demonstrated that nanomaterials found in the soil were
configurable in the early phases of formation and that it later became elastoplastic [18]. Ren used electron
microscopy to examine the impact of nanoscale silica on the mechanical and physical properties of alluvial
clay, as well as a variety of tests to gauge its liquid and plastic limits, uniaxial pressure, specific gravity, and
frost lift. The concentration of nano silica rising Uniaxial compressive strength and the plastic and liquid
boundaries of the slurry both decreased the amount of frost produced. The clay sample's specific gravity did
not alter, though. The composition of the clay sample was unaffected by nanosilica, but it did reduce Its
structure is more uniform because of its average pore size [19]. Changizi and Haddad [20] examined how
adding nano silica particles to soft clay might affect its resistance and mechanical qualities. The Atterberg limit,
optimal moisture level, maximum dry weight, and compressive power were all measured using nanosilica.
Nano silica ratios were 0.5, 0.7, and 0.1% in contrast to soil. The limitation has improved in response to an
increase in nano silica content.
The plasticity index decreased, and contraction limitation increased as ratios increased. According to the
findings, viscose gel got thicker during the first stress before hardening from 70 kN/m2 for soft clay to 300
kN/m2 for stabilized soil with NS 0.7%. Up to 56% more compressive force is now available. The soil's elastic
modulus increased even though the tensile stress decreased as the nanosilica content increased. Based on
the findings, it can be said that the addition of nanosilica enhanced clay's mechanical and resistivity properties
[20]. Nano silica’s impact on clay particle size is depicted in Figures 9 to 11, illustrating Nano silica's effect on
compressive and shear strength, respectively.
Figure 9: Nano silica Impact on clay soil particles [20].
Figure 10: Nano silica impact on compressive strength [20].
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Figure 11: Nano silica impact on compressive strength (UCS) [20].
Ghavami et al. [21] studied the effects of an industrial byproduct, silica fume, and nanosilica on kaolin clay's
geotechnical and microstructural characteristics and soft soil with poor resistance capabilities. Adding silica
fume increased Kaolin clay's strength by 5, 10, and 15%. Additionally, nano silica was used to create stabilized
soils at concentrations of 1, 2, and 3% by weight of soil dry matter. Then, tests for the Atterberg limit, standard
control, unconfined compressive strength, and California bearing ratio were run. Additionally, a scanning
electron microscope SEM was used to detect the tiny structural modifications of soil samples caused by
fixation. According to the findings, silica dust and nanosilica increase the optimum water content while
decreasing the maximum dry density of the stabilized soil. When 15% silica fume and 3% nano-silica were
added to kaolin clay, the unconfined compressive strength rose by up to 70% and 55%, respectively, after 28
days of curing life, as shown in Figure 12.
Figure 12: Effect of the addition of nanosilica on UCS after (a) 7 days (b) 28 days [21].
Clay soils, direct shear testing, Atterberg boundary tests, normal pressure tests, and unconfined
compression strength (UCS) tests have all used nanosilica. Energy-dispersive X-ray spectroscopy, scanning
electron microscopy, and X-ray diffraction have also been used to investigate the mineralogy and
microstructure of core soils and treated samples EDS. The results showed that adding the appropriate amount
of 1% nanosilica to the dispersed clay changed the properties of the non-dispersed soil. Despite silica
nanoparticle treatment, the treated soil samples had a higher liquid limit, plastic limit, and moisture content,
resulting in a lower plasticity index and maximum dry density. After 7 and 28 days of treatment, samples treated
with 1% nanosilica content showed appreciable gains in absorbed energy, secant modulus of elasticity, and
direct shear strength [22].
The effects on the clay of adding lime and nano-silica in various ratios are examined in this study. According
to the findings, a little amount of nanoscale silica was added to the blended clay. When soil is supplemented
with lime, the qualities of plastics, including their strength, swelling, and compression, are significantly
improved. This study also examined the effects of treatment duration; the outcomes indicated that nano-silica
was an effective addition. In a shorter amount of time, it causes the lime-mixed soil to become stronger more
quickly. According to the study's findings, soil engineering properties should be improved for all projects [6].
Only 4% lime is used to prepare the samples for the first group, made from natural soft soil. Additionally,
the second group is created by combining a mixture of soil and 4% lime with CaCO 3 Nanomaterial in varying
amounts (0.25, 0.5, and 10%) as a final step. Using lime and nano CaCO3, the additive created in this work is
designed to treat the qualities of soft clay soil. Soft dirt has been delivered from the Iraqi city of Najaf [23].
Nano-SiO2 was added to the loess soil. The mechanical, structural, and mineralogical properties of loess soil
treated with nano-SiO2 and subjected to different contents and curing times. The mechanical behavior was
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investigated using untreated and treated loess's unconfined compressive strength (UCS). The results reveal
that the UCS increases with increasing SiO2 content and curing time, resulting in coarser particles, denser
packing, and smaller holes in treated loess. The outcomes of mineralogical component analysis further
increase inter-particle interaction [24].
Kalhor et al. [25] added Nano-SiO2 to the fine-grained soils and tested the freezing-defrosting cycles on the
geotechnical properties of the samples treated with Nano-SiO2. Unconfined compressive quality (UCS) was
improved by approximately 63% by adding 2% of nano-SiO2 and a relieving period of 42 days. Additionally,
the samples' conductivity becomes brittle due to the growth in nano-SiO2. Furthermore, the freeze-defrost
cycles reduce the UCS of the sample cycles. On clayey soil blended with nano-SiO2, laboratory tests such as
typical Proctor, Atterberg limits, and unconfined compression tests (UCTs) were performed. Furthermore, the
samples were cured for six weeks. As shown in Figures 13 to 15, when clay soils are stabilized and stabilized
using nano-silica, the maximum dry unit weight and PI decrease while the optimum moisture level, liquid limit
(LL), and plastic limit (PL) increase [25].
Figure 13: Strength variations of clay with nano silica combination (a) content of nanoparticles and (b) curing
period [25].
Figure 14: Impact of nanosilica on the stress-strain behavior [25].
Figure 15: Effect of Nano-SiO2-stabilized specimens on UCS [25].
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Gelsefifi added nanosilica to the soil to test the effectiveness of employing nanomaterials to stabilize weak
soil. Road in North Iran acquired the brittle soil and rated it as low-quality pliability clay. The study involved the
administration of 50 CBR tests. In the initial step, lime's impact on the weak soil's stability was investigated.
The findings demonstrated a negligible impact of lime on soil improvement. The impact of Nano-silica on the
second step. The study looked at soil-lime combination stability. The results showed that nano-silica
substantially impacted the soil-lime mixture, with the CBR strength of the soil and soil-lime being increased by
up to 21 and 7.5 times the combination, respectively. The results of this investigation, which also considered
the impacts of curing time, demonstrated that adding nanomaterial has a quicker effect on the soil-lime
mixture's CBR strength increase. A combination of 5% lime and 3% Nano-silica was the best mixture design
for stabilizing the Boodian poor soil [26].
In order to improve the Earth, scientists introduced two types of additions with the same elemental structure
but differing particle sizes: nano silica and micro silica. Both additions' effects on the mud's geotechnical
properties were compared through experimental studies. After 28 days of processing, samples were analyzed,
and the maximum percentage added for both additives was 6%. Differences in consistency limits, sample
pressure conditions, and the soil's undamaged shear strength were measured for each blend. The findings of
the unconfined compression test revealed that the influence of nano-SiO on the soil's unconfined compressive
strength and modulus of elasticity was more pronounced at dosages less than 2%. The strength of the samples
is brought near to that obtained for stable samples employing microsilica by increasing the amount of
nanomaterials. While microsilica followed a largely steady pattern, samples containing nanosilica showed a
greater rate of strong growth in concentrations of less than 1% nanosilica. The maximum dry density dropped
to 5% with adding additives, revealing a higher ideal moisture content. For both additions, there were some
subtle differences in the plasticity alterations.
In contrast to micro silica, which marginally decreased these parameters, adding more nano-SiO2
increased the liquid limit and soil plasticity index. The soil additions had no discernible impact on the soil's
chemical composition, according to X-ray diffraction (XRD). Furthermore, field emission scanning electron
microscopy (FESEM) analysis revealed that both materials were critical in lowering porosity and establishing
particle integrity [27].
Mohammadi investigated the effects of calcium carbonate nanoparticles on the geotechnical properties of
sand-clay soils. 0.3, 0.7, 1.1, and 1.5% of nano-calcium carbonate were added to SC soil that had been treated
for 7, 14, or 28 days. The soil also contained 10%, 20%, and 30% clay. The neural network Synthetic and
Group technique for data processing was used to quantitatively examine the experimental results. The
microstructure of soil samples supplemented with calcium carbonate nanoparticles was examined using X-ray
diffraction (XRD) examination of the crystalline phases. Nano-calcium carbonate was added to SC samples,
especially those with low clay concentrations, and both the uniaxial and overall compressive strengths
increased. It was shown that the optimal calcium carbonate level for soil with 10% clay content and 20% clay
content was 0.7%, followed by 1.1% for soil with 30% clay content, as in Figures 16 to 18.
Figure 16: Effect of curing time on UCS for stabilized soil with nano silica [28].
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Figure 17: Unconfined compressive strength of soil after 7, 14, and 28 days of curing at Nano-CaCO3 content
0.7% [28].
Figure 18: Unconfined compressive strength of soil after 7, 14, and 28 Days of Curing with Nano-CaCO3
contents 1.1% [28].
The calcium carbonate nanoparticles improved the compressive strength of the soil and intermolecular
recrystallization, according to the XRD patterns. The correlation between experimental results and the
upgraded SC soil's uniaxial compressive strength was found to have an average error of 4% [28]. Yong
investigated whether soil stabilizers made from calcium or magnesium hydroxide nanoparticles created using
the rapid deposition approach may enhance the engineering qualities of the residual tropical soils. Compaction,
Atterberg boundary, falling head permeability, and unconfined compressive strength were among the
geotechnical tests utilized to investigate untreated and nanoparticle-treated soils (UCS) engineering
properties. Researchers explored the fixation mechanisms connected to soil chemical reactions (EDX) utilizing
microscopic technologies such as variable pressure scanning electron microscopy X-ray diffraction (XRD) (VPSEM) and energy-dispersive X-ray spectroscopy. The researchers discovered that calcium hydroxide and
magnesium hydroxide nanoparticles improved the geotechnical properties of the remaining soil by lowering
hydraulic conductivity and increasing UCS. The percentages of soil treated with magnesium hydroxide and
calcium hydroxide particles that showed hydraulic conductivity after seven weeks of permeability were 84.14%
and 98.70%, respectively, compared to untreated soil. Magnesium hydroxide-treated soils experienced a 14day rise in UCS of 148.05%, whereas calcium hydroxide-treated soils experienced an increase of 180.17%
[29], as shown in Figures 19 to 21.
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Figure 19: Compaction curves of untreated [29].
Figure 20: Variations of hydraulic conductivity of soil samples treated with nanomaterials and curing time
[29].
Figure 21: Effect of curing time on unconfined compressive strength for treated and untreated samples [29].
Taipodia and Dutta [30] examined how clay's characteristics were affected by nano-calcium dichloride,
nano-calcium oxide, and nano-potassium nitrate particles and concluded that the use of nanoparticles
increased shear strength, decreased permeability, and decreased compressibility.
4. CONCLUSIONS
The stability and resistance of soil to clay were investigated in this study about the additions of NS, NanoCaCO3, and limestone. Since they have a unique surface and vigorously interact with other soil matrix particles,
these nanoparticles can significantly alter the soil engineering properties even after a brief exposure. This
research demonstrated how nanoparticles impact various attributes, including resistance, density,
permeability, and pressure. For this study, 30 studies looked at the effect of nanomaterials on soil resistivity.
The influence of each type of material can be summarized as follows:
• Lime: Decrease in soil deformation and increase in soil hardness, compressive strength, friction angle,
and cohesion.
• Nanosilica: Lower plasticity index and maximum dry density promote ideal water content and increase
compressive strength.
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Nano CaCO3: Improving the properties of plastic, including its strength, swelling, and compaction.
Nanoclay: Improving the strength, swelling, and compaction and promoting ideal water content.
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Selected Neurophysiological, Psychological, and Behavioral Influences on Subjective Sleep Quality in Nurses: A Structure Equation Model
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Selected Neurophysiological, Psychological, and
Behavioral Influences on Subjective Sleep Quality in
Nurses: A Structure Equation Model
Min-Huey Chung1*, Wen-I Liu2, Hui-Ling Lee3, Nanly Hsu4
1 Graduate Institute of Nursing, College of Nursing, Taipei Medical University, Taipei, Taiwan, 2 School of Nursing, National Taipei University of Nursing and Health
Sciences, Taipei, Taiwan, 3 Department of Nursing, Kang-Ning Junior College of Medical Care and Management, Taipei, Taiwan, 4 Nursing Department, Yuanpei University,
Hsinchu, Taiwan
Abstract
Few studies have examined relationships among neurophysiological, psychological, and behavioral factors with regard to
their effects on sleep quality. We used a structure equation model to investigate behavioral and psychological factors that
influence neurophysiological regulation of sleep in shift workers. Using a cross-sectional study design, we tested the model
with a sample of 338 female nurses working rotating shifts at an urban regional hospital. The Morningness-Eveningness
Questionnaire (MEQ) and short-form Menstrual Distress Questionnaire (MDQ) were used to measure neurophysiological
factors involved in morningness-eveningness and menstrual distress. The Sleep Hygiene Awareness and Practice Scale
(SHAPS) and Profile of Mood States Short Form (POMS-SF) were completed to measure behavioral factors of sleep hygiene
practices and psychological factors of mood states. In addition, the Pittsburgh Sleep Quality Index (PSQI) measured
participant’s self-reported sleep quality. The results revealed that sleep hygiene practices and mood states mediated the
effects of morningness-eveningness and menstrual distress on sleep quality. Our findings provide support for developing
interventions to enhance sleep hygiene and maintain positive mood states to reduce the influence of neurophysiological
factors on sleep quality among shift workers.
Citation: Chung M-H, Liu W-I, Lee H-L, Hsu N (2013) Selected Neurophysiological, Psychological, and Behavioral Influences on Subjective Sleep Quality in Nurses:
A Structure Equation Model. PLoS ONE 8(11): e79529. doi:10.1371/journal.pone.0079529
Editor: Allan Siegel, University of Medicine & Dentistry of NJ - New Jersey Medical School, United States of America
Received July 28, 2013; Accepted September 28, 2013; Published November 20, 2013
Copyright: ß 2013 Chung et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors thank Dr. Chien-Hua Wu for his excellent statistical analysis supported by a grant from the Department of Health, Taiwan (DOH100-TD-B111-003). This study was supported by a grant (NSC99-2314-B-038-012-MY3) from the National Science Council, Taiwan. 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.
* E-mail: minhuey300@tmu.edu.tw
sleep quality, and (H2) a positive association exists between sleep
hygiene practices and mood states.
Psychological factors include reactions to stressful and emotional situations, such as anxiety and mood states, which are
significantly associated with excessive worrying or depression
during shift work [9]. Psychological symptoms related to
neuroticism and depression were found to significantly increase
from the late follicular to the late luteal phase in healthy females
[10]. Mood states may be affected by menstrual distress. In
addition, a previous study showed that poor sleep quality was
significantly associated with negative mood states in a large cohort
of college students [11]. Thus, we also hypothesized the following:
(H3) a positive association exists between mood states and sleep
quality, and (H4) a positive association exists between mood states
and menstrual distress.
Neurophysiological factors can be considered reactions to
circadian variations and menstrual discomfort that are influenced
by biological rhythms of sleeping and waking [12,13]. Morningness and eveningness refer to variations in the circadian phases of a
person’s endogenous ‘‘clock’’ [14]. These diurnal types can be
measured using the Morningness-Eveningness Questionnaire
(MEQ), which distinguishes between morning (M)-type and
evening (E)-type persons based on preferential behavioral rhythms
in their daily activities and habitual sleep patterns [15]. According
Introduction
A considerable amount of evidence indicates that the effectiveness of non-drug treatments for insomnia is comparable to that of
pharmacologic treatments [1,2]. Conceptual models for these nondrug treatments comprise behavioral, psychological, and neurophysiological constructs. Behavioral factors related to sleep
hygiene may interfere with sleep quality [3]. Sleep hygiene is
defined as the establishment of appropriate sleep behaviors that
promote sleep quality [4]. These practices include maintaining a
stable sleep schedule, not using the bed or bedroom for activities
other than sleep, avoiding late-afternoon naps, and avoiding
emotionally, physiologically, or cognitively stimulating activities
before bedtime [4]. Insomnia patients frequently practice poor
sleep hygiene such as smoking or drinking alcohol before bedtime
[5]. Sleep hygiene practices may directly affect sleep quality.
Moreover, certain people tend to experience numerous changes in
their bedtimes, wake-up times, and durations of sleeping periods
[6,7]. Maintaining lifestyle regularity was associated with a
decreased risk of depression [8]. Adopting strategies, such as
habitual sleep patterns, to improve sleep hygiene may reduce
variations in mood. Thus, we hypothesized the following: (H1) a
positive association exists between sleep hygiene practices and
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November 2013 | Volume 8 | Issue 11 | e79529
Sleep and Structure Equation Model
Figure 1. Schematic representation of relationships among exogenous and endogenous variables (solid arrows represent
hypothesized paths) that were examined using the structure equation model.
doi:10.1371/journal.pone.0079529.g001
Taiwan. The inclusion criteria were as follows: (a) a registered
female nurse who provided direct care for patients, (b) a full-time
nurse who had worked rotating 8-h shifts for at least 3 months, and
(c) no history of psychiatric or neurological disorders. Nurses who
were pregnant at any time during the study were excluded. Of the
435 surveys distributed, 359 (82%) were returned, among which
21 were excluded for pregnancy and 2 were censored because of
missing data. Thus, 338 completed questionnaires were analyzed.
The questionnaire data assessed 19 parameters. Estimations of the
response rate and a valid sample size indicated that at least 10,15
participants were required per measured indicator [19]. Our crosssectional analysis of the 338 nurses had a statistical power of 0.8.
to the morningness-eveningness conceptual framework, people
display different sleep hygiene practices. E-types experience
numerous changes in their bedtimes, wake-up times, and durations
of sleeping periods [6,7]. Chung et al. showed that morningnesseveningness is a significant predictor of sleep quality, and
suggested that identifying a nurse’s diurnal type could assist in
correcting poor sleep quality [16]. Furthermore, the morningnesseveningness preference of shift workers significantly influences
their mood states [17]. Thus, we proposed the following
hypotheses: (H5) a negative association exists between morningness-eveningness and sleep hygiene practices; (H6) a negative
association exists between morningness-eveningness and sleep
quality; and (H7) a negative association exists between morningness-eveningness and mood states.
Women with severe menstrual symptoms are significantly more
likely to use tobacco, drink alcohol heavily, and be overweight
than women who do not have menstrual problems [18]. Smoking
and drinking heavily close to bedtime are poor sleep hygiene
practices [5]. Health behaviors related to sleep hygiene may be
related to menstrual distress. In addition, women experiencing
menstrual problems reported significantly increased distress, such
as insomnia and fatigue [18]. Therefore, we proposed two
additional hypotheses: (H8) a positive association exists between
menstrual distress and sleep hygiene practice, and (H9) a positive
association exists between menstrual distress and sleep quality.
To date, most studies of the contributions of neurophysiological,
behavioral, and psychological factors to sleep quality in nurses
examined only isolated factors. Consequently, relationships among
these factors in nurses remain unclear. In the present study, we
used a structure equation model to examine contextual factors
underlying sleep quality in nurses. We examined associations
among behavioral factors of sleep hygiene, psychological factors of
mood states, and neurophysiological factors involved in morningness-eveningness and menstrual distress and their effects on sleep
quality.
Ethical Considerations
Our study was approved by the Institutional Review Board at
Taipei Medical University Hospital. The purpose, content,
duration, and methods of the study, as well as the guidelines for
protecting their fundamental rights, were explained to potential
participants before the commencement of the study during a ward
meeting. All participants provided written consent. Data were
anonymously collected, remained confidential, and were used for
academic research purposes only. If a participant chose to
withdraw during the course of the study, the researcher fully
respected her right to do so, and guaranteed that her interests
would not be harmed as a result.
Instruments
Morningness-Eveningness Questionnaire (MEQ). The
MEQ is the most widely used questionnaire in chronopsychological research. It measures self-reported sleeping and waking
habits [20]. People who display a preference for waking up early in
the day are referred to as M-types. Conversely, E-types prefer
sleeping later, and experience better functioning in the evening
[21]. The MEQ contains 19 items, with possible total scores
ranging 16,86. Our participants’ diurnal preferences were
determined based on their MEQ scores. People who scored
$59 points were categorized as M-types, and those scoring
#41 points were categorized as E-types. Respondents scoring
42,58 points were considered neither type [20]. An acceptable
internal consistency of 0.71 was reported for the Chinese version
Materials and Methods
Participants
We performed a cross-sectional study using a convenience
sample of nurses recruited from an urban regional hospital in
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Sleep and Structure Equation Model
Table 1. Demographic characteristics, circadian types,
menstrual distress, sleep hygiene, mood states, and sleep
quality (N = 338).
Sleep quality (mean ± SD)
p value
$26
8.0963.04
0.206
,26
7.6363.45
Variable
Table 2. Correlations among selected neurophysiological,
psychological, and behavioral factors, and sleep quality
(N = 338).
BF
Age (years)
Nursing experience (years)
$1.79
,1.79
8.0563.02
0.220
7.6163.52
Body-mass index (kg/m2)
$24
7.9263.28
,24
7.3163.32
7.9263.30
Married
7.4263.23
7.8663.36
No
7.7163.01
7.8063.26
No
7.8763.34
7.3963.21
Evening shift
7.8263.62
Night shift
8.6962.94*
6.5362.63
Neither
7.8363.24
Evening
8.3063.57
0.865
,0.05
0.064
Menstrual distress score
$17
8.8063.39
,17
6.8062.83
,0.001
Sleep Hygiene Practice score
$31
9.1263.19
,31
6.4162.77
,0.001
Profile of Mood States score
$46
8.9763.20
,46
6.4062.87
,0.001
The dichotomized group was based on the median of each independent
variable except for the body-mass index; SD, standard deviation of the mean;
*p,0.05 vs day shift.
doi:10.1371/journal.pone.0079529.t001
of the MEQ [22]. Cronbach’s a for our study was acceptable at
0.74, with a split-half reliability of 0.70.
Short-Form Menstrual Distress Questionnaire (MDQ;
MDQ-SF). The MDQ assesses a woman’s feelings during her
menstrual cycle [23]. The questionnaire assesses 8 symptoms
covered by 47 items. The MDQ demonstrated acceptable
reliability and validity in previous studies, and the average a
coefficient for each subscale [24] ranged 0.64,0.88. The MDQSF used in our study contains 22 items that assess 4 symptoms,
including pain, water retention, autonomic reaction, and negative
effects, on a 5-point scale [25]. A higher score indicates greater
PLOS ONE | www.plosone.org
0.32*
NF1 (morningness-eveningness
score)
20.29** 20.15** 1.00
NF2 (menstrual distress score)
0.28*
0.41*
20.13* 1.00
SQ (sleep quality score)
0.49**
0.43*
20.13* 0.36*
1.00
1.00
insomnia patients with those of good sleepers [26]. The sleep
hygiene practice section contains 19 self-reported items that
inquire about how many days per week a participant practices
certain activities, including napping, regular use of sleep medications, waking up at the same time every day, and smoking or
drinking alcohol in the evening. Responses are scored on a 7-point
Likert scale ranging from 0 (very beneficial) to 7 (very disruptive).
Possible total scores range 0,133, and a higher score indicates
poorer sleep hygiene practices [26]. Acceptable test-retest reliability was reported for the SHAPS [27]. In our study, the Chinese
version of the SHAPS had a Cronbach’s a of 0.71 and a split-half
reliability of 0.74 after the ‘‘sleep disturbance through temperature’’ item was discarded.
Short-Form Profile of Mood States (POM-SF). We used
the POMS-SF to assess mood states of participants during the
week prior to data collection [28]. The 30-item questionnaire
consists of tension, depression, anger, fatigue, confusion, and vigor
subscales. Each subscale contains 5 items, each of which is scored
on a 5-point Likert scale ranging from 0 (not at all) to 4
(extremely). Possible global scores range 0,120, and a higher
score represents a higher level of mood disturbance [28,29]. The
internal consistency of the English version of the POMS-SF [28]
was shown to be at least 0.76. The Chinese version of the POMSSF respectively had internal consistency indices of 0.75,0.99 and
0.71,0.91 for patients with and those without pain. The content
validity index was 0.92 [30]. Cronbach’s a for the 6 POMS-SF
subscales in our study ranged 0.88,0.90.
Pittsburgh Sleep Quality Index (PSQI). The PSQI measures self-reported sleep quality and sleep disturbances during the
preceding month [31]. Its 7 components assess subjective sleep
quality, sleep latency, sleep duration, sleep efficiency, sleep
disturbances, use of sleeping medications, and daytime dysfunction. Each component is scored from 0 to 3, yielding a global score
ranging 0,21, with a higher score indicating poorer sleep quality
[31]. The reliability and validity of the PSQI were demonstrated
in primary insomnia patients of various populations [32]. The
PSQI has good internal consistency, with Cronbach’s a ranging
0.77,0.83 [33]. In studies of shift workers, Cronbach’s a for the
PSQI was 0.70, indicating acceptable reliability [34]. The Chinese
version of the PSQI demonstrated an internal consistency of 0.76
Circadian type
Morning
1.00
PF (Mood States score)
Sleep
Hygiene
Awareness
and
Practice
Scale
(SHAPS). The SHAPS compares sleep hygiene practices among
0.726
Shift schedule
Day shift
BF (Sleep Hygiene Practice score)
SQ
discomfort during menstruation. In our study, the MDQ-SF had a
Cronbach’s a of 0.92 and a split-half reliability of 0.93.
Consumption of coffee
Yes
NF2
0.284
Consumption of tea
Yes
NF1
BF, behavioral factors (sleep hygiene practice); PF, psychological factors (mood
states); NF1, neurophysiological factor I (morningness-eveningness); NF2,
neurophysiological factor II (menstrual distress); SQ, sleep quality.
*p,0.05;
**p,0.001.
doi:10.1371/journal.pone.0079529.t002
0.238
Marital status
Single
PF
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Sleep and Structure Equation Model
Figure 2. Model of selected behavioral, psychological, and neurophysiological factors that affect sleep quality. Model fit indices: x2/
d.f. = 2.335, goodness of fit index (GFI) = 0.981, adjusted GFI (AGFI) = 0.947, comparative fit index (CFI) = 0.991, normed-fit index (NFI) = 0.985, root
mean squared error of the approximation (RMSEA) = 0.063. Abbreviations: SHP, Sleep Hygiene Practices; POMS, Profile of Mood States; ME,
morningness-eveningness; MD, menstrual distress; PSQI, Pittsburgh Sleep Quality Index; PSQI_D, PSQI in the day shift; PSQI_E, PSQI in the evening
shift; PSQI_N, PSQI in night shift; error (e). * p,0.05. ** p,0.001.
doi:10.1371/journal.pone.0079529.g002
[35]. In our study, the global PSQI had a Cronbach’s a of 0.71,
indicating acceptable reliability.
to determine significant predictors of sleep quality. The technique
of parceling with an average of continuous multi-item scales was
used to construct item parcels. For model fit and parameter
estimations, a maximum-likelihood (ML) estimation was used, and
measurement errors were included in the model. Measurement
errors were computed for each latent variable as (12a)6d2, where
a is the reliability estimate (Cronbach’s a coefficient) of the
variable, and d2 is the variance of the mean score of the latent
construct as previously described [36]. Parceled data are a few
parameters to be estimated in defining a construct model and are
considered to less likely have correlated residuals or multiple crossloadings [37]. Therefore, parceled data may result in reductions in
various sources of sampling error.
To determine the ML estimation of the model, values for the
goodness-of-fit index (GFI), the adjusted GFI (AGFI), the normedfit index (NFI), the comparative fit index (CFI), and the root mean
squared error of the approximation (RMSEA) were calculated.
Statistical Analysis
All analyses were performed using the SPSS computer software,
vers. 20.0 for Windows (SPSS, Chicago, IL). Results of comparisons with a p value of ,0.05 were considered to represent
statistically significant differences. Scores for the SHAPS, POMSSF, MEQ, MDQ, and PSQI were compared using Student’s t-test,
and their relationships were explored using a Pearson correlation
analysis. Fitness indices of the SPSS AMOS 20.0 (IBM, Armonk,
NY) were used to estimate how well the hypothesized model fit the
actual data. A model that adequately fits the data can provide a
plausible representation of the causal structure of the study. Path
diagrams were used to graphically represent the underlying
structure of the variable effects in the hypothetical model
(Figure 1). We used latent variable modeling with item parceling
Table 3. Direct, indirect, and total effects of dominants on sleep quality.
Variable
Total effect
Direct effect
Indirect effect
BF
PF
SQ
BF
PF
SQ
BF
PF
SQ
NF1 (morningness-eveningness)
0.217
0.062
0.083
0.217
0.028
20.001
-
0.034
0.084
NF2 (menstrual distress)
0.345
0.441
0.364
0.345
0.386
0.139
-
0.055
0.225
BF (Sleep Hygiene Practice)
-
0.158
0.353
-
0.158
0.311
-
-
0.042
PF (Mood States)
-
-
0.267
-
-
0.267
-
-
-
BF, behavioral factors (sleep hygiene practice); PF, psychological factors (mood states); NF1, neurophysiological factor I (morningness-eveningness); NF2,
neurophysiological factor II (menstrual distress); SQ, sleep quality.
doi:10.1371/journal.pone.0079529.t003
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Sleep and Structure Equation Model
Table 4. Results of the mediation effect analysis.
Equation
Relationship
Regression weight
Standard error
Sobel test
95% Asymmetrical coefficient
interval
NF1RBFRSQ
NF1RBF
0.224
0.078
2.40*
(0.06, 0.44)
BFRSQ
1.005
0.230
NF2RBF
0.345
0.068
3.31*
(0.15, 0.62)
BFRSQ
1.005
0.230
NF2RPF
0.386
0.059
3.79*
(0.15, 0.55)
PFRSQ
0.861
0.185
BFRPF
0.158
0.072
1.98*
(0.01, 0.31)
PFRSQ
0.861
0.185
NF2RBFRSQ
NF2RPFRSQ
BFRPFRSQ
BF, behavioral factors (sleep hygiene practice); PF, psychological factors (mood states); NF1, neurophysiological factor I (morningness-eveningness); NF2,
neurophysiological factor II (menstrual distress); SQ, sleep quality.
*p,0.05.
doi:10.1371/journal.pone.0079529.t004
The GFI, AGFI, NFI, and CFI should achieve values of $0.90,
and the RMSEA should be at least 0.08 if the model fits the data
well [38]. We tested the hypothesis that psychological and
behavioral factors predicted sleep quality through interactions
with neurophysiological factors using a previously described
method [39]. The Sobel test was used to estimate the significance
of mediation effects [40]. In addition, the mediation effect on the
product-of-coefficients model was assessed with asymmetric
confidence intervals using the PRODCLIN2 program [41]. The
95% confidence intervals (CIs) did not include 0 to indicate the
existence of mediation effects.
In Figure 2, all path coefficients were significant and supported
all of the hypotheses, except for H6 and H7. The 4 equations of
indirect effects were used to examine mediation effects. As shown
in Table 4, the NF1RBFRSQ, NF2RBFRSQ, and
NF2RPFRSQ equations were used to estimate indirect effects
of NF on sleep quality. Mediating effects exerted through these
paths and the BFRPFRSQ equation were significant (Sobel test,
p,0.05), and the 95% CI did not include 0, indicating mediation
effects of behavioral and psychological factors on sleep quality.
Results
We tested a comprehensive conceptual framework for predicting the sleep quality of nurses. Statistical results supported the
hypothesis that neurophysiological factors influence sleep by
mediating psychological or behavioral regulation of normal sleep.
Because there is limited empirical data from previous studies
supporting the applicability of such a conceptual model, our results
provide evidence of the applicability of the conceptual model
among nurses. This is the first study to use a conceptual model to
investigate behavioral, psychological, and neurophysiological
factors underlying sleep quality in nurses. Our findings warrant
further study of the conceptual model in other types of healthcare
providers, in different clinical settings, and in different cultures.
Our findings demonstrated that behavioral, psychological, and
neurophysiological factors are significant predictors of sleep
quality. In Table 3, our results showed that behavioral factors,
such as sleep hygiene practices, caused direct and indirect effects
on sleep quality. Moreover, behavioral factors appeared to be the
strongest predictors of sleep quality (b = 0.31, p,0.001) as shown
in Figure 2. Behavioral factors may be important predictors of
sleep quality in nurses. Although it contrasts with those of some
previous studies [12,42], this finding is consistent with those of
others [43,44]. Sleep hygiene indicates that one maintains a
regular sleep/wake pattern and controls all environmental factors
that may interfere with sleep [4]. Nurses may know that shift
patterns induce further irregular sleep-wake times; they usually try
to practice good sleep hygiene. If they have difficulty sleeping at
night because of work, they might limit themselves to more hours
in bed at other times to reduce sleepiness on the night shift. The
misalignment of biological rhythms with social rhythms imposed
by one’s work schedule may explain why neurophysiological
factors influence sleep quality through mediation of the regulation
of sleep hygiene practice.
Discussion
Our study included 338 female shift nurses aged 27.8565.15
years, with an average of 3.5264.29 years of professional nursing
experience. The average body-mass index was 20.9063.20 kg/
m2. Most participants were unmarried (82.0%). The majority of
participants regularly drank tea or coffee (78.1% and 61.5%,
respectively). Most of the participants were categorized as neither
circadian type (71.9%). In Table 1, significant differences were
found in sleep quality between groups of shift schedule, menstrual
distress, sleep hygiene practice, and mood states. Therefore, the
PSQI score in the structure equation model was measured by 3
shift groups. Table 2 shows that significant positive correlations
existed for scores of sleep quality with mood states, sleep hygiene,
and menstrual distress. However, a significant negative correlation
was observed for scores of sleep quality with morningnesseveningness.
As shown in Figure 2, the statistical goodness of fit of the
structural model, denoted by the x2 value, was 2.335 (d.f. = 10,
p = 0.010). The GFI was 0.981, the AGFI was 0.947, the NFI was
0.985, the CFI was 0.991, and the RMSEA was 0.063. In the
model, sleep hygiene practice (b = 0.31, p,0.001), menstrual
distress (b = 0.14, p,0.05), and mood states (b = 0.27, p,0.001)
significantly predicted sleep quality. However, sleep quality was
not significantly affected by the variable of morningness-eveningness (b = 0.00, p = 0.919). Table 3 summarizes direct, indirect, and
total effects of dominant factors of sleep quality. According to path
coefficients, sleep hygiene practices exhibited the strongest direct
effects on sleep quality. Menstrual distress, despite showing a
weaker direct effect than sleep hygiene, exhibited the strongest
total effect on sleep quality.
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Sleep and Structure Equation Model
Table 2 shows that mood states were positively correlated with
menstrual distress and the PSQI score, such that poorer mood
states were associated with higher menstrual distress and poorer
sleep quality. Mood states also exerted direct influences on sleep
quality. These results are consistent with those of a previous study,
which showed that negative moods, such as depression and
anxiety, were associated with nighttime sleep [45]. Furthermore,
mood states mediated the effects of sleep hygiene practices on sleep
quality as shown in Figure 2 and Table 4. Mood states may be
affected by environmental and behavioral factors that are
considered part of sleep hygiene practices, such as lighting, noise,
temperature, hunger, caffeine intake, and the use of alcohol and
tobacco. However, determining whether related factors of mood
states are associated with sleep hygiene in predicting sleep quality
requires further study.
Menstrual distress, sleep hygiene, and mood states exerted
direct effects on sleep quality as shown in Table 3. The regulation
of sleep quality may be more affected by menstrual distress (total
effect = 0.372) than by morningness-eveningness (total effect = 0.067; Table 3). Nurses likely experience poor sleep quality
through indirect effects of neurophysiological factors, especially
menstrual distress (b = 0.35, p,0.001; Figure 2). These findings
are consistent with those of previous studies in which higher scores
for menstrual distress correlated with higher PSQI scores,
representing poorer sleep quality [46]. In addition, our results
also showed that menstrual distress affected sleep quality by
mediating mood states, and menstrual distress had stronger total
effects on sleep quality. Menstrual distress indicates the intensity of
symptoms that female nurses experience. Our results support a
previous study [47] which showed that women with menstrual
distress were significantly more likely to report frequent depression
and insomnia than those without menstrual-related problems.
These results imply that the effects of interventions on menstrual
distress are important factors among female shift workers.
The morningness-eveningness characteristics of nurses were
associated with their mood states as shown in Table 2. This result
is consistent with mood changes in morningness-eveningness
chronotypes in healthy individuals [48]. However, our results
showed that mediation effects of mood states were not significant
between morningness-eveningness and sleep quality. One possible
reason is that the neurophysiological factor of menstrual distress
and behavioral factors of sleep hygiene were added to the structure
equation model to decrease mediation effects. In Table 2, negative
correlations were also found between mornings-eveningness and
sleep quality. In regard to morningness-eveningness, participants
who belonged to the E-type had worse sleep quality. This result is
consistent with a previous study [49], which indicated that
eveningness was associated with a shorter time in bed during the
week. However, our results showed that morningness-eveningness
had diminished effects on sleep quality through the mediator of
sleep hygiene practice (b = 0.001, p = 0.919) in Figure 2. The
stronger effects of sleep hygiene practice on sleep quality (b = 0.31,
p,0.001) may explain why our findings were not consistent with
those of a study by Chung et al. [16]. Clarification of the causal
relationship between diurnal type and menstrual distress warrants
further investigation.
Certain limitations to our findings should be considered. Any
causality suggested by the path analysis must be interpreted
cautiously because of the cross-sectional design of our study. The
POMS assesses mood states during the preceding week, whereas
the PSQI assesses sleep quality during the preceding month. Thus,
this difference in time frames may have caused some bias. The
subjective scale of the SHAPS was not designed for shift workers,
and does not address behaviors for promoting sleep that may be
unique to shift workers, such as limited exposure to light during
the night shift. In addition, not all neurophysiological and
psychological factors were evaluated in our study. In future
studies, researchers should consider differences in bedtimes
resulting from habits or behaviors specifically related to the
participants’ working conditions. Night shift workers may drink
relatively high amounts of caffeine to assist them in staying awake
during the night. Therefore, the amount and timing of caffeine
ingestion should be recorded in future studies of shift workers. Our
sample consisted of young nurses with relatively little work
experience, all drawn from a single medical facility. These features
of our study group limit the generalization of the results regarding
the effects of diurnal preference, menstrual distress, mood states,
and sleep hygiene on sleep quality in nurses working rotating shifts.
Conclusions
We examined possible pathways of associations among behavioral, psychological, and neurophysiological factors that mediate
sleep quality in nurses. We evaluated the fitness of a conceptual
model to clarify factors that influence sleep quality in nurses. Our
results support the hypothesis that neurophysiological factors, such
as menstrual distress and morningness-eveningness, influence sleep
quality in nurses by mediating behavioral (sleep hygiene) and
psychological factors (mood states). Future longitudinal studies are
warranted to explore possible causal links between these factors.
The clinical significance of our findings is that the effects of
menstrual distress and morningness-eveningness on sleep quality
may be reduced by practicing good sleep hygiene and maintaining
positive mood states.
Author Contributions
Conceived and designed the experiments: HLL MHC. Performed the
experiments: HLL MHC. Analyzed the data: WIL MHC. Contributed
reagents/materials/analysis tools: NH MHC. Wrote the paper: MHC.
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English
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Novel Loci for Adiponectin Levels and Their Influence on Type 2 Diabetes and Metabolic Traits: A Multi-Ethnic Meta-Analysis of 45,891 Individuals
|
PLOS genetics
| 2,012
|
cc-by
| 25,337
|
Novel Loci for Adiponectin Levels and Their Influence on
Type 2 Diabetes and Metabolic Traits: A Multi-Ethnic
Meta-Analysis of 45,891 Individuals Winkler10, Bruce M. Psaty37,38, Russell P. Tracy39,
Jennifer Brody40, Ida Chen41, Jorma Viikari42, Mika Ka¨ho¨ nen43, Peter P. Pramstaller44,45,46,
David M. Evans4, Beate St. Pourcain47, Naveed Sattar48, Andrew R. Wood6, Stefania Bandinelli49,
Olga D. Carlson50, Josephine M. Egan50, Stefan Bo¨ hringer24, Diana van Heemst51,
Lyudmyla Kedenko52, Kati Kristiansson53, Marja-Liisa Nuotio53, Britt-Marie Loo54, Tamara Harris55,
Melissa Garcia55, Alka Kanaya56, Margot Haun20, Norman Klopp57, H.-Erich Wichmann57,58,59,
Panos Deloukas16, Efi Katsareli60, David J. Couper61, Bruce B. Duncan62,63, Margreet Kloppenburg64,
Linda S. Adair65, Judith B. Borja66, DIAGRAM+ Consortium{, MAGIC Consortium{, GLGC
Investigators{, MuTHER Consortium, James G. Wilson67, Solomon Musani68, Xiuqing Guo69,
Toby Johnson34,70,71, Robert Semple72, Tanya M. Teslovich13, Matthew A. Allison73, Susan Redline74,
Sarah G. Buxbaum75, Karen L. Mohlke25, Ingrid Meulenbelt19, Christie M. Ballantyne76,
George V. Dedoussis60, Frank B. Hu22, Yongmei Liu21, Bernhard Paulweber52, Timothy D. Spector15,
P. Eline Slagboom77, Luigi Ferrucci14, Antti Jula54, Markus Perola53, Olli Raitakari78,
Jose C. Florez30,79,80,81, Veikko Salomaa82", Johan G. Eriksson83,84,85,86,87", Timothy M. Frayling6",
Andrew A. Hicks44", Terho Lehtima¨ki12", George Davey Smith4", David S. Siscovick88",
Florian Kronenberg20", Cornelia van Duijn17,18", Ruth J. F. Loos8", Dawn M. Waterworth7",
James B. Meigs3,30", Josee Dupuis31,89", J. Brent Richards15,90"* Florian Kronenberg20", Cornelia van Duijn17,18", Ruth J. F. Loos8", Dawn M. Waterworth7",
James B. Meigs3,30", Josee Dupuis31,89", J. Novel Loci for Adiponectin Levels and Their Influence on
Type 2 Diabetes and Metabolic Traits: A Multi-Ethnic
Meta-Analysis of 45,891 Individuals Marie-France Hivert2,3., Nicholas Timpson4., John R. B. Perry5,6., Xin Yuan7., Zari Dastani1., Marie-France Hivert2,3., Nicholas Timpson4., John R. B. Perry5,6., Xin Yuan7.,
Robert A. Scott8., Peter Henneman9., Iris M. Heid10., Jorge R. Kizer11., Leo-Pekka Lyytika¨inen12.,
Christian Fuchsberger13., Toshiko Tanaka14, Andrew P. Morris5, Kerrin Small15,16, Aaron Isaacs17,18,
Marian Beekman19, Stefan Coassin20, Kurt Lohman21, Lu Qi22, Stavroula Kanoni16, James S. Pankow23,
Hae-Won Uh24, Ying Wu25, Aurelian Bidulescu26, Laura J. Rasmussen-Torvik27,
Celia M. T. Greenwood28, Martin Ladouceur29, Jonna Grimsby3,30, Alisa K. Manning31, Ching-Ti Liu31,
Jaspal Kooner32, Vincent E. Mooser7, Peter Vollenweider33, Karen A. Kapur34, John Chambers35,
Nicholas J. Wareham8, Claudia Langenberg8, Rune Frants9, Ko Willems-vanDijk9, Ben A. Oostra18,36,
Sara M. Willems17, Claudia Lamina20, Thomas W. Winkler10, Bruce M. Psaty37,38, Russell P. Tracy39,
Jennifer Brody40, Ida Chen41, Jorma Viikari42, Mika Ka¨ho¨ nen43, Peter P. Pramstaller44,45,46,
David M. Evans4, Beate St. Pourcain47, Naveed Sattar48, Andrew R. Wood6, Stefania Bandinelli49,
Olga D. Carlson50, Josephine M. Egan50, Stefan Bo¨ hringer24, Diana van Heemst51,
Lyudmyla Kedenko52, Kati Kristiansson53, Marja-Liisa Nuotio53, Britt-Marie Loo54, Tamara Harris55,
Melissa Garcia55, Alka Kanaya56, Margot Haun20, Norman Klopp57, H.-Erich Wichmann57,58,59,
Panos Deloukas16, Efi Katsareli60, David J. Couper61, Bruce B. Duncan62,63, Margreet Kloppenburg64,
Linda S. Adair65, Judith B. Borja66, DIAGRAM+ Consortium{, MAGIC Consortium{, GLGC
Investigators{, MuTHER Consortium, James G. Wilson67, Solomon Musani68, Xiuqing Guo69,
Toby Johnson34,70,71, Robert Semple72, Tanya M. Teslovich13, Matthew A. Allison73, Susan Redline74,
Sarah G. Buxbaum75, Karen L. Mohlke25, Ingrid Meulenbelt19, Christie M. Ballantyne76,
George V. Dedoussis60, Frank B. Hu22, Yongmei Liu21, Bernhard Paulweber52, Timothy D. Spector15,
P. Eline Slagboom77, Luigi Ferrucci14, Antti Jula54, Markus Perola53, Olli Raitakari78,
Jose C. Florez30,79,80,81, Veikko Salomaa82", Johan G. Eriksson83,84,85,86,87", Timothy M. Frayling6",
Andrew A. Hicks44", Terho Lehtima¨ki12", George Davey Smith4", David S. Siscovick88",
Florian Kronenberg20", Cornelia van Duijn17,18", Ruth J. F. Loos8", Dawn M. Waterworth7",
James B. Meigs3,30", Josee Dupuis31,89", J. Brent Richards15,90"* Zari Dastani1., Marie-France Hivert2,3., Nicholas Timpson4., John R. B. Perry5,6., Xin Yuan7.,
Robert A. Scott8., Peter Henneman9., Iris M. Heid10., Jorge R. Kizer11., Leo-Pekka Lyytika¨inen12.,
Christian Fuchsberger13., Toshiko Tanaka14, Andrew P. Morris5, Kerrin Small15,16, Aaron Isaacs17,18,
Marian Beekman19, Stefan Coassin20, Kurt Lohman21, Lu Qi22, Stavroula Kanoni16, James S. Pankow23,
Hae-Won Uh24, Ying Wu25, Aurelian Bidulescu26, Laura J. Rasmussen-Torvik27,
Celia M. T. Greenwood28, Martin Ladouceur29, Jonna Grimsby3,30, Alisa K. Manning31, Ching-Ti Liu31,
Jaspal Kooner32, Vincent E. Mooser7, Peter Vollenweider33, Karen A. Kapur34, John Chambers35,
Nicholas J. Wareham8, Claudia Langenberg8, Rune Frants9, Ko Willems-vanDijk9, Ben A. Oostra18,36,
Sara M. Willems17, Claudia Lamina20, Thomas W. PLoS Genetics | www.plosgenetics.org Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis Washington, Seattle, Washington, United States of America, 38 Group Health Research Institute, Group Health Cooperative, Seattle, Washington, United States of
America, 39 Departments of Pathology and Biochemistry, University of Vermont, Burlington, Vermont, United States of America, 40 Cardiovascular Health Research
Unit, University of Washington, Seattle, Washington, United States of America, 41 Medical Genetics Research Institute, Cedars Sinai Medical Center, Los Angeles,
California, United States of America, 42 Department of Medicine, University of Turku and Turku University Hospital, Turku, Finland, 43 Department of Clinical
Physiology, University of Tampere and Tampere University Hospital, Tampere, Finland, 44 Center for Biomedicine, European Academy Bozen/Bolzano (EURAC)
(Affiliated Institute of the University of Lu¨beck, Lu¨beck, Germany), Bolzano, Italy, 45 Department of Neurology, General Central Hospital, Bolzano, Italy,
46 Department of Neurology, University of Lu¨beck, Lu¨beck, Germany, 47 School of Social and community medicine, University of Bristol, Bristol, United Kingdom,
48 British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom, 49 Geriatric Unit, Azienda Sanitaria Firenze
(ASF), Florence, Italy, 50 Laboratory of Clinical Investigation, National Institute of Aging, Baltimore, Maryland, United States of America, 51 Gerontology and
Geriatrics, Leiden University Medical Center, Leiden, The Netherlands, 52 First Department of Internal Medicine, St. Adiponectin: Multiethnic Genome-Wide Meta-Analysis Johann Spital, Paracelsus Private Medical
University Salzburg, Salzburg, Austria, 53 Public Health Genomics Unit, Department of Chronic Disease Prevention, National Institute for Health and Welfare, and
Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki, Finland, 54 Population Studies Unit, Department of Chronic Disease Prevention,
National Institute for Health and Welfare, Turku, Finland, 55 Intramural Research Program, Laboratory of Epidemiology, Demography, and Biometry, National
Institute on Aging, National Institutes of Health, Bethesda, Maryland, United States of America, 56 Division of General Internal Medicine, Women’s Health Clinical
Research Center, University of California San Francisco, San Francisco, California, United States of America, 57 Institute of Epidemiology, Helmholtz Zentrum
Mu¨nchen, German Research Center for Environmental Health, Munich, Germany, 58 Institute of Medical Informatics, Biometry and Epidemiology, Ludwig-
Maximilians-Universita¨t, Munich, Germany, 59 Klinikum Großhadern, Munich, Germany, 60 Harokopio University, Athens, Greece, 61 Collaborative Studies
Coordinating Center, Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 62 School of
Medicine, Federal University of Rio Grande do Sul, Porto Alegre, Brazil, 63 Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina, United States of America, 64 Department of Rheumatology and Department of Clinical Epidemiology, Leiden, The Netherlands, 65 Department of
Nutrition, University of North Carolina, Chapel Hill, North Carolina, United States of America, 66 Office of Population Studies Foundation, University of San Carlos,
Cebu City, Philippines, 67 Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi, United States of America,
68 Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi, United States of America, 69 Medical Genetics Institute, Cedars-Sinai
Medical Center, Los Angeles, California, United States of America, 70 University Institute of Social and Preventative Medicine, Centre Hospitalier Universitaire
Vaudois (CHUV) and University of Lausanne, Lausanne, Switzerland, 71 Swiss Institute of Bioinformatics, Lausanne, Switzerland, 72 Metabolic Research
Laboratories, Institute of Metabolic Science, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom, 73 Department of Family and
Preventive Medicine, University of California San Diego, La Jolla, California, United States of America, 74 Brigham and Women’s Hospital, Boston, Massachusetts,
United States of America, 75 Jackson Heart Study Coordinating Center, Jackson State University, Jackson, Mississippi, United States of America, 76 Baylor College
of Medicine and Methodist DeBakey Heart and Vascular Center, Houston, Texas, United States of America, 77 Molecular Epidemiology, Leiden University Medical
Center, Leiden, The Netherlands, 78 Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku and the Department of Clinical
Physiology, Turku University Hospital, Turku, Finland, 79 Program in Medical and Population Genetics, Broad Institute, Cambridge, Massachusetts, United States of
America, 80 Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 81 Diabetes Research Center,
Diabetes Unit, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 82 Chronic Disease Epidemiology and Prevention Unit,
Department of Chronic Disease Prevention, National Institute for Health and Welfare, Helsinki, Finland, 83 Diabetes Prevention Unit, Department of Chronic
Disease Prevention, National Institute for Health and Welfare, Helsinki, Finland, 84 Unit of General Practice, Helsinki University Central Hospital, Helsinki, Finland,
85 Folkhalsan Research Centre, Helsinki, Finland, 86 Vaasa Central Hospital, Vaasa, Finland, 87 Department of General Practice and Primary Health Care, University
of Helsinki, Helsinki, Finland, 88 University of Washington, Seattle, Washington, United States of America, 89 National Heart, Lung, and Blood Institute’s
Framingham Heart Study, Framingham, Massachusetts, United States of America, 90 Departments of Medicine, Human Genetics, Epidemiology, and Biostatistics,
Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Canada Copyright: 2012 Dastani 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. Novel Loci for Adiponectin Levels and Their Influence on
Type 2 Diabetes and Metabolic Traits: A Multi-Ethnic
Meta-Analysis of 45,891 Individuals Brent Richards15,90"* 1 Department of Epidemiology, Biostatistics, and Occupational Health, Jewish General Hospital, Lady Davis Institute, McGill University, Montreal, Canada,
2 Department of Medicine, Universite´ de Sherbrooke, Sherbrooke, Canada, 3 General Medicine Division, Massachusetts General Hospital, Boston, Massachusetts,
United States of America, 4 MRC CAiTE Centre and School of Social and Community Medicine, University of Bristol, Bristol, United Kingdom, 5 Wellcome Trust
Centre for Human Genetics, University of Oxford, Oxford, United Kingdom, 6 Genetics of Complex Traits, Peninsula Medical School, University of Exeter, Exeter,
United Kingdom, 7 Genetics, GlaxoSmithKline, King of Prussia, Pennsylvania, United States of America, 8 MRC Epidemiology Unit, Institute of Metabolic Science,
Addenbrooke’s Hospital, Cambridge, United Kingdom, 9 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands,
10 Department of Epidemiology and Preventive Medicine, Regensburg University Medical Center, Regensburg, Germany, 11 Departments of Medicine and Public
Health, Weill Cornell Medical College, New York, New York, United States of America, 12 Department of Clinical Chemistry, University of Tampere and Tampere
University Hospital, Tampere, Finland, 13 Center for Statistical Genetics, Department of Biostatistics, University of Michigan, Ann Arbor, Michigan, United States of
America, 14 Clinical Research Branch, National Institute on Aging, Baltimore, Maryland, United States of America, 15 Department of Twin Research and Genetic
Epidemiology, King’s College London, London, United Kingdom, 16 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom,
17 Genetic Epidemiology Unit, Department of Epidemiology, Erasmus Medical Center, Rotterdam, The Netherlands, 18 Centre for Medical Systems Biology, Leiden,
The Netherlands, 19 Section of Molecular Epidemiology, Leiden University Medical Center and The Netherlands Genomics Initiative, The Netherlands Consortium
for Healthy Aging, Leiden, The Netherlands, 20 Division of Genetic Epidemiology, Innsbruck Medical University, Innsbruck, Austria, 21 Wake Forest University
School of Medicine, Winston-Salem, North Carolina, United States of America, 22 Harvard School of Public Health, Boston, Massachusetts, United States of America,
23 Division of Epidemiology and Community Health, University of Minnesota, Minneapolis, Minnesota, United States of America, 24 Medical Statistics and
Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands, 25 Department of Genetics, University of North Carolina, Chapel Hill, North Carolina,
United States of America, 26 Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia, United States of America, 27 Department of
Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America, 28 Lady Davis Institute for Medical
Research, Department of Oncology, McGill University, Montreal, Canada, 29 Department of Human Genetics McGill University, Montreal, Canada, 30 Department of
Medicine, Harvard Medical School, Boston, Massachusetts, United States of America, 31 Department of Biostatistics, Boston University School of Public Health,
Boston, Massachusetts, United States of America, 32 Cardiology, Ealing Hospital National Health Service (NHS) Trust, London, United Kingdom, 33 Department of
Internal Medicine, University of Lausanne, Lausanne, Switzerland, 34 Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland,
35 Epidemiology and Biostatistics, Imperial College London, London, United Kingdom, 36 Deptartment of Clinical Genetics and Department of Epidemiology,
Erasmus Medical Center, Rotterdam, The Netherlands, 37 Cardiovascular Health Research Unit, Departments of Medicine and Epidemiology, University of PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 1 Citation: Dastani Z, Hivert M-F, Timpson N, Perry JRB, Yuan X, et al. (2012) Novel Loci for Adiponectin Levels and Their Influence on Type 2 Diabetes and
Metabolic Traits: A Multi-Ethnic Meta-Analysis of 45,891 Individuals. PLoS Genet 8(3): e1002607. doi:10.1371/journal.pgen.1002607 Abstract Salzburg Atherosclerosis Prevention Program in subjects at
High Individual Risk (SHAPIR): Part of this work was funded by the ‘‘Genomics of Lipid-associated Disorders’’ (GOLD) of the ‘‘Austrian Genome Research Programme’’
(GEN-AU) to Florian Kronenberg, and grants from the ‘‘Medizinische Forschungsgesellschaft Salzburg’’ and the ‘‘Kamillo Eisner Stiftung’’ (Switzerland) to Bernhard
Paulweber. THISEAS: The genotyping of the THISEAS study was funded by the Wellcome Trust. The recruitment was partially supported by the General Secretary of
Research and Technology (PENED 03,EPSILON.,DELTA.474). We are grateful to all the volunteers for their time and help, the medical staff of the hospitals and the
field investigators, Eirini Theodoraki, Maria Dimitriou and Kathy Stirrups for her assistance in the genotyping. Cebu Longitudinal Health and Nutrition Survey (CLHNS):
We thank the Office of Population Studies Foundation research and data collection teams and the study participants who generously provided their time for this
study. This work was supported by National Institutes of Health grants DK078150, TW05596, HL085144, RR20649, ES10126, and DK56350. Coordinating Centre: McGill
University. This work was supported by grants from the Canadian Foundation for Innovation, the Canadian Institutes of Health Research (CIHR), Fonds de la recherche
en sante´ du Que´bec, the Lady Davis Institute, the Ministe`re du De´veloppement e´conomique, de l’Innovation et de l’Exportation du Que´bec and the Jewish General
Hospital. JB Richards and Z Dastani are supported by the CIHR. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. Competing Interests: DM Waterworth, X Yuan, and VE Mooser are full-time employees of GlaxoSmithKline. P Vollenweider received grant money from
GlaxoSmithKline to fund the CoLaus study The other authors declare no competing financial interests Competing Interests: DM Waterworth, X Yuan, and VE Mooser are full-time employees of GlaxoSmithKline. P Vollenweider received grant money from
GlaxoSmithKline to fund the CoLaus study. The other authors declare no competing financial interests. * E-mail: brent.richards@mcgill.ca . These authors contributed equally to this work. " These authors also contributed equally to this work. { Memberships of these consortia are provided in the Acknowledgments. clinical parameters including blood glucose, indices of insulin
resistance, proatherogenic dyslipidemia, and risk of type 2 diabetes
(T2D), stroke and coronary artery disease [1,2,3,4]. Collectively
these conditions account for most of the burgeoning pandemic of Abstract Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely
associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide
association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We
identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.561028–
1.2610243). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and
N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples
revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after
accounting for multiple testing (p,361024). We next developed a multi-SNP genotypic risk score to test the association
of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk
score was associated with increased risk of T2D (p = 4.361023, n = 22,044), increased triglycerides (p = 2.6610214,
n = 93,440), increased waist-to-hip ratio (p = 1.861025, n = 77,167), increased glucose two hours post oral glucose
tolerance testing (p = 4.461023, n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL-
cholesterol concentrations (p = 4.5610213, n = 96,748) and decreased BMI (p = 1.461024, n = 121,335). These findings
identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of
insulin resistance. Citation: Dastani Z, Hivert M-F, Timpson N, Perry JRB, Yuan X, et al. (2012) Novel Loci for Adiponectin Levels and Their Influence on Type 2 Diabetes and
Metabolic Traits: A Multi-Ethnic Meta-Analysis of 45,891 Individuals. PLoS Genet 8(3): e1002607. doi:10.1371/journal.pgen.1002607 Editor: Peter M. Visscher, The University of Queensland, Australia Received September 30, 2011; Accepted February 3, 2012; Pub PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 PLoS Genetics | www.plosgenetics.org 2 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis Funding: Baltimore Longitudinal Study of Aging (BLSA): The BLSA was supported in part by the Intramural Research Program of the NIH, National Institute on Aging. A portion of that support was through an R&D contract with MedStar Research Institute. Erasmus Rucphen Family (ERF). Abstract The ERF study was supported by grants from
The Netherlands Organisation for Scientific Research, Erasmus MC and the Centre for Medical Systems Biology (CMSB), and the European Network for Genetic and
Genomic Epidemiology (ENGAGE) consortium. Invecchaire in Chianti (InCHIANTI). JRB Perry is a Sir Henry Wellcome Postdoctoral Research Fellow (092447/Z/10/Z). Framingham Heart Study (FHS): This research was conducted in part using data and resources from the Framingham Heart Study of the National Heart Lung and Blood
Institute of the National Institutes of Health and Boston University School of Medicine. The analyses reflect intellectual input and resource development from the
Framingham Heart Study investigators participating in the SNP Health Association Resource (SHARe) project. This work was partially supported by the National Heart,
Lung, and Blood Institute’s Framingham Heart Study (Contract No. N01-HC-25195) and its contract with Affymetrix, Inc for genotyping services (Contract No. N02-HL-
6-4278). A portion of this research utilized the Linux Cluster for Genetic Analysis (LinGA-II) funded by the Robert Dawson Evans Endowment of the Department of
Medicine at Boston University School of Medicine and Boston Medical Center. The Collaborative Health Research in the Region of Augsburg (KORA F3): This study was
partially funded by the ‘‘Tiroler Wissenschaftsfonds’’ (Project UNI-0407/29) and by the ‘‘Genomics of Lipid-associated Disorders – GOLD’’ of the ‘‘Austrian Genome
Research Programme GEN-AU’’ to F Kronenberg. The MONICA/KORA Augsburg cohort study was financed by the Helmholtz Zentrum Mu¨nchen. It was further funded
by the NIH subcontract from the Children’s Hospital, Boston, US, (H-E Wichmann and IM Heid, prime grant 1 R01 DK075787-01A1 to JN Hirschhorn) and the German
National Genome Research Net NGFN2 and NGFNplus (H-E Wichmann 01GS0823). TwinsUK: Study was funded by the Wellcome Trust, European Commission
Framework (FP7/2007–2013), ENGAGE project HEALTH-F4-2007-201413, and the FP5 GenomEUtwin Project (QLG2-CT-2002-01254). It also receives support from the
Arthritis Research Campaign, Chronic Disease Research Foundation, the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre
award to Guy’s and St Thomas’ NHS Foundation Trust in partnership with King’s College London, and a Biotechnology and Biological Sciences Research Council
project grant (G20234). Cardiovascular Health Study (CHS): The CHS research reported in this article was supported by contract numbers N01-HC-85079 through N01-
HC-85086, N01-HC-35129, N01 HC-15103, N01 HC-55222, N01-HC-75150, N01-HC-45133, grant numbers U01 HL080295 and R01 HL087652, HL105756, and HL094555
from the National Heart, Lung, and Blood Institute, with additional contribution from the National Institute of Neurological Disorders and Stroke. Abstract A full list of principal
CHS investigators and institutions can be found at http://www.chs-nhlbi.org/pi.htm. DNA handling and genotyping was supported in part by National Center for
Research Resources grant M01-RR00425 to the Cedars-Sinai General Clinical Research Center Genotyping core and National Institute of Diabetes and Digestive and
Kidney Diseases grant DK063491 to the Southern California Diabetes Endocrinology Research Center, NHLBI R01-HL085251. Helsinki Birth Cohort Study (HBCS): HBCS
has been supported by grants from Academy of Finland (project numbers 114382, 126775, 127437, 129255, 129306, 130326, 209072, 210595, 213225, 216374), Finnish
Diabetes Research Society, Finnish Foundation for Pediatric Research, Samfundet Folkha¨lsan, Juho Vainio Foundation, Novo Nordisk Foundation, Finska
La¨karesa¨llskapet, Pa¨ivikki and Sakari Sohlberg Foundation, Signe and Ane Gyllenberg Foundation, and Yrjo¨ Jahnsson Foundation. DILGOM survey was funded by the
Finnish Academy, grant number 118065. Cardiovascular Risk in Young Finns (YFS): The Young Finns Study has been financially supported by the Academy of Finland:
grants 126925, 121584, 124282, 129378 (Salve), 117787 (Gendi), and 41071 (Skidi), the Social Insurance Institution of Finland, Kuopio, Tampere and Turku University
Hospital Medical Funds, Juho Vainio Foundation, Paavo Nurmi Foundation, Finnish Foundation of Cardiovascular Research (T.L., OT.R), Tampere Tuberculosis
Foundation (Te.Le., Mik, Ka¨), the Emil Aaltonen Foundation (T.L.) and Finnish Cultural Foundation. The expert technical assistance in the statistical analyses by Irina
Lisinen and Ville Aalto are gratefully acknowledged. Dietary, Lifestyle, and Genetic determinants of Obesity and Metabolic syndrome (DILGOM): K Kristiansson was
supported by the Orion-Farmos Research Foundation and the Academy of Finland (grant no. 125973). M Perola and V Salomaa were supported by the Finnish
Foundation for Cardiovascular Research, the Sigrid Juse´lius Foundation, and the Academy of Finland (grants 129322, 129494 and 139635). JG Eriksson was supported
by the Academy of Finland (grants 126775, 129255, 129907, and 135072). Fenland study: The Fenland Study is funded by the Wellcome Trust and the Medical
Research Council, as well as by the Support for Science Funding programme and CamStrad. We are grateful to all the volunteers for their time and help, and to the
General Practitioners and practice staff for help with recruitment. We thank the Fenland Study co-ordination team and the Field Epidemiology team of the MRC
Epidemiology Unit for recruitment and clinical testing. Multiethnic Study of Atherosclerosis (MESA): The MESA project is conducted and supported by the National
Heart, Lung, and Blood Institute (NHLBI) in collaboration with MESA investigators. Abstract Support is provided by grants and contracts N01 HC-95159, N01-HC-95160, N01-HC-
95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169, and RR-024156. Funding for CARe
genotyping was provided by NHLBI Contract N01-HC-65226. Jackson Heart Study (JHS): The Jackson Heart Study is supported by the National Heart, Lung, and Blood
Institute, through contracts with Jackson State University (N01-HC-95170), the University of Mississippi Medical Center (N01-HC-95171), and Tougaloo College (N01-
HC-95172). Adiponectin measurements used in the current study were funded by PHS Award UL1 RR025008 from the Clinical and Translational Science Award
program, National Institutes of Health, National Center for Research Resources (NCRR). Health ABC: This research was supported by NIA contracts N01AG62101,
N01AG62103, and N01AG62106. The genome-wide association study was funded by NIA grant 1R01AG032098-01A1 to Wake Forest University Health Sciences and
genotyping services were provided by the Center for Inherited Disease Research (CIDR). CIDR is fully funded through a federal contract from the National Institutes of
Health to The Johns Hopkins University, contract number HHSN268200782096C. This research was supported in part by the Intramural Research Program of the NIH,
National Institute on Aging. Genetics, Arthrosis , and Progression) study (GARP): This study was supported the Leiden University Medical Centre and the Dutch Arthritis
Association. Pfizer, Groton, CT, USA supported the inclusion of the GARP study. The genotypic work was supported by the Netherlands Organization of Scientific
Research (MW 904-61-095, 911-03-016, 917 66344 and 911-03-012), Leiden University Medical Centre and the Centre of Medical System Biology and Netherlands
Consortium for Healthy Aging both in the framework of the Netherlands Genomics Initiative (NGI). The adiponectin measurements were supported by TI-Pharma. Atherosclerosis Risk in Communities (ARIC): The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung,
and Blood Institute contracts N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, N01-HC-55022, R01HL087641,
R01HL59367, R01HL086694, and RC2 HL102419; National Human Genome Research Institute contract U01HG004402; National Institutes of Health contract
HHSN268200625226C; and National Institute of Diabetes and Digestive and Kidney Diseases R01DK056918. Infrastructure was partly supported by Grant Number
UL1RR025005,a component of the National Institutes of Health and NIH Roadmap for Medical Research. Competing Interests: DM Waterworth, X Yuan, and VE Mooser are full-time employees of GlaxoSmithKline. P Vollenweider received grant money from
GlaxoSmithKline to fund the CoLaus study. The other authors declare no competing financial interests. Results of Meta-Analysis of GWAS The meta-analysis was divided into four phases 1) Discovery
phase, which involved cohorts providing GWAS results, 2) In-
silico replication phase which included additional GWAS cohorts
joining our meta-analysis after the completion of the discovery
phase,
3)
De-novo
genotyping
in
cohorts
without
GWAS
genotyping and 4) Multi-Ethnic meta-analysis applying a novel
method for complex trait mapping using different ethnicities. Discovery
phase
in
individuals
of
white
European
origin. The meta-analysis of sex-combined data from 16
GWAS (n = 29,347) of individuals of white European descent
identified ten loci associated with adiponectin levels at p#5.061028
(Table 1 and Figure 1A and Figure S1, Table S2). These results
include the previously described associations with adiponectin at
ADIPOQ (rs6810075[T]; ß = 0.06, p-value = 3.60610241), KNG1
(rs2062632[T]; ß = 0.05, p-value = 2.52610219) on 3q27.3, and
CDH13 (rs12922394[T; ß = 20.1, p = 3.16610218) on 16q23.3
(Table 1). Furthermore, we identified variants that showed genome-
wide significant association in eight novel independent loci
including rs9853056 (within the STAB1 gene, rs4282054 (within
the NT5DC2 gene), rs13083798 (within the PBRM1 gene), rs1108842
(within the GNL3 gene), rs11235 (within the NEK4 gene), rs2710323
(within the ITIH1 gene), rs3617 (within the ITIH3 gene), and
rs2535627 (within 200 Kb of ITIH4 gene) at 3p21.1; rs1597466
(within 1 Mb of TSC22D2 gene) at 3q25.1; rs2980879 (within 1 Mb
of TRIB1 gene) at 8q24.13; rs7955516 (within 1.3 Mb PDE3A gene)
at 12p12.2; rs601339 (within the GPR109A gene) at 12q24.31;
rs6488898 (within the ATP6V0A2 gene), rs7133378 (within the
DNAH10 gene), rs7305864 (within the CCDC92 gene), and
rs7978610 (within the ZNF664 gene at 12q24.31, which is 1.3 Mb
away from GPR109A); rs2925979 (within the CMIP at 16q23.2
gene); and rs731839 (within the PEPD gene) at 19q13.11. (Figure 2A–
2E, Table 1). obesity-related morbidity and mortality that poses a severe and
global healthcare challenge [5]. Murine studies suggest that
adiponectin plays a mediating role in at least some of these obesity-
related complications, and although less clearly established in
humans, this suggests that understanding the pathophysiology of
adiponectin may uncover novel therapeutic targets in major,
highly prevalent human disease.[6,7]. Twins and family studies have revealed moderate to high
estimates of heritability (30–70%) for plasma adiponectin levels
[8,9,10,11]. However, until recently, few genes associated with
adiponectin levels have been identified. Candidate and genome-
wide
association
studies
(GWAS)
have
shown
pronounced
associations between common polymorphisms in the adiponectin
gene (ADIPOQ) and adiponectin levels [12,13,14,15]. Results of Meta-Analysis of GWAS A recent
meta-analysis of three GWAS for adiponectin levels identified
variants in a novel candidate gene, ARL15, that were associated
with adiponectin levels, coronary heart disease (CHD), T2D and
other metabolic traits [16]. Furthermore, CDH13 and KNG1 genes
were found to be associated with adiponectin levels in two studies
involving East Asian populations [17,18]. Although part of the
variance explained by the ADIPOQ locus, most of the heritability of
adiponectin levels remains unaccounted for. Therefore, we sought
to identify novel common variants influencing adiponectin levels
and test their association with risk of T2D and related metabolic
traits within the framework of a large multi-ethnic consortium of
GWAS. In our analysis a common variant (rs601339, MAF = 0.18, allele
G) downstream of the GPR109A gene (the putative niacin receptor)
was associated with adiponectin (ß = 0.04, p = 7.94610210) and
HDL-C (ß = 0.03, p = 5.5961027) in the global lipid analysis. In a
coincident candidate gene analysis 11 SNPs were typed in
GPR109A/B in CoLaus and LOLIPOP cohorts, containing
individuals of European descent. A single nominally significant
coding SNP R311C (rs7314976, MAF = 0.14) within the GPR109A
gene was taken forward for replication and found to be consistently
associated with adiponectin in the three cohorts (CoLaus, Fenland
and MRC Ely study, n = 8285, p = 4.661028) and HDL-cholesterol
(HDL-C) in four cohorts (CoLaus, Fenland, Ely study and Lolipop,
n = 18425, p = 2.961028) (Figure S2A, S2B). However R311C and
rs601339 were not in linkage disequilibirium with each other
(r2 = 0.04). Therefore the two variants represent two independent
signals from the same locus but with similar effects on HDL-
cholesterol and adiponectin. We combined genome-wide association results of 35,355
individuals from three different ethnicities (white Europeans
(n = 29,347), African American s(n = 4,232) and East Asians
(n = 1,776)), applying a novel meta-analytic method to allow for
heterogeneity in allelic effects between populations of different
ethnic backgrounds. We next examined whether identified
genome-wide significant single nucleotide polymorphisms (SNPs)
also associated with expression of their nearest gene in human
adipocytes, the main source of adiponectin. Since adiponectin has
been associated with T2D, insulin resistance and metabolic traits
we next investigated whether a multi-SNP genotypic risk,
comprising genome-wide significant SNPs for adiponectin levels,
also influenced risk of T2D and related traits measured in the
DIAbetes Genetics Replication and Meta-analysis (DIAGRAM+) In silico follow-up phase. Author Summary Serum adiponectin levels are highly heritable and are
inversely correlated with the risk of type 2 diabetes (T2D),
coronary artery disease, stroke, and several metabolic
traits. To identify common genetic variants associated with
adiponectin levels and risk of T2D and metabolic traits, we
conducted a meta-analysis of genome-wide association
studies of 45,891 multi-ethnic individuals. In addition to
confirming that variants at the ADIPOQ and CDH13 loci
influence adiponectin levels, our analyses revealed that 10
new loci also affecting circulating adiponectin levels. We
demonstrated that expression levels of several genes in
these
candidate
regions
are
associated
with
serum
adiponectin levels. Using a powerful novel method to
assess the contribution of the identified variants with other
traits using summary-level results from large-scale GWAS
consortia, we provide evidence that the risk alleles for
adiponectin are associated with deleterious changes in
T2D risk and metabolic syndrome traits (triglycerides, HDL,
post-prandial glucose, insulin, and waist-to-hip ratio),
demonstrating that the identified loci, taken together,
impact upon metabolic disease. Introduction Adiponectin is a highly abundant adipocyte-derived plasma
protein whose levels correlate inversely with a range of important PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 PLoS Genetics | www.plosgenetics.org 3 Adiponectin: Multiethnic Genome-Wide Meta-Analysis [19], Meta-Analysis of Glucose and Insulin Related Traits
Consortium (MAGIC) [20], Genetic Investigation of ANthropo-
metric measures Traits (GIANT) [21] , Global Lipids Genetic
Consortium (GLGC) [22], and Body Fat GWAS consortia [23]. PLoS Genetics | www.plosgenetics.org Results of Meta-Analysis of GWAS In the in-silico follow-up phase
468 SNPs demonstrating genome-wide significant (p,561028 ) or
suggestive
(p,561026)
association
with
adiponectin
in the
discovery
phase
were
tested
for
association
in
additional
European cohorts. (Table S3). These SNPs were tested in seven
additional GWAS datasets (n = 6,623 from NHS, HPFS, HABC,
ERF2, LLS, GARP and ARIC studies) and the combined meta- March 2012 | Volume 8 | Issue 3 | e1002607 4 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Table 1. Lead SNP per Locus for Genome-Wide Significant SNPs Arising from the Sex-Combined Meta-Analysis in European
Populations. Table 1. Lead SNP per Locus for Genome-Wide Significant SNPs Arising from the Sex-Combined Meta-Analysis in European
Populations. Nearest**
Gene
Lead SNP{
Region
Chr/position{
EA/NEA"
EAF""
Beta1
SE
P
I2
n
Beta1
SE
P
I2
n
Discovery Phase Results
Joint Analysis Phase*
LYPLAL1
rs3001032
1q41
1/217794402
T/C
0.7
20.02
0.005
1.98E-06 0
29,321
20.02
0.004
3.60E-08 0
35,930
GNL3
rs1108842
3p21.1
3/52695120
C/A
0.50
0.03
0.004
3.66E-11 0.33
29,338
0.03
0.004
1.39E-13 0.2
35,962
TSC22D2
rs1597466
3q25.1
3/151538251
T/G
0.1
20.04
0.008
1.88E-08 0
29,319
20.03
0.007
1.62E-06 0.1
35,794
ADIPOQ
rs6810075
3q27.3
3/188031259
T/C
0.6
0.06
0.005
3.60E-41 0
29,140
0.06
0.004
1.19E-43 0
35,749
VEGFA
rs998584
6q21.1
6/43865874
C/A
0.5
0.03
0.005
5.84E-08 0.3
28,167
0.03
0.005
3.25E-08 0.2
34,108
TRIB1
rs2980879
8q24.13
8/126550657
T/A
0.7
0.03
0.005
1.08E-08 0
24,084
0.03
0.005
7.13E-09 0
30,708
PDE3A
rs7955516
12q12.2
12/20389303
C/A
0.4
0.03
0.005
2.43E-08 0.1
29,178
0.02
0.004
4.45E-08 0
38,276
GPR109A
rs601339
12q24.31
12/121740696
G/A
0.2
0.04
0.006
3.87E-11 0
29,325
0.03
0.005
7.81E-10 0.3
35,947
DNAH10
rs7133378
12q24.31
12/122975455
G/A
0.7
20.03
0.005
1.29E-09 0
29,223
20.02
0.004
6.21E-07 0.5
35,697
CMIP
rs2925979
16q23.2
16/80092291
T/C
0.3
20.04
0.005
1.87E-18 0
29,347
20.04
0.005
1.21E-20 0
35,970
CDH13
rs12922394
16q23.3
16/81229828
T/C
0.1
20.10
0.011
3.16E-18 0.3
24,466
20.08
0.010
1.99E-15 0.4
31,089
PEPD
rs731839
19q13.11
19/38590905
G/A
0.35
20.04
0.005
2.20E-13 0.03
29,166
20.03
0.004
7.97E-12 0.4
35,771
All SNPs achieving genome-wide significance in the joint analysis phase are marked in italics. *Joint analysis indicates results from the meta-analysis of discovery and follow-up in-silico and de-novo phases. **When possible, plausible biological candidate genes have been listed; otherwise, the closest gene is designated. {Lead SNP is the SNP with the lowest p-value for each locus. 1Betas are estimated from models using the natural log transformed adiponectin. "EA: Effect allele, NEA: Non-effect allele. Results of Meta-Analysis of GWAS ""EAF: Effect allele frequency. doi:10.1371/journal.pgen.1002607.t001 Table 1. Lead SNP per Locus for Genome-Wide Significant SNPs Arising from the Sex-Combined Meta-Analysis in European
Populations. Locus for Genome-Wide Significant SNPs Arising from the Sex-Combined Meta-Analysis in European (n = 12,662) separately (Figure S2A, S2B, Tables S5 and S6). Although no novel loci reached genome-wide significance in
women or men separately, three loci (chromosome 3p, 8 and 12)
associated with adiponectin levels in the sex-combined analysis
showed evidence of association (p value,561028) in women
(Figure
S3). Since
different
assays
were
used
to
measure
adiponectin levels, we next tested whether stratification by assay
rendered similar results and found the results were highly
concordant
with
the
combined
analysis. GWAS
for
high
molecular weight adiponectin in the CHS study (n = 2,718)
identified 2 SNPs in ADIPOQ (rs17300539, p = 3.0610216) and
CMIP (rs2927307, p = 2.761028). These two genes are located
within the loci identified in our discovery meta-analysis of
adiponectin levels. analysis of the discovery and follow-up in-silico GWAS datasets
detected additional loci on chromosomes 1q41 near the LYPLAL1
gene (rs3001032, p = 3.661028) and chromosome 6p21.1 near the
VEGFA gene (rs998584, p = 5.8610212) that reached genome-wide
significance. While we confirmed seven loci that had reached
genome-wide significance at the discovery stage (Table 1, Figure 2F
and 2G, Table S2), two identified loci (3q25.1 and 12q24.31) did
not remain genome-wide significant in the joint analysis of
discovery and follow-up results. De novo follow-up phase. Next, in the de-novo genotyping
follow-up phase, we genotyped 10 SNPs with suggestive evidence
of association (561028,p,561026) from the meta-analysis of the
discovery and in-silico follow-up phases in an additional 3,913
individuals. Meta-analyzing the discovery and 2 follow-up stages
identified
a
SNP
in
ARL15
(rs6450176
[G];
ß = 0.026,
p = 5.861028), which was initially described in a previous
GWAS for adiponectin levels (Table S3) [16]. Gene Expression Studies Through gene expression studies we sought to address two
questions: First, are any of the SNPs that were genome-wide
significant for adiponectin levels associated with expression of their
nearest transcripts (cis-eQTLs) and second, whether mRNA levels
of loci identified through the GWAS for adiponectin levels are
associated with circulating adiponectin levels. To address the first
question, we examined whether SNPs within 1 Mb of the SNPs
achieving genome-wide significance in the discovery stage were
associated with the expression levels of nearby genes in human
adipocytes from 776 participants of the MuTHER Consortium
[25]. We identified 74 SNPs in three eQTLs to be associated with
the expression of five genes in adipocytes, using an array-wide level
of statistical significance for eQTLs (P,5.161025. See Materials
and Methods for details). These genes included: NT5DC2 on
chromosome 3; CCDC92, GPR109A, and ZNF664 on chromo-
some12; and PEPD on chromosome 19 (Table 3). The cis-eQTL Multi-ethnic meta-analysis. To identify loci influencing
adiponectin levels in non-European individuals we performed an
additional analysis in 4,232 individuals from an African American
population and 1,776 individuals from an East Asian population. In the African American populations, only associations at the
ADIPOQ locus reached genome-wide significance, while in the
East Asian population there was evidence of association at the
ADIPOQ and CDH13 loci (Table S4). Subsequently, we performed
a meta-analysis that combined all available GWAS including those
of white European origin, African American and East Asian
ancestry using novel method MANTRA [24]. This analysis
identified two additional loci in or near IRS1 gene on 2q36.3 and
at 6q24.1 within a gene desert. (Table 2, Figure 1B). In the African American populations, only associations at the
ADIPOQ locus reached genome-wide significance, while in the
East Asian population there was evidence of association at the
ADIPOQ and CDH13 loci (Table S4). Subsequently, we performed
a meta-analysis that combined all available GWAS including those
of white European origin, African American and East Asian
ancestry using novel method MANTRA [24]. This analysis
identified two additional loci in or near IRS1 gene on 2q36.3 and
at 6q24.1 within a gene desert. (Table 2, Figure 1B). Secondary GWAS analyses. We next performed meta-
analysis of the GWAS data in women (n = 16,685) and men Secondary GWAS analyses. Gene Expression Studies We next performed meta-
analysis of the GWAS data in women (n = 16,685) and men PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 5 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Figure 1. Manhattan plots for meta-analyses in the discovery phase. A) Combined sex analysis in European populations, B) Meta-Analysis of
Multiple Ethnicities. The Manhattan plots show 2Log10 (p-value) measures for association between single nucleotide polymorphisms (SNPs) and
chromosomal position. The SNPs that achieved genome-wide significance are highlighted in green. doi:10.1371/journal.pgen.1002607.g001
Adiponectin: Multiethnic Genome Wide Meta Analysis Figure 1. Manhattan plots for meta-analyses in the discovery phase. A) Combined sex analysis in European populations, B) Meta-Analysis of
Multiple Ethnicities. The Manhattan plots show 2Log10 (p-value) measures for association between single nucleotide polymorphisms (SNPs) and
chromosomal position. The SNPs that achieved genome-wide significance are highlighted in green. doi:10.1371/journal.pgen.1002607.g001 SNPs often are proxies for the lead SNPs from the GWAS,
however, this relationship may also be influenced through
mechanisms that are independent from gene expression, such as
gene function. number of statistically independent SNPs (p-value threshold of
561024) among the SNPs that were genome-wide significant for
adiponectin. These include associations with HDL-C (n= 104
SNPs), triglycerides (TG) (n = 65 SNPs), total cholesterol (TC,
n = 12 SNPs), LDL-cholesterol (LDL–C, n = 11 SNPs), and waist-
hip ratio (WHR) (n= 65 SNPs) [26]. (However, we note that since
sample sizes are different across different consortia power to identify
associations is not consistent.) Among these, coding and intronic
variants in STAB1 and NT5DC2 genes were associated with WHR
and HDL-C, while the variants 1 Mb near TRIB1 were associated
with all lipid traits. The coding and intronic variants ariants in the
locus on chromosome 12 harboring ZNF664, CCDC92, and
DNAH10 showed evidence of association with WHR, HDL-C, and
TG. Finally, variants in the PEPD gene were associated with TG. We next identified that mRNA levels of 18 genes arising from
six candidate loci were correlated with circulating adiponectin
levels (Table 4). Since circulating adiponectin levels may be
associated with a surplus of adipocyte transcripts we next tested
for enrichment of signal from the candidate loci. Gene Expression Studies There were 133
transcripts in the identified candidate regions, of which 8.2% (11/
133) were associated with adiponectin levels at an array-wide
level of significance (p,261026), while 7.5% of the 24k probes
on the entire array exceeded the same p-value threshold,
indicating there was therefore no additional enrichment of signal
at these candidate loci. We next calculated a multi-SNP genotypic risk score based
genome-wide significant SNPs from the discovery phase. This
multi-SNP genotypic risk score explained 5% of the variance of
natural log-transformed adiponectin levels. We then tested the
association of this risk score with risk of T2D and metabolic related
traits. The multi-SNP genotypic risk score was associated with
increased risk for T2D (ß = 0.3, p = 4.361023), where ß is the
average additive effect of adiponectin-decreasing risk alleles on the PLoS Genetics | www.plosgenetics.org Biological Relevance of the GWAS Loci In the first step toward understanding the biological relevance of
the identified regions, we examined the genes harbored by the
associated loci using human disease and animal databases. Although some of the genes in these loci do not have a known
function, several signify diverse biological functions. On chromosome 1, the lead SNP was located 300 kb from the
LYPLAL1, a protein that regulates phospholipids on cellular
membranes. Independent efforts have also identified this locus in
other metabolic/obesity related traits GWAS: first with WHR
(rs2605100; r2 = 0.49 [21] and rs4846567; r2 = 0.55 [27] respectively
with the lead adiponectin SNP, rs3001032), and more recently with
fasting insulin by a joint meta-analysis including the interaction
between SNP and BMI (MF Hivert for the MAGIC investigators,
personal communication). In the same report by MAGIC, variants
near IRS1 (insulin receptor substrate 1) and PEPD (a protein that
hydrolyzes dipeptides and tripeptides) have also been associated
with fasting insulin at genome wide significant levels, demonstrating
a close link between adiponectin regulation and insulin resistance
pathways. Moreover, both IRS1 and PEPD have been associated
with T2D (IRS1 in DIAGRAM [28] and PEPD in a Japanese
population [29]; p = 9.3610212 and p = 1.461025, respectively). All
three
homologous
genes
GPR109A/B/81
located
on
chromosome 12 are predominantly expressed in adipocytes and
mediate antilipolytic effects [45]. Our eQTL results (Table 3) and
the correlation between mRNA and adiponectin levels (Table 4)
argue strongly for a role of GPR109A at this locus. GPR109A (also
known as NIACR1) is a receptor with a high-affinity, concentra-
tion-dependent response to nicotinic acid (niacin) [45]. Treatment
by niacin increases serum adiponectin levels by up to 94% in obese
men with metabolic syndrome in a time- and dose-dependent
manner [46]. Functional studies in GPR109A receptor knockout
mice demonstrate that niacin increases serum total and HMW
adiponectin
concentrations and decreases lipolysis following
GPR109A receptor activation [47]. Moreover, a recent meta-
analysis on cohorts containing extremes of HDL-C provided
evidence suggestive of association in GPR109A/B/81 [48]. The lead SNP at 3p21.1 falls within GNL3 that is located in a
genomic region containing many genes which could have potential
functions
in
metabolism. Our
data
provide
evidence
that
adiponectin levels were correlated with human adipocyte mRNA
levels of many genes in this region (GLYCTK, SEMA3G, STAB1,
PBRM1, SFMBT1; see Table 4). However, this association does
not imply a direct influence of these genes on adiponectin level. Biological Relevance of the GWAS Loci Among those genes, STAB1 encodes for stabilin 1, described as an
endocytic receptor for advanced glycation end products and may
have
a function
in angiogenesis,
lymphocyte
homing, cell
adhesion, or receptor scavenging for acetylated low-density
lipoprotein [30]. Finally, variants in ZNF664 have been associated with CHD,
HDL-C and TG levels in a large meta-analysis of over 100,000
individuals of European ancestry [22]. The sex heterogeneity
observed in this study is comparable to our finding that the more
loci associated with adiponectin at genome wide significance level
have been shown in female stratified analysis. Taken together, the loci identified in this large-scale GWAS for
adiponectin levels highlight many genes with demonstrated
relationships with metabolic disease. T2D and Metabolic Traits doi:10.1371/journal.pgen.1002607.g002 log odds ratio of T2D), increased TG (ß = 0.25, p = 2.6610214),
increased WHR adjusted for BMI (ß = 0.18, p = 1.861025),
increased post-prandial glucose (ß = 0.25, p = 0.01), increased
fasting insulin (ß = 0.05, p = 0.01), homeostatic model assessment-
insulin resistance (HOMA-IR) (ß = 0.04, p = 0.047), and with
lower HDL-C concentrations (ß = 20.24, p = 4.5610213) and
decreased BMI (ß = 20.16, p = 1.461024). (Table 5). is the vascular endothelial growth factor A gene, a known gene in a
variety of vascular endothelial cell functions, such as angiogenesis
and maintenance of the glomerular endothelium in nephrons [32]. Variants in this gene are also associated with diabetic retinopathy
and WHR [27,33]. Moreover, the product of VEGFA interacts
with resveratrol, which has been shown to have a beneficial
influence in some metabolic traits, including diabetes [34]. Rodent
studies show that resveratrol decreases blood glucose, blood
insulin, and glycated hemoglobin, as well as increases insulin
sensitivity in animals with hyperglycemia (reviewed in [35]). Resveratrol also inhibits TNF-a-induced reductions in adiponectin
levels in 3T3-L1 adipocytes [36]. Furthermore, it has been shown
that resveratrol modulates adiponectin expression and improves
insulin sensitivity, likely through the inhibition of inflammatory-
like response in adipocytes [37]. At this locus, VEGFA mRNA
levels in adipocytes were the strongest association with adiponectin
levels (Table 4). Also likely involved in vascular biology, TRIB1
encodes a G protein-coupled receptor-induced protein interacting
with MAP kinases that regulates proliferation and chemotaxis of
vascular smooth muscle cells [38]. TRIB1 expression was shown to
be elevated in human atherosclerotic arteries [39]. Several variants
(rs2954029, rs2954021, rs17321515; all in moderate LD with our
lead SNP) in the TRIB1 gene have been associated with HDL-C,
LDL-C and CHD risk in European and Asian populations
[22,40,41,42,43]. These two loci (TRIB1 and VEGFA) argue for
the importance of vascular biology in adiponectin regulation as
underlined previously by findings of adiponectin levels associated
with variants near CDH13 (a receptor for adiponectin expressed by
endothelial smooth muscle) [44]. PLoS Genetics | www.plosgenetics.org Discussion In this comprehensive multi-ethnic analysis of the genetic
influences on adiponectin levels and their impact on metabolic
traits and T2D, we have identified 10 novel loci and confirmed the
associations of variants in the ADIPOQ and CDH13 loci with
adiponectin levels. The adiponectin risk alleles were associated
with T2D and related metabolic traits such as BMI, WHR, TG,
HDL-C, 2-hour glucose, HOMA-IR and fasting insulin. These
findings demonstrate that adiponectin, T2D and metabolic
syndrome have a shared allelic architecture. T2D and Metabolic Traits Using data from several large-scale GWAS consortia, some of the
significantly
associated
variants
identified
here
demonstrated
associations with T2D and its related traits (Table S7A, S7B, S7C,
and S7D). Several individual SNPs showed evidence for association
with T2D and various metabolic traits after accounting for the March 2012 | Volume 8 | Issue 3 | e1002607 6 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Figure 2. Regional plots of eight newly discovered genome-wide significant chromosomal regions associated with adiponectin
concentrations in European populations. A) chromosome 16q23.2, B) chromosome 19 q13.11 C) Chromosome 3p21.1, D) two loci on
chromosome 12q24.31, E) chromosome 8q24.13, F) chromosome 6p21.1, and G) chromosome 1q41. In each panel, purple diamonds indicate the top
PLoS Genetics | www.plosgenetics.org
7
March 2012 | Volume 8 | Issue 3 | e1002607 Figure 2. Regional plots of eight newly discovered genome-wide significant chromosomal regions associated with adiponectin
concentrations in European populations. A) chromosome 16q23.2, B) chromosome 19 q13.11 C) Chromosome 3p21.1, D) two loci on
chromosome 12q24.31, E) chromosome 8q24.13, F) chromosome 6p21.1, and G) chromosome 1q41. In each panel, purple diamonds indicate the top PLoS Genetics | www.plosgenetics.org PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 7 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis SNPs, which have the strongest evidence of association. Each circle shows a SNP with a color scale relating the r2 value for that SNP and the top SNP
from HapMap CEU. Blue lines indicate estimated recombination rates from HapMap. The bottom panels illustrate the relative position of genes near
each locus. Candidate genes are indicated by red ovals. doi:10.1371/journal.pgen.1002607.g002 SNPs, which have the strongest evidence of association. Each circle shows a SNP with a color scale relating the r2 value for that SNP and the top SNP
from HapMap CEU. Blue lines indicate estimated recombination rates from HapMap. The bottom panels illustrate the relative position of genes near
each locus. Candidate genes are indicated by red ovals. PLoS Genetics | www.plosgenetics.org Shared Allelic Architecture of Adiponectin Levels and
Metabolic Traits Interestingly, several of the genes near lead genome-wide
significant SNPs have been implicated in angiogenesis, which
might be important for adipose tissue expansion, highlighting the
recurring theme of ‘‘adipose tissue expandability’’ in the genetic
origins of obesity-related complications [31]. For example, VEGFA Using a multi-SNP genotypic risk score we attempted to
understand if the allelic architecture of adiponectin levels was
shared with T2D and metabolic traits. This risk score was March 2012 | Volume 8 | Issue 3 | e1002607 8 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Table 2. Genome-Wide Significant SNPs from the Sex-Combined Multi-Ethnic Meta-Analysis. Nearby*
Gene
Lead SNP{
Gene region chr/position{
EA/NEA"
EAF"" (CEU/EA/AA)
Multi-Ethnic Fixed Effects Meta-analysis
Multi-Ethnic Random Effects
Meta-analysis
MANTRA
N
Beta (SE)
pvalue
Q-Value
I2
Beta (SE)
pvalue
BF1
phet{{
LYPLAL1
rs2791553
1q41
1/217742665
G/A
0.6/0.46/0.54
20.02(0.004)
4.91E-07
25.18
0
20.02(0.004)
4.91E-07
6.3
0.06
37,665
IRS1
rs925735
2q36.3
2/226887874
G/C
0.64/0.89/0.74
20.02(0.004)
1.88E-08
22.15
0.01
20.02(0.004)
2.12E-08
8.1
0.06
37,638
GNL3
rs2590838
3p21.1
3/52597126
G/A
0.5 1/0.34/0.54
20.03(0.004)
4.08E-15
28.85
0.06
20.03(0.004)
1.88E-13
14.1
0.05
37,680
ADIPOQ
rs6810075
3q27.3
3/188031259
T/C
0.93/1/0.86
0.06(0.004)
1.10E-43
27.44
0.02
0.06(0.004)
2.41E-42
43.6
0.16
31,533
-
rs592423
6q24.1
6/139882386
C/A
0.54/0.36/0.41
0.02(0.004)
3.59E-07
15.46
0
0.02(0.004)
3.59E-07
6.5
0.03
37,430
TRIB1
rs2980879
8q24.13
8/126550657
T/A
0.69/0.77/0.67
0.03(0.004)
9.91E-10
21.08
0
0.03(0.004)
9.91E-10
8.2
0.04
32,426
GPR109A
rs601339
12q24.31
12/121740696
G/A
0.19/0.39/0.31
0.03(0.005)
3.77E-09
36.11
0.25
0.03(0.006)
4.31E-06
8.3
0.09
37666
CMIP
rs2925979
16q23.2
16/80092291
T/C
0.3 0/0.43/0.31
20.04(0.004)
3.12E-21
23.12
0
20.04(0.004)
3.12E-21
19.8
0.31
37,687
CDH13
rs12051272
16q23.3
16/81220789
T/G
0.03/0.33/0.03
20.26(0.017)
4.74E-51
39.17
0.62
20.26(0.032)
1.10E-14
66.0
1.00
24,216
PEPD
rs4805885
19q13.11
19/38597963
T/C
0.39/0.64/0.41
20.03(0.004)
1.65E-11
34.94
0.23
20.03(0.005)
2.05E-08
9.9
0.05
37,479
The novel loci identified using Multi-Ethnic Meta-analysis (that were not identified in the European only analysis) are listed in bold. *When possible, plausible biological candidate genes have been listed; otherwise, the closest gene is designated. {Lead SNP is the SNP with the lowest p-value for each locus. {Positions are relative to Human Genome NCBI Build 36. 1log10 Bayes factor (BF) from the MANTRA analysis. A log10 BF of 6 and higher was considered as a conservative threshold for genome-wide significance. {{The posterior probability of heterogeneity between studies. "EA: effect allele, NEA: non-effect allele. ""EAF: Frequency of effect allele in CEU, East Asian, and AA, populations respectively. PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 Shared Allelic Architecture of Adiponectin Levels and
Metabolic Traits Nonetheless, in aggregate, these results provide strong
evidence that the genetic determinants of adiponectin levels are
shared with metabolic disease, and in particular, traits related to
insulin resistance. Table 4. The Association of mRNA Levels from Genes in
Candidate Loci in Human Adipocytes with Circulating
Adiponectin Levels. Gene
Gene region
GeneStart
GeneEnd
Beta1
Pvalue
GLYCTK
3p21.1
52296875
52304311
0.060
1.77E-20
SEMA3G
3p21.1
52442307
52454083
20.018
9.28E-06
STAB1
3p21.1
52504395
52533551
20.039
2.26E-14
PBRM1
3p21.1
52554407
52688779
0.007
2.49E-04
SFMBT1
3p21.1
52913666
53055110
0.010
2.53E-08
DNAJB11
3q27.3
187771160
187786283
20.014
3.31E-07
EIF4A2
3q27.3
187984054
187990379
0.021
1.53E-08
ADIPOQ
3q27.3
188043156
188058944
0.054
1.03E-13
MAD2L1BP
6q21.1
43711554
43716666
0.009
4.09E-04
VEGFA
6q21.1
43845923
43862199
0.012
2.15E-09
ZCCHC8
12q24.31*
121523387
121551471
0.011
2.60E-04
GPR109B
12q24.31
121765255
121767392
0.010
3.74E-06
GPR109A
12q24.31
121778105
121781082
0.026
1.80E-11
PITPNM2
12q24.31*
122033979
122160928
20.010
5.09E-06
U1SNRNPBP
12q24.31
122508604
122516894
0.011
1.72E-04
ATP6V0A2
12q24.31
122762817
122812252
20.008
2.86E-04
ZNF664
12q24.31
123023622
123065922
0.010
8.28E-06
SLC7A10
19q13.11
38391409
38408596
0.072
1.66E-14
1Betas are estimated from log transformed and quantile-quantile normalized
values. *These two loci are independent loci. doi:10.1371/journal.pgen.1002607.t004 Table 4. The Association of mRNA Levels from Genes in
Candidate Loci in Human Adipocytes with Circulating
Adiponectin Levels. We note that there are several strengths and limitations of this
study. Our main findings, identifying genetic determinants of
adiponectin levels, are based on the largest meta-analysis to date
and include results from three ethnicities. The availability of
expression data
from human adipose tissue permitted the
association of identified SNPs with mRNA levels at candidate
genes and, in turn, correlation of these mRNA levels with
circulating adiponectin itself. While access to the data from large
consortia permitted assessment of the relevance of the identified
SNPs to T2D and components of the metabolic syndrome, we
note that a subset of the cohorts included in our GWAS were also
included in these external consortia. However, we note that even if
we assume that all ADIPOGen study participants were included in
the external consortia, for cohorts participating in both studies,
that the majority of data in these external consortia still arises from
study participants not present in ADIPOGen (minimum percent of
non-overlapping subects: 86.8%, 85.5%, 86.4% and 82.5% for
MAGIC, GLGC, GIANT, and DIAGRAM+ consortia, respec-
tively). Shared Allelic Architecture of Adiponectin Levels and
Metabolic Traits adiponectin risk alleles at ZNF664 and PEPD are of considerable
interest since they impart deleterious changes on aspects of the
metabolic syndrome (increased TC, TG, LDL-C and WHR and
decreased HDL-C), but also act to decrease BMI and percent fat. Our data do not provide direct evidence as to whether the
genetic determinants of adiponectin levels influence these traits
through adiponectin itself, or through pleiotropic pathways and
therefore do not constitute a Mendelian randomization study. These
findings
provide
a
note
of
caution
for
Mendelian
randomization studies,
which may
be
prone to
erroneous
conclusions if pleiotropic effects of tested variants are not
considered. Nonetheless, in aggregate, these results provide strong
evidence that the genetic determinants of adiponectin levels are
shared with metabolic disease, and in particular, traits related to
insulin resistance. associated with increased risk of T2D and traits associated with
insulin
resistance
and
the
metabolic
syndrome. However,
unexpectedly, adiponectin decreasing alleles were associated with
a decrease in BMI. In our adiponectin GWAS, BMI was included
as a covariate in order to avoid direct identification of obesity
SNPs since BMI is strongly related to adiponectin levels [49,50]. Furthermore, this unexpected direction of effect was entirely
explained by SNPs at the ZNF664 and PEPD loci; when these loci
were removed from the analysis, the association of the genotypic
risk score with BMI disappeared (results not shown). Therefore, associated with increased risk of T2D and traits associated with
insulin
resistance
and
the
metabolic
syndrome. However,
unexpectedly, adiponectin decreasing alleles were associated with
a decrease in BMI. In our adiponectin GWAS, BMI was included
as a covariate in order to avoid direct identification of obesity
SNPs since BMI is strongly related to adiponectin levels [49,50]. Furthermore, this unexpected direction of effect was entirely
explained by SNPs at the ZNF664 and PEPD loci; when these loci
were removed from the analysis, the association of the genotypic
risk score with BMI disappeared (results not shown). Therefore, Our data do not provide direct evidence as to whether the
genetic determinants of adiponectin levels influence these traits
through adiponectin itself, or through pleiotropic pathways and
therefore do not constitute a Mendelian randomization study. These
findings
provide
a
note
of
caution
for
Mendelian
randomization studies,
which may
be
prone to
erroneous
conclusions if pleiotropic effects of tested variants are not
considered. Shared Allelic Architecture of Adiponectin Levels and
Metabolic Traits doi:10.1371/journal.pgen.1002607.t002
osgenetics.org
9
March 2012 | Volume 8 | PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 9 associated with increased risk of T2D and
insulin
resistance
and
the
metabolic
s
unexpectedly, adiponectin decreasing alleles
a decrease in BMI. In our adiponectin GWA
as a covariate in order to avoid direct ide
SNPs since BMI is strongly related to adipo
Furthermore, this unexpected direction o
explained by SNPs at the ZNF664 and PEPD
were removed from the analysis, the associa
risk score with BMI disappeared (results no
Table 3. The Association of Lead Genom
Gene
Lead SNP-
Cis-eQTL{
Chr
Transcript
Start Site
NT5DC2
rs13081028
3
52533424
GPR109A
rs2454722*
12
121778105
CCDC92
rs10773049
12
122986907
ZNF664
rs825453
12
123074711
PEPD
rs8182584
19
38569694
{Lead SNP is the SNP with the lowest p-value for ea
{{Lead SNP is the SNP with the lowest p-value for e
"EA: Effect allele. ""EAF: Frequency of effect allele. 1Betas are estimated expression levels of the genes
*P value for lead SNP is the SNP in gene expression
**P value for lead SNP in meta-analysis from discove
$r2 LD between lead SNP from expression and lead
doi:10.1371/journal.pgen.1002607.t003
Table 4. The Association of mRNA Leve
Candidate Loci in Human Adipocytes wit
Adiponectin Levels. Gene
Gene region
GeneStart
GeneE
GLYCTK
3p21.1
52296875
523043
SEMA3G
3p21.1
52442307
5245408
STAB1
3p21.1
52504395
5253355
PBRM1
3p21.1
52554407
5268877
SFMBT1
3p21.1
52913666
530551
DNAJB11
3q27.3
187771160
1877862
EIF4A2
3q27.3
187984054
1879903
ADIPOQ
3q27.3
188043156
1880589
MAD2L1BP
6q21.1
43711554
4371666
VEGFA
6q21.1
43845923
4386219
ZCCHC8
12q24.31*
121523387
1215514
GPR109B
12q24.31
121765255
1217673
GPR109A
12q24.31
121778105
1217810
PITPNM2
12q24.31*
122033979
1221609
U1SNRNPBP
12q24.31
122508604
1225168
ATP6V0A2
12q24.31
122762817
1228122
ZNF664
12q24.31
123023622
1230659
SLC7A10
19q13.11
38391409
3840859
1Betas are estimated from log transformed and qua
values. *These two loci are independent loci. doi:10.1371/journal.pgen.1002607.t004 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Table 3. The Association of Lead Genome-Wide Significant SNPs for Adiponectin with mRNA Levels of Their Nearest Gene. Table 3. The Association of Lead Genome-Wide Significant SNPs for Adiponectin with mRNA Levels of Their Nearest Gene. Table 3. The Association of Lead Genome-Wide Significant SNPs for Adiponectin with mRNA Le Table 3. The Association of Lead Genome-Wide Significant SNPs for Adiponectin with mRNA Levels of Their Nearest Gene. Shared Allelic Architecture of Adiponectin Levels and
Metabolic Traits Gene
Lead SNP-
Cis-eQTL{
Chr
Transcript
Start Site
Transcript
End Site
EA"
EAF""
Beta (SE)1
P-Exp*
P-GWAS**
lead SNP-
GWAS{{
r2$
NT5DC2
rs13081028
3
52533424
52544133
G
0.444
0.14(0.02)
1.32E-19
1.05E-09
rs1108842
0.84
GPR109A
rs2454722*
12
121778105
121781082
G
0.166
20.15(0.03)
1.71E-09
3.87E-11
rs601339
1
CCDC92
rs10773049
12
122986907
123023116
T
0.611
0.15(0.02)
8.09E-22
2.67E-08
rs7133378
0.02
ZNF664
rs825453
12
123074711
123065922
T
0.615
20.04(0.01)
4.51E-05
4.03E-08
rs7978610
0.03
PEPD
rs8182584
19
38569694
38704639
T
0.364
20.13(0.02)
9.96E-10
6.64E-11
rs731839
1
{Lead SNP is the SNP with the lowest p-value for each gene in gene expression data. {{Lead SNP is the SNP with the lowest p-value for each locus in meta-analysis from discovery phase. "EA Eff
t
ll l Gene
Lead SNP-
Cis-eQTL{
Chr
Transcript
Start Site
Transcript
End Site
EA"
EAF""
Beta (SE)1
P-Exp*
P-GWAS**
lead SNP-
GWAS{{
r2$
NT5DC2
rs13081028
3
52533424
52544133
G
0.444
0.14(0.02)
1.32E-19
1.05E-09
rs1108842
0.84
GPR109A
rs2454722*
12
121778105
121781082
G
0.166
20.15(0.03)
1.71E-09
3.87E-11
rs601339
1
CCDC92
rs10773049
12
122986907
123023116
T
0.611
0.15(0.02)
8.09E-22
2.67E-08
rs7133378
0.02
ZNF664
rs825453
12
123074711
123065922
T
0.615
20.04(0.01)
4.51E-05
4.03E-08
rs7978610
0.03
PEPD
rs8182584
19
38569694
38704639
T
0.364
20.13(0.02)
9.96E-10
6.64E-11
rs731839
1
{Lead SNP is the SNP with the lowest p-value for each gene in gene expression data. {{Lead SNP is the SNP with the lowest p-value for each locus in meta-analysis from discovery phase. "EA: Effect allele. ""EAF: Frequency of effect allele. 1Betas are estimated expression levels of the genes. *P value for lead SNP is the SNP in gene expression data. **P value for lead SNP in meta-analysis from discovery phase. $r2 LD between lead SNP from expression and lead SNP from meta-analysis. doi:10.1371/journal.pgen.1002607.t003 adiponectin risk alleles at ZNF664 and PEPD are of considerable
interest since they impart deleterious changes on aspects of the
metabolic syndrome (increased TC, TG, LDL-C and WHR and
decreased HDL-C), but also act to decrease BMI and percent fat. adiponectin risk alleles at ZNF664 and PEPD are of considerable
interest since they impart deleterious changes on aspects of the
metabolic syndrome (increased TC, TG, LDL-C and WHR and
decreased HDL-C), but also act to decrease BMI and percent fat. Shared Allelic Architecture of Adiponectin Levels and
Metabolic Traits Therefore, since a substantial majority of participants are
independent between ADIPOGen and these consortia, it is
unlikely that our findings demonstrating a shared allelic architec-
ture between adiponectin levels and these traits are spurious. Further, we suggest that locus, 6q24.1, identified only through
multi-ethnic meta-analysis using MANTRA and not confirmed
through fixed and random effects meta-analysis, be replicated for
confirmation of this finding. In conclusion, the data presented in this study provide strong
evidence of association for 10 novel loci for adiponectin levels. March 2012 | Volume 8 | Issue 3 | e1002607 10 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Table 5. Results of Association of Multi-SNP Genotypic Risk Score with Diabetes and Related Traits Table 5. Results of Association of Multi-SNP Genotypic Risk Score with Diabetes and Related Traits. Trait
N
Effect1 (95% CI)
P
Consortium
T2D**
22,044
0.301 (0.09, 0.51)
4.3E-03
DIAGRAM+
BMI (SD units)
121,335
20.162 (20.25, 20.08)
1.4E-04
GIANT
WHR*
77,167
0.177 (0.1, 0.26)
1.8E-05
GIANT
Percent Fat
34,853
20.052 (20.15, 0.05)
0.31
Body Fat Percent
Fasting Glucose (mmol/L)
46,186
0.011 (20.03, 0.05)
0.58
MAGIC
Fasting Insulin**(pmol/L)
38,238
0.05 (0.01, 0.09)
1.5E-02
MAGIC
HomaB
36,466
0.033 (0, 0.07)
5.1E-02
MAGIC
Homa IR
37,037
0.042 (0, 0.08)
4.7E-02
MAGIC
2hr Glucose**(mmol/L)
15,234
0.245 (0.06, 0.44)
1.1E-02
MAGIC
HbA1C (%)
35,908
20.002 (20.04, 0.03)
0.91
MAGIC
TG**(SD units)
93,440
0.248 (0.18, 0.31)
2.6E-14
GLGC
HDL-C** (SD units)
96,748
20.243 (20.31, 20.18)
4.5E-13
GLGC
LDL-C (SD units)
92,348
0.023 (20.05, 0.09)
0.52
GLGC
TC (SD units)
97,021
0.0003 (20.07, 0.07)
0.99
GLGC
T2D: Type 2 diabetes, BMI: Body mass Index, WHR: Waist to hip ratio, HbA1C: hemoglobin A1C, TG: Triglyceride, HDL-C: High Density Lipoprotein Cholesterol, LDL-C:
Low Density Lipoprotein Cholesterol, TC: Total Cholesterol. 1Effect is mean change in trait or disease per adiponectin-decreasing allele. *Waist to hip ratio adjusted for BMI. **Significantly associated trait is coded in bold. doi:10.1371/journal.pgen.1002607.t005 SNPs of the first stage also associate with mRNA levels of nearest
gene(s) expressed using adipose tissue of 776 European women. We also tested for association between adiponectin levels and
mRNA levels of the genes in our candidate loci in adipose tissue of
a subgroup of 436 individuals [25]. Third, we calculated a multi-
SNP genotypic risk score using genome-wide significant adipo-
nectin-lowering alleles and tested the association of this risk score
with T2D and related metabolic traits. Figure 3 shows a flow chart
detailing the study design. Study Design Our study consisted of three stages. First, in the discovery stage
we performed a meta-analysis of the GWAS summary statistics of
16 studies involving 29,347 participants of white European origin
to detect SNPs that are associated with adiponectin levels. All
signals with p,561026 were followed up in seven additional
cohorts (n = 6,623) with GWAS data (in-silico phase) that later
joined the consortium and then a subset of SNPs (n = 10) by de-novo
genotyping in 3,913 additional participants from three cohorts
(n = 39,883 for the combined analysis in Europeans). We also
performed a multi-ethnic meta-analysis by combining summary
statistics from the 16 studies of individuals of white European
discovery cohorts (n = 29,347) with those of five cohort studies that
included African Americans subjects (n = 4,232) and one East
Asian cohort (n = 1,776) to obtain a total 35,355 individuals for the
GWAS meta-analysis involving different ethnicities. After identi-
fying variation near two genes of pharmaceutical importance
(GPR109A and GPR109B), which encode the putative niacin
receptors, we typed additional rare coding and tagging variants in
a subset of cohorts. Second, we examined whether the identified Ethical Consideration In total, 45,891 individuals from 26 European and 7 non-
European cohorts participated in the different phases of this meta-
analysis. Participating
cohorts
were
either
population-based
(n = 23), family-based (n = 4), or case-control (n = 4) studies. The
age of participants ranged from 10 to 95 years. Adiponectin levels
were measured using ELISA or RIA methods. More details on the
study cohorts and adiponectin measurement are presented in the
Text S1 and Table S1. In addition, genotyping of four coding and
tagging SNPs in the candidate genes, GRP109A and GPR109B,
was undertaken in samples from the Lausanne, Lolipop, MRC
Ely, and Fenland cohorts. All participants provided informed written consent. The
research protocol of all studies were reviewed and approved by
institutional ethics review committees at the involved institutions. Shared Allelic Architecture of Adiponectin Levels and
Metabolic Traits Further analyses confirmed that the level of expression of some of
these candidate genes in human adipocytes correlated directly with
adiponectin levels. A multi-SNP genotypic risk score, and several
of the identified variants, directly influence parameters of the
metabolic syndrome and, in particular, markers of insulin
resistance. These findings identify novel genetic determinants of
adiponectin levels, which, taken together, influence risk of T2D
and markers of insulin resistance. PLoS Genetics | www.plosgenetics.org Genotyping and Imputation —In silico follow-up:
468 SNPs with p,561026 from the
discovery phase (which includes both genome-wide significant
[n = 196, p,561028] and ‘‘suggestive’’ [n =272, 561028,p,
561026] SNPs Table S3) were tested for their association in 6,623
individuals from seven additional cohorts with GWAS data that
joined the consortium after the discovery stage had been finalized. —De novo follow-up:
We next selected the lead SNP arising
from selected loci from the joint analysis of the discovery and in-
silico follow-up phase with p-values greater than 561028 but less
than 561026 and genotyped 10 SNPs in 3,164 samples from the
SAPHIR cohort and an additional subgroup of the KORA cohort. Finally, these same SNPs, or their proxy SNPs (n = 2), were tested
for association in the THISEAS cohort (n = 738), which had been
genotyped using the Metabochip [55]. Study-level summary
statistics from the follow-up phases were meta-analyzed with the
data from the discovery phase. Eleven coding and tagging variants in two candidate genes of
pharmaceutical
importance
(GPR109A
encoding
the
niacin
receptor and GPR109B) were genotyped in a parallel study in
Lausanne, Lolipop, MRC Ely, and Fenland white subjects. Genotyping was performed using a KASPar-On-Demand SNP
Genotyping Assay (KBioscience Ltd., Hoddesdon, UK). In
Lausanne and Lolipop samples the genotyping assay was carried
out on 3.75 ng of genomic DNA in 1 ml 1536-well plate reactions,
dispensed with a Meridian, microfluidic dispenser (KBioscience
Ltd.,
Hoddesdon, UK), thermocycled
using
a Hydrocycler
(KBioscience Ltd., Hoddesdon, UK). A Pherastar (BMG GmbH,
Germany) was used for end-point detection and Kraken-LIMS
(KBioscience Ltd., Hoddesdon, UK) was used for automated allele
calling. In MRC Ely and Fenland samples, the genotyping assay
was carried out on 10 ng of genomic DNA in 5 ml 384-well plate
reactions using a G-Storm GS4 Thermal Cycler (GRI, Rayne,
UK). The ABI PRISM 7900HT Sequence Detection System
(Applied Biosystems, Warrington, UK) was used for end-point
detection and allele calling. y p
Multi-ethnic meta-analysis. In order to perform a meta-
analysis
of
GWAS
data
from
cohorts
of
different
ethnic
backgrounds, we utilized the novel MANTRA (Meta-ANalysis of
Trans-ethnic Association studies) software [24]. This method
combines
GWAS
from
different
ethnic
groups
by
taking
advantage of the expected similarity in allelic effects between the
most
closely
related
populations. Fixed-effects
meta-analysis
assumes the allelic effect to be the same in all populations, and
cannot
account
for
heterogeneity
between
ethnic
groups. Genotyping and Imputation Populations are then
clustered according to their similarity in terms of relatedness as
measured by the mean allele frequency difference at 10,000
independent SNPs, and to their allelic effects at the variant. If all
populations are assigned to the same cluster, this is equivalent to a
fixed allelic effect across all populations (i.e. no trans-ethnic
heterogeneity). The posterior distribution of the allelic effect in
each
population
under
the
Bayesian
partition
model
is
approximated
by
means
of
a
Monte-Carlo
Markov
chain
algorithm. Evidence in favor of association of the trait with the
variant was assessed by means of a Bayes’ factor (BF). A log10 BF of
6 or higher is considered a relatively conservative threshold for
genome-wide significance. We also performed meta-analysis by
using both random and fixed effects models including all ethnicities. Those loci that achieved both a BF.6 in MANTRA and a P-value
less than 561027 in multiethnic analysis are presented in Table 2. Genotyping and Imputation All cohorts were genotyped using commercially available
Affymetrix or Illumina genome-wide genotyping arrays. Quality
control was performed for each study independently and genotype
imputation was carried out using IMPUTE, MACH, BimBam or
Beagle with reference to either the Phase II CEU, CEU+YRI, or
CHB+JPT+CEU HapMap according to the origin of population. Imputation of East Asian genotypes was undertaken by first
masking genotypes of 200 SNPs and then imputing them based on
the CEU+CHB+JPT panel from HapMap. This resulted in an
allelic concordance rate of ,96.7%. For the African Americans, a
combined CEU+YRI reference panel was created. This panel
included SNPs segregating in both CEU and YRI, as well as SNPs PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 11 Adiponectin: Multiethnic Genome-Wide Meta-Analysis segregating in one panel and monomorphic and non-missing in
the other (2.74 million SNPs). Due to the overlap of African
American individuals on the Affymetrix 6.0 and IBC arrays [51], it
was possible to analyze imputation performance at SNPs not
genotyped on Affymetrix 6.0. For imputation based on Affymetrix
data, the use of the CEU+YRI panel resulted in an allelic
concordance rate of ,95.6% (calculated as 120.5 * [imputed_
dosage– chip_dosage]). This rate is comparable to rates calculated
for individuals of African descent imputed with the HapMap 2
YRI individuals. Table S1 summarizes the genotyping methods
used for each cohort, genotype-calling algorithms, imputation
algorithms and exclusion thresholds. SNP-level quality control
metrics were applied prior to meta-analysis for each cohort. These
were: call rate $95%, minor allele frequency (MAF)$1%, Hardy-
Weinberg equilibrium (HWE) p.1026, and quality measures for
imputed SNPs (r2$0.3, or proper info $0.4, for cohorts imputing
their data with MACH and IMPUTE, respectively). pean and non-European populations separately, using the same
methods as described above. Presence of heterogeneity in the meta-analysis was assessed by
the
I2
statistic
and
Q-test
[54]. Since
cohorts
measured
adiponectin concentrations using either RIA or ELISA methods,
we also performed a GWA meta-analysis stratified by the method
of measurement to test whether this contributed to heterogeneity Follow-up phase. The follow-up phase comprised two stages;
in-silico follow-up and de-novo follow-up. Statistical Analysis Genome-wide association studies. All cohorts indepen-
dently tested for the additive genetic association of common
(MAF.1%) genotyped and imputed SNPs with natural log
transformed adiponectin levels, while adjusting for age, sex,
body mass index (BMI), principal components of population
stratification and study site (where appropriate), and for family
structure in cohorts with family members [49,50,52]. The analyses
were performed for men and women combined, as well as for men
and women separately. The Cardiovascular Health Study cohort
(CHS) also provided GWA results for high molecular weight
(HMW) adiponectin using the same methods as described above. Meta-analysis of GWAS. The meta-analysis was performed
by two analysts independently each using different methods;
inverse variance-weighted methods using both fixed and random
effect models available through either the METAL (http://www. sph.umich.edu/csg/abecasis/metal/) or GWAMA version 2.0.5
(http://www.well.ox.ac.uk/gwama/)
software
packages
[53]. Summary statistics were crosschecked to ensure consistency of
results. Prior to the meta-analysis, study-specific summary statistics
were corrected using genomic control (lambda range = 0.99–1.25)
and the overall meta-analytic results were additionally corrected
for
genomic
control
(lambda = 1.06). To
examine
whether
associations with adiponectin were sex-specific, we performed
meta-analyses for men and women separately. A p-value threshold
of 561028 was considered to be genome-wide significant. Ethnicity-specific meta-analyses were performed for white Euro- Genotyping and Imputation Conversely, random effects meta-analysis assumes that each
population has a different underlying allelic effect, however,
populations
from
the
same
ethnic
group
would
be
more
homogeneous than those that are more distantly related. To
address this challenge we accounted for the expected similarity in
allelic effects between the most closely related populations by means
of a Bayesian partition model. For each variant, allelic effects and
corresponding standard errors are estimated within each population
under the assumption of an additive model. Populations are then
clustered according to their similarity in terms of relatedness as
measured by the mean allele frequency difference at 10,000
independent SNPs, and to their allelic effects at the variant. If all
populations are assigned to the same cluster, this is equivalent to a
fixed allelic effect across all populations (i.e. no trans-ethnic
heterogeneity). The posterior distribution of the allelic effect in
each
population
under
the
Bayesian
partition
model
is
approximated
by
means
of
a
Monte-Carlo
Markov
chain
algorithm. Evidence in favor of association of the trait with the
variant was assessed by means of a Bayes’ factor (BF). A log10 BF of
6 or higher is considered a relatively conservative threshold for
genome-wide significance. We also performed meta-analysis by
using both random and fixed effects models including all ethnicities. Those loci that achieved both a BF.6 in MANTRA and a P-value
less than 561027 in multiethnic analysis are presented in Table 2. Multi-ethnic meta-analysis. In order to perform a meta-
analysis
of
GWAS
data
from
cohorts
of
different
ethnic
backgrounds, we utilized the novel MANTRA (Meta-ANalysis of
Trans-ethnic Association studies) software [24]. This method
combines
GWAS
from
different
ethnic
groups
by
taking
advantage of the expected similarity in allelic effects between the
most
closely
related
populations. Fixed-effects
meta-analysis
assumes the allelic effect to be the same in all populations, and
cannot
account
for
heterogeneity
between
ethnic
groups. Conversely, random effects meta-analysis assumes that each
population has a different underlying allelic effect, however,
populations
from
the
same
ethnic
group
would
be
more
homogeneous than those that are more distantly related. To
address this challenge we accounted for the expected similarity in
allelic effects between the most closely related populations by means
of a Bayesian partition model. For each variant, allelic effects and
corresponding standard errors are estimated within each population
under the assumption of an additive model. Association of Genome-Wide Significant SNPs with Gene
Expression (Stage 2) In order to identify cis-expression quantitative trait loci (cis-
eQTLs) and test whether mRNA levels of candidate genes arising
from our GWAS were associated with adiponectin levels, we used PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 12 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Figure 3. Flow chart of study design. doi:10.1371/journal.pgen.1002607.g003 Figure 3. Flow chart of study design. doi:10.1371/journal.pgen.1002607.g003 expression profiles in human adipocytes from the Multiple Tissue
Human Expression Resource (MuTHER) Consortium, (856
female twins from the UK) [25]. mRNA expression profiles from
subcutaneous fat and genome-wide genotypes were available for
776 individuals and circulating adiponectin levels for 436 of these
women. We note that while adiponectin levels were measured at
an earlier time point than the fat biopsies, the BMI at time of
adipose
expression
measurement
and
time
of
adiponectin
measurement was highly correlated (r2 = 0.9). SNPs (which were estimated to be equivalent to 96 independent
statistical tests due to linkage disequilibrium [LD]) [26] were tested
for
their
association,
we
employed
a
Bonferroni-corrected
threshold of a = 0.0005 (where 0.0005 = 0.05/96) to define the
threshold of association for any individual SNP association with
T2D and related traits. While any individual SNP may demonstrate a relationship with
T2D or related traits, it can be more informative to test whether a
multi-SNP genotypic risk score is associated with the outcome of
interest. In the absence of pleiotropic effects arising from loci other
than ADIPOQ, such a multi-SNP genotypic risk score would enable
testing of whether adiponectin levels are causally related to risk of
T2D or metabolic traits through a Mendelian randomization
framework. Since most of the SNPs that we identified to be
genome-wide significant for adiponectin levels were not in the
ADIPOQ locus, the presence of such pleiotropy precluded a formal
Mendelian randomization study. To create a multi-SNP genotypic
risk score we implemented a novel method that approximates the
average effect of adiponectin decreasing alleles on T2D or related
traits. Further, this method allows the use of consortium-level
meta-analytic results for a set of SNPs, rather than requiring the
re-analysis of individual-level data in each cohort, thereby
providing more accurate effects of each allele (due to the larger
sample size in the consortium-level meta-analysis). Association of Genome-Wide Significant SNPs with Gene
Expression (Stage 2) The weighted
sum of the individual SNP coefficients leads not only to an
estimate of the average combined allelic effect, but also to an
approximate estimate of the explained variance (when scaled by
the inverse of the total meta-analysis sample size) from a
multivariate regression model containing these SNPs. cis-eQTLs were defined as associations between SNPs and a
transcript within 1 Mb of the identified SNP. To correct for
multiple testing, we used QVALUE software [56], and estimated
that a genome-wide false discovery rate of 1% corresponds to a p-
value threshold of 5.0661025 (this conservative threshold accounts
for all multiple arising from the use of the array, rather than multiple
testing arising from assessing only transcripts in the genome-wide
significant regions). To test whether mRNA levels of candidate
genes identified in the GWAS meta-analysis are associated with
circulating adiponectin levels, we applied a Bonferoni corrected
threshold of p,361024 (where 361024 = 0.05/133 and 133 was
the number of transcripts tested at the candidate loci). PLoS Genetics | www.plosgenetics.org Table S1
Cohort characteristics.
(XLSX) Table S2
Comparing the Genome-Wide Significant SNPS from
fixed effect model with random effect model. *SNP with I2 less
than 0.5 are listed in bold, EA: Effect Allele, NEA: Non-Effect
Allele. (PDF) ^a%
Pm
i~1 wi^bisi{2
Pm
i~1 w2
i s{2
i (PDF) Table S3
Association Results of SNPs achieving p#561026 in
the Discovery phase in European Populations (Sex-Combined
Analysis). *Denotes SNPs typed in the de-novo follow-up phase. (PDF) with a standard error estimate of se(^a)%
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1
Pm
i~1 w2
i s{2
i
s se(^a)%
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1
Pm
i~1 w2
i s{2
i
s Table S4
Genome-Wide Significant SNPs (p,561028) Associ-
ated with Adiponectin Levels in Non-Europeans Populations. EA:
Effect Allele, NEA: Non-Effect Allele, EA-Freq: Frequency of
Effect Allele. (PDF) Therefore, under the assumption of uncorrelated SNPs, their
joint effect can be estimated in external data by a weighted mean
of the individual SNP effects, weighted by the estimates from the
discovery data. All these quantities can be obtained from meta-
analysis or summary data, so that individual-level data are not
required to obtain these results. Table S5
SNPs associated with adiponectin at genome-wide
significant levels (p,561028) using the fixed-effect model in
women only in European populations (including Discovery and
Follow-Up phases). (PDF) To implement this method, we first selected LD-independent
adiponectin associated alleles by LD pruning the set of genome-
wide significant adiponectin SNPs from the discovery phase with
an LD threshold of r2#0.05 in the HapMap CEU population,
yielding 20 independent LD blocks from the 196 SNPs in Table
S2. (We also applied the method using an LD threshold of
r2#0.01 and found no relevant change in results). Since many
SNPs from the same independent blocks were associated with
adiponectin, we selected the SNP from the LD block that
explained the most variance in adiponectin levels. Next, we
approximated the effect of the multi-SNP genetic risk score using
b and its standard error as derived from the consortium-level
meta-analysis in DIAGRAM+, MAGIC, GLGC, GIANT and
Body Fat GWAS consortium. Table S6
SNPs associated with adiponectin at genome-wide
significant levels (p,561028) using fixed-effect models in men
only in Euopean populations. (PDF) Table S7
Association results of nominally significant SNPs with
Type 2 Diabetes in the DIAGRAM+ Consortium. EA: Effect
Allele, NEA: Non-Effect Allele. B) Association results of nominally
significant SNPs with diabetes-related traits in the MAGIC
Consortium. Association of Genome-Wide Significant SNPs with T2D
and Metabolic Traits (Stage 3) The DIAGRAM+ (effective n = 22,044) [19], MAGIC (n = up
to 46,186) [20], GLGC (n = up to 97,021) [22], GIANT (n = up to
121,335) [21], and Body Fat GWAS (n = up to 36,625) consortia
provided summary statistics for the association of each SNP that
was genome-wide significant in the discovery phase. Since 196 PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 13 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Specifically, suppose m SNPs have shown association in the
discovery phase, and effects are denoted wi. However, suppose that
the goal of interest is to estimate the joint effect of these SNPs on
an outcome of interest, y. Let j index the individuals in the
outcome of interest dataset and let 2½ln L(M1){ ln L(M0)%nR2 If the SNPs are uncorrelated, and if the total percentage of
variance explained is small, then the change in log likelihood can
be approximated by C{
X
m
i~1
bi{^bi
2
2s2
i where bi now refers to the effect of SNP i in the outcome data, ^bi
is the outcome data estimate, and si is the associated standard
error estimate. Assuming that this log likelihood difference
approximation is maximized with an appropriate value of C,
then it can be shown that a can be estimated by: where bi now refers to the effect of SNP i in the outcome data, ^bi
is the outcome data estimate, and si is the associated standard
error estimate. Assuming that this log likelihood difference
approximation is maximized with an appropriate value of C,
then it can be shown that a can be estimated by: Table S1
Cohort characteristics. (XLSX) Supporting Information Figure S1
The comparison between two independent meta-
analyses performed in different centers for quality control
purposes. The 2log10 p-value of all SNPS with MAF$0.01 in
the first analysis are plotted against the 2log10 p-value from the
second analysis. (TIF) sj~
Xm
i~1 wixij Figure S2
The Manhattan plots of sex-stratified meta-analyses
in the discovery phase in the European population. The meta-
analysis shown in panel a) is stratified for women and that in panel
b) is stratified for men. Manhattan plots demonstrate 2Log 10(p-
value) measures for association between single nucleotide poly-
morphisms (SNPs) and chromosomal position. The SNPs that
achieved genome-wide significance are highlighted in green in the
plots. The red ovals identify loci found only in women. (TIF) be a risk score based on the discovery data SNPs, and their
associated parameter estimates wi. Therefore, the desired goal is
to
estimate
the
parameter
in
the
following
equation:
yj~y0zasjzej
in
the
outcome
of
interest
dataset. The
proportion of variance in y explained by the previous equation,
(i.e. the R2) attributable to the risk score can be estimated. Standard linear model theory shows that the change in log
likelihood is proportional to the R2, Figure S3
Association Results Near Peaks for Sex-specific
Analysis of Adiponectin. SNPs in regions near peak associations
are shown for a) chromosome 8 female, b) chromosome 8 males, c)
chromosome 12 females and d) chromosome 12 males. Purple
diamonds indicate the top SNPs, which have the strongest
evidence of association in women. Each circle shows a SNP with
a color scale proportional to the r2 value for that SNP and the top
SNP from HapMap CEU. Blue lines show the estimated
recombination rates from HapMap. The bottom panels illustrate
the relative position of each gene in the locus. Consortia Authors’ list:
DIAGRAM+: 15. Ontario Institute for Cancer Research, 101 College Street, Suite
800, Toronto, Ontario M5G 0A3, Canada Benjamin F Voight1,2,3, Laura J Scott4, Valgerdur Steinthorsdottir5,
Andrew P Morris6, Christian Dina7,8, Ryan P Welch9, Eleftheria
Zeggini6,10, Cornelia Huth11,12, Yurii S Aulchenko13, Gudmar Thorleifs-
son5, Laura J McCulloch14, Teresa Ferreira6, Harald Grallert11,12, Najaf
Amin13, Guanming Wu15, Cristen J Willer4, Soumya Raychaudhuri1,2,16,
Steve A McCarroll1,17, Claudia Langenberg18, Oliver M Hofmann19, Jose´e
Dupuis20,21, Lu Qi22–24, Ayellet V Segre`1,2,17, Mandy van Hoek25, Pau
Navarro26, Kristin Ardlie1, Beverley Balkau27,28, Rafn Benediktsson29,30,
Amanda J Bennett14, Roza Blagieva31, Eric Boerwinkle32, Lori L
Bonnycastle33, Kristina Bengtsson Bostro¨m34, Bert Bravenboer35, Suzan-
nah Bumpstead10, Noe¨l P Burtt1, Guillaume Charpentier36, Peter S
Chines33, Marilyn Cornelis24, David J Couper37, Gabe Crawford1, Alex SF
Doney38,39, Katherine S Elliott6, Amanda L Elliott1,17,40, Michael R
Erdos33, Caroline S Fox21,41, Christopher S Franklin42, Martha Ganser4,
Christian Gieger11, Niels Grarup43, Todd Green1,2, Simon Griffin18,
Christopher J Groves14, Candace Guiducci1, Samy Hadjadj44, Neelam
Hassanali14, Christian Herder45, Bo Isomaa46,47, Anne U Jackson4, Paul
RV Johnson48, Torben Jørgensen49,50, Wen HL Kao51,52, Norman
Klopp11, Augustine Kong5, Peter Kraft22,23, Johanna Kuusisto53, Torsten
Lauritzen54, Man Li51, Aloysius Lieverse55, Cecilia M Lindgren6, Valeriya
Lyssenko56, Michel Marre57,58, Thomas Meitinger59,60, Kristian Mid-
thjell61, Mario A Morken33, Narisu Narisu33, Peter Nilsson56, Katharine R
Owen14, Felicity Payne10, John RB Perry62,63, Ann-Kristin Petersen11,
Carl Platou61, Christine Proenc¸a7, Inga Prokopenko6,14, Wolfgang
Rathmann64, N William Rayner6,14, Neil R Robertson6,14, Ghislain
Rocheleau65–67, Michael Roden45,68, Michael J Sampson69, Richa
Saxena1,2,40, Beverley M Shields62,63, Peter Shrader3,70, Gunnar Sigurds-
son29,30, Thomas Sparsø43, Klaus Strassburger64, Heather M Stringham4,
Qi Sun22,23, Amy J Swift33, Barbara Thorand11, Jean Tichet71, Tiinamaija
Tuomi46,72, Rob M van Dam24, Timon W van Haeften73, Thijs van
Herpt25,55, Jana V van Vliet-Ostaptchouk74, G Bragi Walters5, Michael N
Weedon62,63, Cisca Wijmenga75, Jacqueline Witteman13, Richard N
Bergman76, Stephane Cauchi7, Francis S Collins77, Anna L Gloyn14, Ulf
Gyllensten78, Torben Hansen43,79, Winston A Hide19, Graham A
Hitman80, Albert Hofman13, David J Hunter22,23, Kristian Hveem61,81,
Markku Laakso53, Karen L Mohlke82, Andrew D Morris38,39, Colin NA
Palmer38,39, Peter P Pramstaller83, Igor Rudan42,84,85, Eric Sijbrands25,
Lincoln D Stein15, Jaakko Tuomilehto86, Andre Uitterlinden25, Mark
Walker87, Nicholas J Wareham18, Richard M Watanabe76,88, Goncalo R
Abecasis4, Bernhard O Boehm31, Harry Campbell42, Mark J Daly1,2,
Andrew T Hattersley62,63, Frank B Hu22–24, James B Meigs3,70, James S
Pankow89, Oluf Pedersen43,90,91, H.-Erich Wichmann11,12,92, Ineˆs Bar-
roso10, Jose C Florez1,2,3,93, Timothy M Frayling62,63, Leif Groop56,72, Rob
Sladek65–67, Unnur Thorsteinsdottir5,94, James F Wilson42, Thomas Illig11, 16. Table S1
Cohort characteristics.
(XLSX) Fasting glucose and 2 h glucose in mmol/L; Insulin
in pmol/L, EA: Effect Allele, NEA: Non-Effect Allele. C)
Association results of nominally significant SNPs with diabetes- PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 14 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Philippe Froguel7,95, Cornelia M van Duijn13, Kari Stefansson5,94, David
Altshuler1,2,3,17,40,93, Michael Boehnke4, Mark I McCarthy6,14,96. Philippe Froguel7,95, Cornelia M van Duijn13, Kari Stefansson5,94, David
Altshuler1,2,3,17,40,93, Michael Boehnke4, Mark I McCarthy6,14,96. related traits in the GIANT and Body fat GWAS consortia. The
beta expressed in inverse normally transformed BMI units (i.e. interpretable as SD or Z-score), shows the change in BMI per
additional effect allele.,*Results that are statistically significant,
accounting for the number of independent SNPs, are highlighted
in bold., EA: Effect Allele, NEA: Non-Effect Allele, EA-Freq:
Frequency of Effect Allele. D) Association results of nominally
significant SNPs with lipid traits in the GLGC Consortium. For
these traits the effect size is in SD units, based on standard error-
weighted meta-analysis. *Results that are statistically significant,
accounting for the number of independent SNPs are highlighted in
bold., EA: Effect Allele, NEA: Non-Effect Allele, EA-Freq:
Frequency of Effect Allele. (PDF) y
1. Broad Institute of Harvard and Massachusetts Institute of Technology
(MIT), Cambridge, Massachusetts 02142, USA 2. Center for Human Genetic Research, Massachusetts General
Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA 3. Department of Medicine, Harvard Medical School, Boston,
Massachusetts 02115, USA 4. Department of Biostatistics, University of Michigan, Ann Arbor,
Michigan 48109-2029, USA 5. deCODE Genetics, 101 Reykjavik, Iceland 6. Wellcome Trust Centre for Human Genetics, University of Oxford,
Oxford, OX3 7BN, UK 7. CNRS-UMR-8090, Institute of Biology and Lille 2 University,
Pasteur Institute, F-59019 Lille, France 8. INSERM UMR915 CNRS ERL3147 F-44007 Nantes, France 9. Bioinformatics Program, University of Michigan, Ann Arbor MI USA
48109 Text S1
Supplemental data include description of study cohorts
and funding. (DOCX) Text S1
Supplemental data include description of study cohorts
and funding. (DOCX) 10. Wellcome Trust Sanger Institute, Hinxton, CB10 1HH, UK 11. Institute of Epidemiology, Helmholtz Zentrum Muenchen, 85764
Neuherberg, Germany 12. Institute of Medical Informatics, Biometry and Epidemiology,
Ludwig-Maximilians-Universita¨t, 81377 Munich, Germany Acknowledgments g
y
13. Department of Epidemiology, Erasmus University Medical Center,
P O B
CA R
d
h
h
l
d We thank all study participants, volunteers, and study personnel that made
this consortium possible. We would also like to thank Ms. Renee Atallah
for her efforts with the writing and correction of the manuscript. 13. Department of Epidemiology, Erasmus University
P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. 13. Department of Epidemiology, Erasmus University P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. 14. Oxford Centre for Diabetes, Endocrinology and Metabolism,
University of Oxford, OX3 7LJ, UK Adiponectin: Multiethnic Genome-Wide Meta-Analysis Faculty of Health Science, University of Copenhagen, 2200
Copenhagen, Denmark 84. Croatian Centre for Global Health, Faculty of Medicine, University
of Split, Soltanska 2, 21000 Split, Croatia 84. Croatian Centre for Global Health, F
of Split, Soltanska 2, 21000 Split, Croatia 51. Department of Epidemiology, Johns Hopkins University, Baltimore,
Maryland 21287, USA 85. Institute for Clinical Medical Research, University Hospital ‘‘Sestre
Milosrdnice’’, Vinogradska 29, 10000 Zagreb, Croatia 52. Department of Medicine, and Welch Center for Prevention,
Epidemiology, and Clinical Research, Johns Hopkins University, Balti-
more, Maryland 21287, USA 86. Department of Chronic Disease Prevention, National Institute for
Health and Welfare, Helsinki FIN-00300, Finland, 87. Diabetes Research Group, Institute of Cellular Medicine, Newcastle
University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK 53. Department of Medicine, University of Kuopio and Kuopio
University Hospital, FIN-70211 Kuopio, Finland y
g
p
y
88. Department of Preventitive Medicine, Keck Medical School,
University of Southern California, Los Angeles, CA, 90089-9001, USA 54. Department of General Medical Practice, University of Aarhus, DK-
8000 Aarhus, Denmark 89. Division of Epidemiology and Community Health, University of
Minnesota, Minneapolis, Minnesota 55454, USA 55. Department of Internal Medicine, Maxima MC, PO-Box 90052,
5600 PD Eindhoven, The Netherlands p
90. Department of Biomedical Science, Panum, Faculty of Health
Science, University of Copenhagen, 2200 Copenhagen, Denmark 56. Department of Clinical Sciences, Diabetes and Endocrinology
Research Unit, University Hospital Malmo¨, Lund University, 205 02
Malmo¨, Sweden 91. Faculty of Health Science, University of Aarhus, DK–8000 Aarhus,
Denmark 57. Department of Endocrinology, Diabetology and Nutrition, Bichat-
Claude Bernard University Hospital, Assistance Publique des Hoˆpitaux de
Paris, 75870 Paris Cedex 18, France 92. Klinikum Grosshadern, 81377 Munich, Germany 93. Diabetes Unit, Massachusetts General Hospital, Boston, Massachu-
setts 02144, USA 58. INSERM U695, Universite´ Paris 7, 75018 Paris, France 94. Faculty of Medicine, University of Iceland, 101 Reykjavı´k, Iceland
95. Genomic Medicine, Imperial College London, Hammersmith
Hospital, W12 0NN, London, UK 59. Institute of Human Genetics, Helmholtz Zentrum Muenchen, 85764
Neuherberg, Germany 60. Institute of Human Genetics, Klinikum rechts der Isar, Technische
Universita¨t Mu¨nchen, 81675 Muenchen, Germany p
96. Oxford National Institute for Health Research Biomedical Research
Centre, Churchill Hospital, Old Road Headington, Oxford, OX3 7LJ, UK 96. Oxford National Institute for Health Research Biomedical Research
Centre, Churchill Hospital, Old Road Headington, Oxford, OX3 7LJ, UK
MAGIC Consortium: 61. Consortia Authors’ list:
DIAGRAM+: Division of Rheumatology, Immunology and Allergy, Brigham and
Women’s Hospital, Harvard Medical School, Boston, Massachusetts
02115, USA 17. Department of Molecular Biology, Harvard Medical School, Boston,
Massachusetts 02115, USA 18. MRC Epidemiology Unit, Institute of Metabolic Science, Adden-
brooke’s Hospital, Cambridge CB2 0QQ, UK 19. Department of Biostatistics, Harvard School of Public Health,
Boston, Massachusetts 02115, USA 20. Department of Biostatistics, Boston University School of Public
Health, Boston, Massachusetts 02118, USA 21. National Heart, Lung, and Blood Institute’s Framingham Heart
Study, Framingham, Massachusetts 01702, USA 21. National Heart, Lung, and Blood Institute’s
Study, Framingham, Massachusetts 01702, USA y
g
22. Department of Nutrition, Harvard School of Public Health, 665
Huntington Ave, Boston, MA 02115, USA 23. Department of Epidemiology, Harvard School of Public Health, 665
Huntington Ave, Boston, MA 02115, USA 24. Channing Laboratory, Dept. of Medicine, Brigham and Women’s
Hospital and Harvard Medical School, 181 Longwood Ave, Boston, MA
02115, USA 25. Department of Internal Medicine, Erasmus University Medical
Centre, PO-Box 2040, 3000 CA Rotterdam, The Netherlands 26. MRC Human Genetics Unit, Institute of Genetics and Molecular 26. MRC Human Genetics Unit, Institute of Genetics and Molecular
Medicine, Western General Hospital, Edinburgh, EH4 2XU, UK Medicine, Western General Hospital, Edinburgh, EH4 2XU, UK 27. INSERM U780, F-94807 Villejuif. France 28. University Paris-Sud, F-91405 Orsay, France 29. Landspitali University Hospital, 101 Reykjavik, Iceland 30. Icelandic Heart Association, 201 Kopavogur, Iceland 31. Division of Endocrinology, Diabetes and Metabolism, Ulm
University, 89081 Ulm, Germany 32. The Human Genetics Center and Institute of Molecular Medicine,
University of Texas Health Science Center, Houston, Texas 77030, USA 32. The Human Genetics Center and Institute of Molecular Medicine,
U i
it
f T
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77030 USA 33. National Human Genome Research Institute, National Institute of
Health, Bethesda, Maryland 20892, USA y
34. R&D Centre, Skaraborg Primary Care, 541 30 Sko¨vde, Sweden 35. Department of Internal Medicine, Catharina Hospital, PO-Box
1350, 5602 ZA Eindhoven, The Netherlands 36. Endocrinology-Diabetology Unit, Corbeil-Essonnes Hospital, F-
91100 Corbeil-Essonnes, France 37. Department of Biostatistics and Collaborative Studies Coordinating
Center, University of North Carolina at Chapel Hill, Chapel Hill, North
Carolina, 27599, USA 38. Diabetes Research Centre, Biomedical Research Institute, University
of Dundee, Ninewells Hospital, Dundee DD1 9SY, UK PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 15 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis 39. Pharmacogenomics Centre, Biomedical Research Institute, Univer-
sity of Dundee, Ninewells Hospital, Dundee DD1 9SY, UK 71. Institut interre´gional pour la Sante´ (IRSA), F-37521 La Riche,
France 72. Department of Medicine, Helsinki University Hospital, University of
Helsinki, FIN-00290 Helsinki, Finland 40. Department of Genetics, Harvard Medical School, Boston,
Massachusetts 02115, USA 73. Department of Internal Medicine, University Medical Center
Utrecht, 3584 CG Utrecht,The Netherlands 41. Division of Endocrinology, Diabetes, and Hypertension, Brigham
and Women’s Hospital, Harvard Medical School, Boston, Massachusetts
02115, USA 74. Molecular Genetics, Medical Biology Section, Department of
Pathology and Medical Biology, University Medical Center Groningen
and University of Groningen, 9700 RB Groningen, The Netherlands 42. Centre for Population Health Sciences, University of Edinburgh,
Teviot Place, Edinburgh, EH8 9AG, UK 75. Department of Genetics, University Medical Center Groningen an
University of Groningen, 9713 EX Groningen, The Netherlands 75. Department of Genetics, University Medical Center Groningen and
University of Groningen, 9713 EX Groningen, The Netherlands
76. Department of Physiology and Biophysics, University of Southern
California School of Medicine, Los Angeles, California 90033, USA 43. Hagedorn Research Institute, DK-2820 Gentofte, Denmark 44. Centre Hospitalier Universitaire de Poitiers, Endocrinologie
Diabetologie, CIC INSERM 0801, INSERM U927, Universite´ de Poitiers,
UFR, Me´decine Pharmacie, 86021 Poitiers Cedex, France y
g
g
76. Department of Physiology and Biophysics, University of Southern
California School of Medicine, Los Angeles, California 90033, USA 7. National Institute of Health, Bethesda, Maryland 20892, U 45. Institute for Clinical Diabetology, German Diabetes Center, Leibniz
Center for Diabetes Research at Heinrich Heine University Du¨sseldorf,
40225 Du¨sseldorf, Germany 78. Department of Genetics and Pathology, Rudbeck Laboratory,
Uppsala University, S-751 85 Uppsala, Sweden. 46. Folkha¨lsan Research Center, FIN-00014 Helsinki, Finland y
,
,
80. Centre for Diabetes, Barts and The London School of Medicine and
Dentistry, Queen Mary University of London, London E1 2AT, UK 47. Malmska Municipal Health Center and Hospital, 68601 Jakobstad,
Finland 81. Department of Medicine, The Hospital of Levanger, N-7600
Levanger, Norway 48. Diabetes Research and Wellness Foundation Human Islet Isolation
Facility and Oxford Islet Transplant Programme, University of Oxford,
Old Road, Headington, Oxford, OX3 7LJ, UK 82. Department of Genetics, University of North Carolina, Chapel Hill,
North Carolina 27599, USA 49. Research Centre for Prevention and Health, Glostrup University
Hospital, DK-2600 Glostrup, Denmark 83. Institute of Genetic Medicine, European Academy Bozen/Bolzano
(EURAC), Viale Druso 1, 39100 Bolzano, Italy 50. Adiponectin: Multiethnic Genome-Wide Meta-Analysis Nord-Trøndelag Health Study (HUNT) Research Center, Depart-
ment of Community Medicine and General Practice, Norwegian
University of Science and Technology, NO-7491 Trondheim, Norway MAGIC Consortium:
Jose´e Dupuis1,2,178, Claudia Langenberg3,178, Inga Prokopenko4,5,178,
Richa Saxena6,7,178, Nicole Soranzo8,9,178, Anne U Jackson10, Eleanor
Wheeler11, Nicole LGlazer12, Nabila Bouatia-Naji13, Anna LGloyn4,
Cecilia MLindgren4,5, Reedik Ma¨gi4,5, Andrew P Morris5, Joshua
Randall5, Toby Johnson14–16, Paul Elliott17,176, Denis Rybin18, Gudmar
Thorleifsson19, Valgerdur Steinthorsdottir19, Peter Henneman20, Harald
Grallert21, Abbas Dehghan22, Jouke Jan Hottenga23, Christopher SFrank-
lin24, Pau Navarro25, Kijoung Song26, Anuj Goel5,27, John R B Perry28,
Josephine MEgan29, Taina Lajunen30, Niels Grarup31, Thomas Sparsø31,
Alex Doney32, Benjamin F Voight6,7, Heather MStringham10, Man Li33,
Stavroula Kanoni34, Peter Shrader35, Christine Cavalcanti-Proenc¸a13,
Meena Kumari36, Lu Qi37, Nicholas J Timpson38, Christian Gieger21,
Carina Zabena39, Ghislain Rocheleau40,41, Erik Ingelsson42,43, Ping An44,
Jeffrey O’Connell45, Jian’an Luan3, Amanda Elliott6,7, Steven A McCar-
roll6,7, Felicity Payne11, Rosa Maria Roccasecca11, Franc¸ois Pattou46,
Praveen Sethupathy47, Kristin Ardlie48, Yavuz Ariyurek49, Beverley
Balkau50, Philip Barter51, John P Beilby52,53, Yoav Ben-Shlomo54, Rafn
Benediktsson55,56, Amanda J Bennett4, Sven Bergmann14,16, Murielle
Bochud15, Eric Boerwinkle57, Ame´lie Bonnefond13, Lori LBonnycastle47,
Knut Borch-Johnsen58,59, Yvonne Bo¨ttcher60, Eric Brunner36, Suzannah J
Bumpstead8, Guillaume Charpentier61, Yii-Der Ida Chen62, Peter
Chines47, Robert Clarke63, Lachlan J MCoin17, Matthew NCooper64,
Marilyn Cornelis37, Gabe Crawford6, Laura Crisponi65, Ian NMDay38, y
gy
y
62. Genetics of Complex Traits, Institute of Biomedical and Clinical
S i
P
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it
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62. Genetics of Complex Traits, Institute of Biomedical and Clinical
Science, Peninsula Medical School, University of Exeter, Magdalen Road, Science, Peninsula Medical School, University of Exeter, Magdalen Road,
Exeter EX1 2LU, UK
63. Diabetes Genetics, Institute of Biomedical and Clinical Science, Exeter EX1 2LU, UK
63. Diabetes Genetics, Institute of Biomedical and Clinical Science,
P
i
l
M di
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l U i
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B
k R
d E Exeter EX1 2LU, UK
63. Diabetes Genetics, Institute of Biomedical and Clinical Science,
Peninsula Medical School, University of Exeter, Barrack Road, Exeter
EX2 5DW, UK
64. Institute of Biometrics and Epidemiology, German Diabetes Center, Peninsula Medical School, University of Exeter, Barrack Road, Exeter
EX2 5DW, UK
64. Institute of Biometrics and Epidemiology, German Diabetes Center, EX2 5DW, UK
64. Institute of Biometrics and Epidemiology, German Diabetes Center, ,
64. Adiponectin: Multiethnic Genome-Wide Meta-Analysis Eco J Cde Geus23, Jerome Delplanque13, Christian Dina13, Michael R
Erdos47, Annette CFedson64,66, Antje Fischer-Rosinsky67,68, Nita GFor-
ouhi3, Caroline SFox2,69, Rune Frants70, Maria Grazia Franzosi71, Pilar
Galan72, Mark OGoodarzi62, Ju¨rgen Graessler73, Christopher J Groves4,
Scott Grundy74, Rhian Gwilliam8, Ulf Gyllensten75, Samy Hadjadj76,
Go¨ran Hallmans77, Naomi Hammond8, Xijing Han10, Anna-Liisa
Hartikainen78, Neelam Hassanali4, Caroline Hayward25, Simon CHeath79,
Serge Hercberg80, Christian Herder81, Andrew A Hicks82, David R
Hillman66,83, Aroon DHingorani36, Albert Hofman22, Jennie Hui52,84, Joe
Hung85,86, Bo Isomaa87,88, Paul R V Johnson4,89, Torben Jørgensen90,91,
Antti Jula92, Marika Kaakinen93, Jaakko Kaprio94–96, Y Antero Kesa-
niemi97, Mika Kivimaki36, Beatrice Knight98, Seppo Koskinen99, Peter
Kovacs100, Kirsten Ohm Kyvik101, GMark Lathrop79, Debbie A Lawlor38,
Olivier Le Bacquer13, Ce´cile Lecoeur13, Yun Li10, Valeriya Lyssenko102,
Robert Mahley103, Massimo Mangino9, Alisa KManning1, Marı´a Teresa
Martı´nez-Larrad39, Jarred B McAteer6,104,105, Laura J McCulloch4, Ruth
McPherson106, Christa Meisinger21, David Melzer28, David Meyre13,
Braxton DMitchell45, Mario A Morken47, Sutapa Mukherjee66,83, Silvia
Naitza65, Narisu Narisu47, Matthew J Neville4,107, Ben A Oostra108, Marco
Orru`65, Ruth Pakyz45, Colin NA Palmer109, Giuseppe Paolisso110, Cristian
Pattaro82, Daniel Pearson47, John F Peden5,27, Nancy LPedersen42,
Markus Perola96,111,112, Andreas F H Pfeiffer67,68, Irene Pichler82, Ozren
Polasek113, Danielle Posthuma23,114, Simon CPotter8, Anneli Pouta115,
Michael A Province44, Bruce MPsaty116,117, Wolfgang Rathmann118, Nigel
WRayner4,5, Kenneth Rice119, Samuli Ripatti96,111, Fernando Rivade-
neira22,120, Michael Roden81,121, Olov Rolandsson122, Annelli Sand-
baek123, Manjinder Sandhu3,124, Serena Sanna65, Avan Aihie Sayer125,
Paul Scheet126, Laura J Scott10, Udo Seedorf127, Stephen J Sharp3,
Beverley Shields98, Gunnar Sigurðsson55,56, Eric J GSijbrands22,120,
Angela Silveira128, Laila Simpson64,66, Andrew Singleton129, Nicholas
LSmith130,131, Ulla Sovio17, Amy Swift47, Holly Syddall125, Ann-Christine
Syva¨nen132, Toshiko Tanaka133,134, Barbara Thorand21, Jean Tichet135,
Anke To¨njes60,136, Tiinamaija Tuomi87,137, Andre´ GUitterlinden22,120, Ko
Willems van Dijk70,138, Mandy van Hoek120, Dhiraj Varma8, Sophie
Visvikis-Siest139, Veronique Vitart25, Nicole Vogelzangs140, Ge´rard
Waeber141, Peter J Wagner96,111, Andrew Walley142, GBragi Walters19,
Kim LWard64,66, Hugh Watkins5,27, Michael NWeedon28, Sarah H
Wild24, Gonneke Willemsen23, Jaqueline CMWitteman22, John WGYar-
nell143, Eleftheria Zeggini5,8, Diana Zelenika79, Bjo¨rn Zethelius43,144,
Guangju Zhai9, Jing Hua Zhao3, MCarola Zillikens120, DIAGRAMCon-
sortium145, GIANTConsortium145, Global BPgen Consortium145,Ingrid B
Borecki44, Ruth J F Loos3, Pierre Meneton80, Patrik KEMagnusson42,
David MNathan104,105, Gordon H Williams69,105, Andrew THattersley98,
Kaisa Silander96,111, Veikko Salomaa146, George Davey Smith38, Stefan R
Bornstein73, Peter Schwarz73, Joachim Spranger67,68, Fredrik Karpe4,107,
Alan R Shuldiner45, Cyrus Cooper125, George V Dedoussis34, Manuel
Serrano-Rı´os39, Andrew DMorris109, Lars Lind132, Lyle J Palmer64,66,84,
Frank B Hu147,148, Paul WFranks149, Shah Ebrahim150, Michael
Marmot36, WH Linda Kao33,151,152, James SPankow153, Michael J
Sampson154, Johanna Kuusisto155, Markku Laakso155, Torben Han-
sen31,156, Oluf Pedersen31,59,157, Peter Paul Pramstaller82,158,159, H Erich
Wichmann21,160,161, Thomas Illig21, Igor Rudan24,162,163, Alan F Wright25,
Michael Stumvoll60, Harry Campbell24, James F Wilson24, Anders
Hamsten on behalf of Procardis Consortium128, Richard NBergman164,
Thomas A Buchanan164,165, Francis SCollins47, Karen LMohlke166, Jaakko
Tuomilehto94,167, 168, Timo TValle167, David Altshuler6,7,104,105, Jerome I
Rotter62, David SSiscovick169, Brenda WJ H Penninx140, Dorret I
Boomsma23, Panos Deloukas8, Timothy DSpector8,9, Timothy MFray-
ling28, Luigi Ferrucci170, Augustine Kong19, Unnur Thorsteinsdottir19,171,
Kari Stefansson19,171, Cornelia Mvan Duijn22, Yurii SAulchenko22,
Antonio Cao65, Angelo Scuteri172,177, David Schlessinger47, Manuela
Uda65, Aimo Ruokonen173, Marjo-Riitta Jarvelin17,93, 174, Dawn MWater-
worth26, Peter Vollenweider141, Leena Peltonen8,48,96,111,112, Vincent
Mooser26, Goncalo R Abecasis10, Nicholas J Wareham3, Robert
Sladek40,41, Philippe Froguel13,142, Richard MWatanabe164,175, James B
Meigs35,105, Leif Groop102, Michael Boehnke10, Mark I McCarthy4,5,107,
Jose CFlorez6,7,104,105 & Ineˆs Barroso11 for the MAGIC investigators
1 D
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f P bli 4 Oxford Centre for Diabetes, Endocrinology and Metabolism,
University of Oxford, Oxford, UK. Adiponectin: Multiethnic Genome-Wide Meta-Analysis Institute of Biometrics and Epidemiology, German Diabetes Center, Leibniz Center for Diabetes Research at Heinrich Heine University
Du¨sseldorf, 40225 Du¨sseldorf, Germany 65. Department of Human Genetics, McGill University, Montreal H3H
1P3, Canada 66. Department of Medicine, Faculty of Medicine, McGill University,
Montreal, H3A 1A4, Canada 67. McGill University and Genome Quebec Innovation Centre,
Montreal, H3A 1A4. Canada 68. Department of Metabolic Diseases, Heinrich Heine University
Du¨sseldorf, 40225 Du¨sseldorf, Germany 69. Department of Endocrinology and Diabetes, Norfolk and Norwich
University Hospital NHS Trust , Norwich, NR1 7UY, UK. 70. General Medicine Division, Massachusetts General Hospital,
Boston, Massachusetts, USA PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 PLoS Genetics | www.plosgenetics.org 16 Adiponectin: Multiethnic Genome-Wide Meta-Analysis 1 Department of Biostatistics, Boston University School of Public
Health, Boston, Massachusetts, USA. Adiponectin: Multiethnic Genome-Wide Meta-Analysis y
5 Wellcome Trust Centre for Human Genetics, University of Oxford,
Oxford, UK. 6 Program in Medical and Population Genetics, Broad Institute,
Cambridge, Massachusetts, USA. 7 Center for Human Genetic Research, Massachusetts General
Hospital, Boston, Massachusetts, USA. p
8 Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. 9 Twin Research and Genetic Epidemiology Department, King’s
College London, St Thomas’ Hospital Campus, London, UK. 10 Center for Statistical Genetics, Department of Biostatistics, University
of Michigan School of Public Health, Ann Arbor, Michigan, USA. 11 Metabolic Disease Group, Wellcome Trust Sanger Institute,
Hinxton, Cambridge, UK. 12 Cardiovascular Health Research Unit and Department of Medicine,
University of Washington, Seattle, Washington, USA. 13 Centre National de la Recherche Scientifique–Unite´ Mixte de
Recherche 8090 , Pasteur Institute, Lille 2 –Droit et Sante´ University, Lille,
France. 14 Department of Medical Genetics, University of Lausanne, Lausanne,
Switzerland. 15 University Institute of Social and Preventative Medicine, Centre
Hospitalier Universitaire Vaudois (CHUV) and University of Lausanne,
Lausanne, Switzerland. 16 Swiss Institute of Bioinformatics, Lausanne, Switzerland. 17 Department of Epidemiology and Public Health, Imperial College
London, Faculty of Medicine, Norfolk Place, London, UK. 18 Boston University Data Coordinating Center, Boston, Massachusetts,
USA. 19 deCODE Genetics, Reykjavik, Iceland. 20 Department of Human Genetics, Leiden University Medical Centre,
Leiden, The Netherlands. 21 Institute of Epidemiology, Helmholtz Zentrum Muenchen, German
Research Center for Environmental Health, Neuherberg, Germany. 22 Department of Epidemiology, Erasmus Medical College, Rotterdam,
The Netherlands. 23 Department of Biological Psychology, VU University Amsterdam,
Amsterdam, The Netherlands. 24 Centre for Population Health Sciences, University of Edinburgh,
Edinburgh, UK. 25 MRC Human Genetics Unit, Institute of Genetics and Molecular
Medicine, Edinburgh, UK. 26 Division of Genetics, Research and Development, GlaxoSmithKline,
King of Prussia, Pennsylvania, USA. 27 Department of Cardiovascular Medicine, University of Oxford,
Oxford, UK. 28 Genetics of Complex Traits, Institute of Biomedical and Clinical
Sciences, Peninsula College of Medicine and Dentistry, University of
Exeter, Exeter, UK. 29 National Institute of Aging, Baltimore, Maryland, US 30 Unit for Child and Adolescent Health and Welfare, National Institute
for Health and Welfare, Biocenter Oulu, University of Oulu, Oulu,
Finland. 31 Hagedorn Research Institute, Gentofte, Denmark. 32 Department of Medicine and Therapeutics, Level 7, Ninewells
Hospital and Medical School, Dundee, UK. p
33 Department of Epidemiology, Bloomberg School of Public Health,
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Johns Hopkins University, Baltimore, Maryland, USA. 2 National Heart, Lung, and Blood Institute’s Framingham Heart Study,
Framingham, Massachusetts, USA. Adiponectin: Multiethnic Genome-Wide Meta-Analysis 41 Genome Quebec Innovation Centre, Montreal, Canada. 79 Centre National de Ge´notypage/Institut de ge´nomique/Commis-
sariat a` l’e´nergie atomique, Evry Cedex, France. 80 INSERM U872,
Faculte´ de Me´decine Paris Descartes, Paris Cedex, France. 42 Department of Medical Epidemiology and Biostatistics, Karolinska
Institutet, Stockholm, Sweden. Faculte´ de Me´decine Paris Descartes, Paris Cedex, France. 81 Institute for Clinical Diabetology, German Diabetes Center, Leibniz
Center for Diabetes Research at Heinrich Heine University Du¨sseldorf,
Du¨sseldorf, Germany. 43 Department of Public Health and Caring Sciences, Uppsala
University, Uppsala, Sweden. 44 Division of Statistical Genomics, Department of Genetics, Washing-
ton University School of Medicine, St. Louis, Missouri, USA. 82 Institute of Genetic Medicine, European Academy Bozen/Bolzano
(EURAC), Viale Druso, Bolzano, Italy, Affiliated Institute of the University
Lu¨beck, Lu¨beck, Germany. 45 Division of Endocrinology, Diabetes and Nutrition, University of
Maryland School of Medicine, Baltimore, Maryland, USA. 83 Department of Pulmonary Physiology, Sir Charles Gairdner
Hospital, Perth, Australia. y
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46 INSERM U 859 , Universite de Lille-Nord de France, Lille, France. 47 Genome Technology Branch, National Human Genome Research
Institute, Bethesda, Maryland, USA. 84 Busselton Population Medical Research Foundation, Sir Charles
Gairdner Hospital, Perth, Australia. 48 The Broad Institute, Cambridge, Massachusetts, USA. p
85 Heart Institute of Western Australia, Sir Charles Gairdner Hospital,
Nedlands West Australia, Australia. 49 Leiden Genome Technology Center, Leiden University Medical
Center, Leiden, The Netherlands. Nedlands West Australia, Australia. ,
86 School of Medicine and Pharmacology, University of Western
Australia, Nedlands West Australia, Australia. 86 School of Medicine and Pharmacology, University of Western 50 INSERM U 780 , Paris Sud University, Villejuif, France. Australia, Nedlands West Australia, Australia. Australia, Nedlands West Australia, Australia. 87 Folkhalsan Research Centre, Helsinki, Finland. 51 The Heart Research Institute, Sydney, New South Wales, Australia. ,
,
87 Folkhalsan Research Centre, Helsinki, Finland. 52 PathWest Laboratory of Western Australia, Department of Molecular
Genetics, J Block, QEII Medical Centre, Nedlands West Australia,
Australia. 88 Malmska Municipal Health Care Center and Hospital, Jakobstad,
Finland. 89 Nuffield Department of Surgery, University of Oxford, Oxford, UK. 53 School of Surgery and Pathology, University of Western Australia,
Nedlands West Australia, Australia. 90 Research Centre for Prevention and Health, Glostrup University
Hospital, Glostrup, Denmark. 54 Department of Social Medicine, University of Bristol, Bristol, UK. 54 Department of Social Medicine, University of Bristol, 91 Faculty of Health Science, University of Copenhagen, Copenhagen,
Denmark. 55 Landspitali University Hospital, Reykjavik, Iceland. 56 Icelandic Heart Association, Kopavogur, Iceland. Adiponectin: Multiethnic Genome-Wide Meta-Analysis 92 National Institute for Health and Welfare, Unit of Population
Studies, Turku, Finland. 57 The Human Genetics Center and Institute of Molecular Medicine
University of Texas Health Science Center, Houston, Texas, USA. 93 Institute of Health Sciences and Biocenter Oulu, University of Oulu,
Oulu, Finland. 58 Steno Diabetes Center, Gentofte, Denmark. 59 Faculty of Health Science, University of Aarhus, Aarhus, Denmark. 94 Department of Public Health, Faculty of Medicine, University of
Helsinki, Helsinki, Finland. 59 Faculty of Health Science, University of Aarhus, Aarhus, Denmark. 60 Department of Medicine, University of Leipzig, Leipzig, Germany. 60 Department of Medicine, University of Leipzig, Leipzig, Germany. 95 National Institute for Health and Welfare, Unit for Child and
Adolescent Mental Health, Helsinki, Finland. 61 Endocrinology–Diabetology Unit, Corbeil-Essonnes Hospital, Es-
sonnes, France. 96 Institute for Molecular Medicine Finland (FIMM), University of
Helsinki, Helsinki, Finland. 62 Medical Genetics Institute, Cedars-Sinai Medical Center, Los
Angeles, California, USA. 97 Department of Internal Medicine and Biocenter Oulu, Oulu,
Finland. 63 Clinical Trial Service Unit and Epidemiological Studies Unit,
University of Oxford, Oxford, UK. 98 Diabetes Genetics, Institute of Biomedical and Clinical Science,
Peninsula College of Medicine and Dentistry, University of Exeter, Exeter,
UK. 64 Centre for Genetic Epidemiology and Biostatistics, University of
Western Australia, Perth, Australia. ,
,
65 Istituto di Neurogenetica e Neurofarmacologia (INN), Consiglio 99 National Institute for Health and Welfare, Unit of Living Conditions,
Health and Wellbeing, Helsinki, Finland. Nazionale delle Ricerche, c/o Cittadella Universitaria di Monserrato,
Monserrato, Cagliari, Italy. g
100 Interdisciplinary Centre for Clinical Research, University of
Leipzig, Leipzig, Germany. 66 Western Australian Sleep Disorders Research Institute, Queen
Elizabeth Medical Centre II, Perth, Australia. p g
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101 The Danish Twin Registry, Epidemiology, Institute of Public
Health, University of Southern Denmark, Odense, Denmark. 67 Department of Endocrinology, Diabetes and Nutrition, Charite-
Universitaetsmedizin Berlin, Berlin, Germany. 102 Department of Clinical Sciences, Diabetes and Endocrinology,
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Lund University, University Hospital Malmo¨, Malmo¨, Sweden. 102 Department of Clinical Sciences, Diabetes and En 68 Department of Clinical Nutrition, German Institute of Human
Nutrition Potsdam-Rehbruecke, Nuthetal, Germany. 103 Gladstone Institute of Cardiovascular Disease, University of
California, San Francisco, California, USA. 69 Division of Endocrinology, Diabetes, and Hypertension, Brigham
and Women’s Hospital, Harvard Medical School, Boston, Massachusetts,
USA. Adiponectin: Multiethnic Genome-Wide Meta-Analysis 33 Department of Epidemiology, Bloomberg School Johns Hopkins University, Baltimore, Maryland, USA. 34 Department of Nutrition–Dietetics, Harokopio University, Athens,
Greece. 35 General Medicine Division, Massachusetts General Hospital, Boston,
Massachusetts, USA. 36 Department of Epidemiology and Public Health, University College
London, London, UK. 37 Departments of Nutrition and Epidemiology, Harvard School of
Public Health, Boston, Massachusetts, USA. 1 Department of Biostatistics, Boston University School of Public
Health, Boston, Massachusetts, USA. 38 MRC Centre for Causal Analyses in Translational Epidemiology,
University of Bristol, Bristol, UK. University of Bristol, Bristol, UK. 2 National Heart, Lung, and Blood Institute’s Framingham Heart Study,
Framingham, Massachusetts, USA. 39 Fundacio´n para la Investigacio´n Biome´dica del Hospital Clı´nico San
Carlos, Madrid, Spain. Carlos, Madrid, Spain. 3 Medical Research Council (MRC), Epidemiology Unit, Institute of
Metabolic Science, Addenbrooke’s Hospital, Cambridge, UK. 40 Departments of Medicine and Human Genetics, McGill University,
Montreal, Canada. PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 17 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis y
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125 MRC Epidemiology Resource Centre, University of Southampton, y
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125 MRC Epidemiology Resource Centre, University of Southampton,
Southampton General Hospital, Southampton, UK. 125 MRC Epidemiology Resource Centre, Univers Southampton General Hospital, Southampton, UK. Southampton General Hospital, Southampton, UK. 126 Department of Epidemiology, University of Texas, M.D. Anderson
Cancer Center, Houston, Texas, USA. 126 Department of Epidemiology, University of Texas, M.D. Anderson
Cancer Center Houston Texas USA 165 Department of Medicine, Division of Endocrinology, Keck School
of Medicine, University of Southern California, Los Angeles, California,
USA. 127 Leibniz-Institut fu¨r Arterioskleroseforschung an der Universita¨t
Mu¨nster, Mu¨nster, Germany. 166 Department of Genetics, University of North Carolina, Chapel Hill,
North Carolina, USA. 128 Atherosclerosis Research Unit, Department of Medicine, Karolinska
Institutet, Stockholm, Sweden. 167 National Institute for Health and Welfare, Unit of Diabetes
Prevention, Helsinki, Finland. 129 Laboratory of Neurogenetics, National Institute on Aging, Bethesda,
Maryland, USA. 168 South Ostrobothnia Central Hospital, Seinajoki, Finland. 130 Department of Epidemiology, University of Washington, Seattle,
Washington, USA. 169 Departments of Medicine and Epidemiology, University of
Washington, Seattle, Washington, USA. 131 Seattle Epidemiologic Research and Information Center, Depart-
ment of Veterans Affairs Office of Research and Development, Seattle,
Washington, USA. 170 Longitudinal Studies Section, Clinical Research Branch, National
Institute on Aging, NIH, Baltimore, Maryland, USA. 171 Faculty of Medicine, University of Iceland, Reykjavı´k, Iceland. 172 Lab of Cardiovascular Sciences, National Institute on Aging 132 Department of Medical Sciences, Uppsala University, Uppsala,
Sweden. 171 Faculty of Medicine, University of Iceland, Reykjavık, Ice
172 Lab of Cardiovascular Sciences, National Institute on
National Institutes of Health, Baltimore, Maryland, USA. 171 Faculty of Medicine, University of Iceland, Reykjavık, Iceland. 172 Lab of Cardiovascular Sciences, National Institute on Aging
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National Institutes of Health, Baltimore, Maryland, USA. 133 Medstar Research Institute, Baltimore, Maryland, USA. 173 Department of Clinical Sciences/Clinical Chemistry, University of
Oulu, University of Oulu, Oulu, Finland. 134 Clinical Research Branch, National Institute on Aging, Baltimore,
Maryland, USA. Maryland, USA. 135 Institut interre´gional pour la sante´ (IRSA), La Riche, France. 136 Coordination Centre for Clinical Trials, University of Leipzig,
Leipzig, Germany. y
174 National Institute of Health and Welfare, Oulu, Finland. GLGC Consortium:
1 1 GLGC Consortium:
Tanya M. Teslovich,1, 118 , Kiran Musunuru,2, 3, 4, 5, 6, 118 , Albert V. Smith,7, 8 , Andrew C. Edmondson,9, 10 , Ioannis M. Stylianou,10 ,
Masahiro Koseki,11 , James P. Pirruccello,2, 5, 6 , Samuli Ripatti,12, 13 ,
4 14
1
15 GLGC Consortium:
Tanya M. Teslovich,1, 118 , Kiran Musunuru,2, 3, 4, 5, 6, 118 , Albert V. Smith,7, 8 , Andrew C. Edmondson,9, 10 , Ioannis M. Stylianou,10 ,
Masahiro Koseki,11 , James P. Pirruccello,2, 5, 6 , Samuli Ripatti,12, 13 ,
Daniel I. Chasman,4, 14 , Cristen J. Willer,1 , Christopher T. Johansen,15 ,
Sigrid W. Fouchier,16 , Aaron Isaacs,17 , Gina M. Peloso,18, 19 , Maja
Barbalic,20 , Sally L. Ricketts,21 , Joshua C. Bis,22 , Yurii S. Aulchenko,17 ,
Gudmar Thorleifsson,23 , Mary F. Feitosa,24 , John Chambers,25 , Marju
Orho-Melander,26 , Olle Melander,26 , Toby Johnson,27 , Xiaohui Li,28 ,
Xiuqing Guo,28 , Mingyao Li,9, 10 , Yoon Shin Cho,29 , Min Jin Go,29 ,
Young Jin Kim,29 , Jong-Young Lee,29 , Taesung Park,30, 31 , Kyunga
Kim,32 , Xueling Sim,33 , Rick Twee-Hee Ong,34 , Damien C. Croteau-
Chonka,35 , Leslie A. Lange,35 , Joshua D. Smith,36 , Kijoung Song,37 , Jing
Hua Zhao,38 , Xin Yuan,37 , Jian’an Luan,38 , Claudia Lamina,39 ,
Andreas Ziegler,40 , Weihua Zhang,25 , Robert Y. L. Zee,4, 14 , Alan F. Wright,41 , Jacqueline C. M. Witteman,17, 42 , James F. Wilson,43 ,
Gonneke Willemsen,44 , H.-Erich Wichmann,45 , John B. Whitfield,46 ,
Dawn M. Waterworth,37 , Nicholas J. Wareham,38 , Ge´rard Waeber,47 ,
Peter Vollenweider,47 , Benjamin F. Voight,2, 5 , Veronique Vitart,41 ,
Andre G. Uitterlinden,17, 42, 48 , Manuela Uda,49 , Jaakko Tuomilehto,50 ,
John R. Thompson,51 , Toshiko Tanaka,52, 53 , Ida Surakka,12, 13 , Heather 141 Department of Internal Medicine, Centre Hospitalier Universitaire
Vaudois, Lausanne, Switzerland. 142 Genomic Medicine, Imperial College London, Hammersmith
Hospital, London, UK. 143 Epidemiology and Public Health, Queen’s University Belfast,
Belfast, UK. 144 Medical Products Agency, Uppsala, Sweden. 145 See Supplementary Note for a full list of autho 146 National Institute for Health and Welfare, Unit of Chronic Disease
Epidemiology and Prevention, Helsinki, Finland. 147 Departments of Nutrition and Epidemiology, Harvard School of
Public Health, Boston, Massachusetts, USA. 148 Channing Laboratory, Brigham and Women’s Hospital and
Harvard Medical School, Boston, Massachusetts, USA. Adiponectin: Multiethnic Genome-Wide Meta-Analysis 115 Department of Obstetrics and Gynaecology, Oulu University
Hospital, Oulu, Finland. 152 The Welch Center for Prevention, Epidemiology, and Clinical
Research, School of Medicine and Bloomberg School of Public Health,
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Johns Hopkins University, Baltimore, Maryland, USA 116 Departments of Medicine, Epidemiology and Health Services,
University of Washington, Seattle, Washington, USA. J
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153 Division of Epidemiology and Community Health, School of Public 153 Division of Epidemiology and Community Healt 153 Division of Epidemiology and Community Health, School
Health, University of Minnesota, Minneapolis, Minnesota, USA. 153 Division of Epidemiology and Community Health, School o
Health, University of Minnesota, Minneapolis, Minnesota, USA. 117 Group Health Research Institute, Group Health Cooperative,
Seattle, Washington, USA. y
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154 Department of Endocrinology and Diabetes, Norfolk and Norwich University Hospital National Health Service Trust, Norwich, UK. 118 Institute of Biometrics and Epidemiology, German Diabetes Centre, 155 Department of Medicine, University of Kuopio and Kuopio 155 Department of Medicine, University of Kuopio and Kuopio
University Hospital, Kuopio, Finland. Leibniz Centre at Heinrich Heine University Du¨sseldorf, Du¨sseldorf,
Germany. 156 Faculty of Health Science, University of Southern Denmark,
Odense, Denmark. 119 Department of Biostatistics, University of Washington, Seattle,
Washington, USA. 157 Institute of Biomedical Science, Faculty of Health Science,
University of Copenhagen, Copenhagen, Denmark. 120 Department of Internal Medicine, Erasmus Medical College,
Rotterdam, The Netherlands. 158 Department of Neurology, General Central Hospital, Bolzano,
Italy. 121 Department of Metabolic Diseases, Heinrich Heine University
Du¨sseldorf, Du¨sseldorf, Germany. y
159 Department of Neurology, University of Lu¨beck, Lu¨beck, Germany. 122 Department of Public Health and Clinical Medicine, Section for
Family Medicine, Umea˚ University, Umea˚, Sweden. 160 Institute of Medical Informatics, Biometry and Epidemiology,
Ludwig-Maximilians-Universita¨t, Munich, Germany. 160 Institute of Medical Informatics, Biometry
Ludwig-Maximilians-Universita¨t, Munich, Germany. 123 School of Public Health, Department of General Practice,
University of Aarhus, Aarhus, Denmark. 161 Klinikum Grosshadern, Munich, Germany. 162 School of Medicine, University of Split, 162 School of Medicine, University of Split, Split, Croatia. y
124 Department of Public Health and Primary Care, Strangeways
Research Laboratory, University of Cambridge, Cambridge, UK. 163 Gen-Info Ltd., Zagreb, Croatia. 163 Gen-Info Ltd., Zagreb, Croatia. p
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Research Laboratory, University of Cambridge, Cambridge, UK. 164 Department of Physiology and Biophysics, Keck School of
Medicine, University of Southern California, Los Angeles, California,
USA. Adiponectin: Multiethnic Genome-Wide Meta-Analysis 104 Diabetes Research Center, Diabetes Unit, Massachusetts General
Hospital, Boston, Massachusetts, USA. 70 Department of Human Genetics, Leiden University Medical Centre,
Leiden, The Netherlands. 105 Department of Medicine, Harvard Medical School, Boston,
Massachusetts, USA. 71 Department of Cardiovascular Research, Istituto di Ricerche
Farmacologiche ‘Mario Negri’, Milan, Italy. 106 Division of Cardiology, University of Ottawa Heart Institute,
Ottawa, Ontario, Canada. 72 Institut National de la Sante´ et de la Recherche Me´dicale, Institut
National de la Recherche Agronomique, Universite´ Paris 107 Oxford National Institute for Health Research, Biomedical
Research Centre, Churchill Hospital, Oxford, UK. 13 , Bobigny Cedex, France. 108 Department of Clinical Genetics, Erasmus Medical College,
Rotterdam, The Netherlands. 73 Department of Medicine III, Division Prevention and Care of
Diabetes, University of Dresden, Dresden, Germany. 109 Biomedical Research Institute, University of Dundee, Ninewells
Hospital and Medical School, Dundee, UK. 74 Center for Human Nutrition, University of Texas Southwestern
Medical Center, Dallas, Texas, USA. 110 Department of Geriatric Medicine and Metabolic Disease, Second
University of Naples, Naples, Italy. 75 Department of Genetics and Pathology, Rudbeck Laboratory,
Uppsala University, Uppsala, Sweden. 111 National Institute for Health and Welfare, Unit of Public Health
Genomics, Helsinki, Finland. 76 Centre Hospitalier Universitaire, de Poitiers, Endocrinologie
Diabetologie, CIC INSERM 0802, INSERM U927, Universite´ de Poitiers,
Unite´ de Formation et de Recherche, Me´decine Pharmacie, Poitiers,
France. 112 Department of Medical Genetics, University of Helsinki, Helsinki,
Finland. 113 Department of Medical Statistics, Epidemiology and Medical
Informatics, Andrija Stampar School of Public Health, Medical School, 77 Department of Public Health and Clinical Medicine, Section for
Nutritional Research, Umea˚ University, Umea˚, Sweden. University of Zagreb, Rockefellerova, Zagreb, Croatia. 78 Department of Clinical Sciences, Obstetrics and Gynecology,
University of Oulu, University of Oulu, Finland. 114 Department of Clinical Genetics, VU University and Medical
Center, Amsterdam, The Netherlands. PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 March 2012 | Volume 8 | Issue 3 | e1002607 18 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis 35 Institut interre´gional pour la sante´ (IRSA), La Riche, Fran 175 Department of Preventive Medicine, Keck School of Medicine,
University of Southern California, Los Angeles, California, USA. 136 Coordination Centre for Clinical Trials, University of Leipzig,
Leipzig, Germany. 176 MRC–Health Protection Agency Centre for Environment and
Health, Imperial College London, London, UK. 137 Department of Medicine, Helsinki University Hospital, University
of Helsinki, Helsinki, Finland. 177 UOC Geriatria, Istituto Nazionale Ricovero e cura per Anziani
(INRCA) IRCCS, Rome, Italy. 178 These authors contributed equally to this work. Correspondence
should be addressed to M.B. (boehnke@umich.edu), M.I.M. (mark.mccarthy@drl.ox.ac.uk), J.C.F. (jcflorez@partners.org) or I.B. (ib1
@sanger.ac.uk). 138 Department of Internal Medicine, Leiden University Medical
Centre, Leiden, The Netherlands. 139 Research Unit, Cardiovascular Genetics, Nancy University Henri
Poincare´, Nancy, France. 140 EMGO Institute for Health and Care Research, Department of
Psychiatry, VU University Medical Center, Amsterdam, The Netherlands. Adiponectin: Multiethnic Genome-Wide Meta-Analysis 18 Department of Biostatistics, Boston University School of Public
Health, Boston, Massachusetts 02118, USA. 19 National Heart, Lung and Blood Institute’s Framingham Heart
Study, Framingham, Massachusetts 01702, USA. 20 Human Genetics Center, University of Texas Health Science Center
at Houston, Houston, Texas 77030, USA. 21 Department of Public Health and Primary Care, Strangeways
Research Laboratory, University of Cambridge, Cambridge CB1 8RN,
UK. 22 Cardiovascular Health Research Unit and Department of Medicine,
University of Washington, Seattle, Washington 98101, USA. 23 deCODE Genetics, 101 Reykjavik, Iceland. 24 Division of Statistical Genomics in the Center for Genome Sciences,
Washington University School of Medicine, St Louis, Missouri 63108,
USA. 25 Department of Epidemiology and Public Health, Imperial College
London, London W2 1PG, UK. 26 Department of Clinical Sciences, Lund University, SE-20502,
Malmo¨, Sweden. 27 Clinical Pharmacology and Barts and the London Genome Centre,
William Harvey Research Institute, Barts and the London School of
Medicine, Queen Mary University of London, London EC1M 6BQ, UK. Yii-der I. Chen,28 , Mark J. Caulfield,27 , Harry Campbell,43 , Noel P. Burtt,5 , Lori L. Bonnycastle,99 , Dorret I. Boomsma,44 , S. Matthijs
Boekholdt,100 , Richard N. Bergman,101 , Ineˆs Barroso,55 , Stefania
Bandinelli,102 , Christie M. Ballantyne,103 , Themistocles L. Assimes,104 , 28 Medical Genetics Institute, Cedars-Sinai Medical Center, Los
Angeles, California 90048, USA. 29 Center for Genome Science, National Institute of Health, Seoul 122-
701, Republic of Korea. 30 Interdisciplinary Program in Bioinformatics, College of Natural
Sciences, Seoul National University, Seoul 151-742, Republic of Korea. 31 Department of Statistics, College of Natural Sciences, Seoul National
University, Seoul 151-742, Republic of Korea. 32 Department of Statistics, Sookmyung Women’s University, Seoul
140-742, Republic of Korea. 33 Centre for Molecular Epidemiology, National University of
Singapore, Singapore, 117597, Republic of Singapore. 34 Genome Institute of Singapore, Singapore 138672, Republic of
Singapore. 35 Department of Genetics, University of North Carolina, Chapel Hill,
North Carolina 27599, USA. 36 Department of Genome Sciences, University of Washington, Seattle,
Washington 98195, USA. 1 Center for Statistical Genetics, Department of Biostatistics, University
of Michigan, Ann Arbor, Michigan 48109, USA. 37 Genetics Division, GlaxoSmithKline R&D, King of Prussia,
Pennsylvania 19406, USA. 2 Center for Human Genetic Research, Massachusetts General
Hospital, Boston, Massachusetts 02114, USA. 38 MRC Epidemiology Unit, Institute of Metabolic Science, Adden-
brooke’s Hospital, Cambridge CB2 0QQ, UK. 3 Cardiovascular Research Center, Massachusetts General Hospital,
Boston, Massachusetts 02114, USA. Adiponectin: Multiethnic Genome-Wide Meta-Analysis Kees
Hovingh,16 , Jouke-Jan Hottenga,44 , Albert Hofman,17, 42 , Andrew A. Hicks,65 , Christian Hengstenberg,88 , Iris M. Heid,45,
89 , Caroline
Hayward,41 , Aki S. Havulinna,50, 90 , Nicholas D. Hastie,41 , Tamara B. Harris,80 , Talin Haritunians,28 , Alistair S. Hall,91 , Ulf Gyllensten,85 ,
Candace Guiducci,5 , Leif C. Groop,26, 92 , Elena Gonzalez,5 , Christian
Gieger,45 , Nelson B. Freimer,93 , Luigi Ferrucci,94 , Jeanette Erdmann,95 ,
Paul Elliott,86, 96 , Kenechi G. Ejebe,5 , Angela Do¨ring,45 , Anna F. Dominiczak,97 , Serkalem Demissie,18, 19 , Panagiotis Deloukas,55 , Eco J. C. de Geus,44 , Ulf de Faire,98 , Gabriel Crawford,5 , Francis S. Collins,99 ,
Yii-der I. Chen,28 , Mark J. Caulfield,27 , Harry Campbell,43 , Noel P. Burtt,5 , Lori L. Bonnycastle,99 , Dorret I. Boomsma,44 , S. Matthijs
Boekholdt,100 , Richard N. Bergman,101 , Ineˆs Barroso,55 , Stefania
Bandinelli,102 , Christie M. Ballantyne,103 , Themistocles L. Assimes,104 ,
Thomas Quertermous,104 , David Altshuler,2, 4, 5 , Mark Seielstad,34 , Tien
Y. Wong,105 , E-Shyong Tai,106 , Alan B. Feranil,107 , Christopher W. Kuzawa,108 , Linda S. Adair,109 , Herman A. Taylor Jr,110 , Ingrid B. Borecki,24 , Stacey B. Gabriel,5 , James G. Wilson,110 , Hilma Holm,23 ,
Unnur Thorsteinsdottir,8, 23 , Vilmundur Gudnason,7, 8 , Ronald M. Krauss,111 , Karen L. Mohlke,35 , Jose M. Ordovas,112, 113 , Patricia B. Munroe,114 , Jaspal S. Kooner,59 , Alan R. Tall,11 , Robert A. Hegele,15 ,
John J.P. Kastelein,16 , Eric E. Schadt,115 , Jerome I. Rotter,28 , Eric
Boerwinkle,20 , David P. Strachan,116 , Vincent Mooser,37 , Kari
Stefansson,8, 23 , Muredach P. Reilly,9, 10 , Nilesh J Samani,117 , Heribert
Schunkert,95 , L. Adrienne Cupples,18, 19, 118 , Manjinder S. Sandhu,21, 38,
55, 118 , Paul M Ridker,4, 14, 118 , Daniel J. Rader,9, 10, 118 , Cornelia M. van
Duijn,17, 42, 118 , Leena Peltonen,119 , Gonc¸alo R. Abecasis,1, 118 , Michael
Boehnke1, 118 , & Sekar Kathiresan, 15 Robarts Research Institute, University of Western Ontario, London,
Ontario N6A 5K8, Canada. 16 Department of Vascular Medicine, Academic Medical Centre at the
University of Amsterdam, 1105 AZ Amsterdam, The Netherlands. niversity of Amsterdam, 1105 AZ Amsterdam, The Netherlan 17 Department of Epidemiology, Erasmus University Medical Center,
P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. GLGC Consortium:
1 1 149 Genetic Epidemiology and Clinical Research Group, Department of Public Health and Clinical Medicine, Section for Medicine, Umea˚
University Hospital, Umea˚, Sweden. y
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150 London School of Hygiene and Tropical Medicine, London, UK. 151 Department of Medicine, School of Medicine, Johns Hopkins
University, Baltimore, Maryland, USA. PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 19 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis Wong,105 , E-Shyong Tai,106 , Alan B. Feranil,107 , Christopher W. Kuzawa,108 , Linda S. Adair,109 , Herman A. Taylor Jr,110 , Ingrid B. Borecki,24 , Stacey B. Gabriel,5 , James G. Wilson,110 , Hilma Holm,23 ,
Unnur Thorsteinsdottir,8, 23 , Vilmundur Gudnason,7, 8 , Ronald M. Krauss,111 , Karen L. Mohlke,35 , Jose M. Ordovas,112, 113 , Patricia B. Munroe,114 , Jaspal S. Kooner,59 , Alan R. Tall,11 , Robert A. Hegele,15 ,
John J.P. Kastelein,16 , Eric E. Schadt,115 , Jerome I. Rotter,28 , Eric
Boerwinkle,20 , David P. Strachan,116 , Vincent Mooser,37 , Kari
Stefansson,8, 23 , Muredach P. Reilly,9, 10 , Nilesh J Samani,117 , Heribert
Schunkert,95 , L. Adrienne Cupples,18, 19, 118 , Manjinder S. Sandhu,21, 38,
55, 118 , Paul M Ridker,4, 14, 118 , Daniel J. Rader,9, 10, 118 , Cornelia M. van
Duijn,17, 42, 118 , Leena Peltonen,119 , Gonc¸alo R. Abecasis,1, 118 , Michael
Boehnke1, 118 , & Sekar Kathiresan, M. Stringham,1 , Tim D. Spector,54 , Nicole Soranzo,54, 55 , Johannes H. Smit,56 , Juha Sinisalo,57 , Kaisa Silander,12, 13 , Eric J. G. Sijbrands,17, 48 ,
Angelo Scuteri,58 , James Scott,59 , David Schlessinger,60 , Serena Sanna,49
, Veikko Salomaa,50 , Juha Saharinen,12 , Chiara Sabatti,61 , Aimo
Ruokonen,62 , Igor Rudan,43 , Lynda M. Rose,14 , Robert Roberts,63 ,
Mark Rieder,36 , Bruce M. Psaty,64 , Peter P. Pramstaller,65 , Irene
Pichler,65 , Markus Perola,12, 13 , Brenda W. J. H. Penninx,56 , Nancy L. Pedersen,66 , Cristian Pattaro,65 , Alex N. Parker,67 , Guillaume Pare,68 ,
Ben A. Oostra,69 , Christopher J. O’Donnell,4, 19 , Markku S. Nieminen,57
, Deborah A. Nickerson,36 , Grant W. Montgomery,46 , Thomas
Meitinger,70, 71 , Ruth McPherson,63 , Mark I. McCarthy,72, 73, 74 ,
Wendy McArdle,75 , David Masson,11 , Nicholas G. Martin,46 , Fabio
Marroni,76 , Massimo Mangino,54 , Patrik K. E. Magnusson,66 , Gavin
Lucas,77 , Robert Luben,21 , Ruth J. F. Loos,38 , Marja-Liisa Lokki,78 ,
Guillaume Lettre,79 , Claudia Langenberg,38 , Lenore J. Launer,80 ,
Edward G. Lakatta,60 , Reijo Laaksonen,81 , Kirsten O. Kyvik,82 , Florian
Kronenberg,39 , Inke R. Ko¨nig,40 , Kay-Tee Khaw,21 , Jaakko Kaprio,12,
13, 83 , Lee M. Kaplan,84 , A˚ sa Johansson,85 , Marjo-Riitta Jarvelin,86, 87 ,
A. Cecile J. W. Janssens,17 , Erik Ingelsson,66 , Wilmar Igl,85 , G. Adiponectin: Multiethnic Genome-Wide Meta-Analysis M. Stringham,1 , Tim D. Spector,54 , Nicole Soranzo,54, 55 , Johannes H. Smit,56 , Juha Sinisalo,57 , Kaisa Silander,12, 13 , Eric J. G. Sijbrands,17, 48 ,
Angelo Scuteri,58 , James Scott,59 , David Schlessinger,60 , Serena Sanna,49
, Veikko Salomaa,50 , Juha Saharinen,12 , Chiara Sabatti,61 , Aimo
Ruokonen,62 , Igor Rudan,43 , Lynda M. Rose,14 , Robert Roberts,63 ,
Mark Rieder,36 , Bruce M. Psaty,64 , Peter P. Pramstaller,65 , Irene
Pichler,65 , Markus Perola,12, 13 , Brenda W. J. H. Penninx,56 , Nancy L. Pedersen,66 , Cristian Pattaro,65 , Alex N. Parker,67 , Guillaume Pare,68 ,
Ben A. Oostra,69 , Christopher J. O’Donnell,4, 19 , Markku S. Nieminen,57
, Deborah A. Nickerson,36 , Grant W. Montgomery,46 , Thomas
Meitinger,70, 71 , Ruth McPherson,63 , Mark I. McCarthy,72, 73, 74 ,
Wendy McArdle,75 , David Masson,11 , Nicholas G. Martin,46 , Fabio
Marroni,76 , Massimo Mangino,54 , Patrik K. E. Magnusson,66 , Gavin
Lucas,77 , Robert Luben,21 , Ruth J. F. Loos,38 , Marja-Liisa Lokki,78 ,
Guillaume Lettre,79 , Claudia Langenberg,38 , Lenore J. Launer,80 ,
Edward G. Lakatta,60 , Reijo Laaksonen,81 , Kirsten O. Kyvik,82 , Florian
Kronenberg,39 , Inke R. Ko¨nig,40 , Kay-Tee Khaw,21 , Jaakko Kaprio,12,
13, 83 , Lee M. Kaplan,84 , A˚ sa Johansson,85 , Marjo-Riitta Jarvelin,86, 87 ,
A. Cecile J. W. Janssens,17 , Erik Ingelsson,66 , Wilmar Igl,85 , G. Kees
Hovingh,16 , Jouke-Jan Hottenga,44 , Albert Hofman,17, 42 , Andrew A. Hicks,65 , Christian Hengstenberg,88 , Iris M. Heid,45,
89 , Caroline
Hayward,41 , Aki S. Havulinna,50, 90 , Nicholas D. Hastie,41 , Tamara B. Harris,80 , Talin Haritunians,28 , Alistair S. Hall,91 , Ulf Gyllensten,85 ,
Candace Guiducci,5 , Leif C. Groop,26, 92 , Elena Gonzalez,5 , Christian
Gieger,45 , Nelson B. Freimer,93 , Luigi Ferrucci,94 , Jeanette Erdmann,95 ,
Paul Elliott,86, 96 , Kenechi G. Ejebe,5 , Angela Do¨ring,45 , Anna F. Dominiczak,97 , Serkalem Demissie,18, 19 , Panagiotis Deloukas,55 , Eco J. C. de Geus,44 , Ulf de Faire,98 , Gabriel Crawford,5 , Francis S. Collins,99 ,
Yii-der I. Chen,28 , Mark J. Caulfield,27 , Harry Campbell,43 , Noel P. Burtt,5 , Lori L. Bonnycastle,99 , Dorret I. Boomsma,44 , S. Matthijs
Boekholdt,100 , Richard N. Bergman,101 , Ineˆs Barroso,55 , Stefania
Bandinelli,102 , Christie M. Ballantyne,103 , Themistocles L. Assimes,104 ,
Thomas Quertermous,104 , David Altshuler,2, 4, 5 , Mark Seielstad,34 , Tien
Y. Adiponectin: Multiethnic Genome-Wide Meta-Analysis 49 Istituto di Neurogenetica e Neurofarmacologia (INN), Consiglio
Nazionale delle Ricerche, c/o Cittadella Universitaria di Monserrato,
Monserrato, Cagliari 09042, Italy. 83 Faculty of Medicine, Department of Public Health, University of
Helsinki, FI-00014 Helsinki, Finland. 84 Massachusetts General Hospital Weight Center, Boston, Massachu-
setts 02114, USA. g
y
50 Department of Chronic Disease Prevention, National Institute for
Health and Welfare, FI-00271 Helsinki, Finland. 85 Department of Genetics and Pathology, Rudbeck Laboratory,
University of Uppsala, SE-75185 Uppsala, Sweden. 51 Department of Health Sciences, University of Leicester, Leicester
LE1 6TP, UK. 86 Department of Epidemiology & Biostatistics, Imperial College
London, St Mary’s Campus, Norfolk Place, London W2 1PG, UK. 52 Clinical Research Branch, National Institute on Aging, National
Institutes of Health, Baltimore, Maryland 21225, USA. 87 Department of Public Health Science and General Practice,
University of Oulu, FI-90220 Oulu, Finland. y
53 Medstar Research Institute, Baltimore, Maryland 21218, USA. y
88 Klinik und Poliklinik fu¨r Innere Medizin II, Universita¨t Regensburg,
93053 Regensburg, Germany. 54 Department of Twin Research and Genetic Epidemiology, King’s
College London, London SE1 7EH, UK. g
55 Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA,
UK. g
g
y
89 Department of Epidemiology and Preventive Medicine Regensburg
University Medical Center Franz-Josef-Strauss-Allee 11, 93053 Regens-
burg, Germany. 56 Department of Psychiatry, EMGO Institute, Neuroscience Campus
Amsterdam, VU University Medical Center, 1007 MB Amsterdam, The
Netherlands. 90 Department of Biomedical Engineering and Computational Science,
Aalto University School of Science and Technology, FI-00076 Aalto,
Finland. 57 Division of Cardiology, Department of Medicine, Helsinki University
Central Hospital (HUCH), FI-00029 Helsinki, Finland. 91 LIGHT Research Institute, Faculty of Medicine and Health,
University of Leeds, Leeds LS2 9JT, UK. 58 Unita Operativa Geriatria, Istituto Nazionale Ricovero e Cura
Anziani (INRCA), Istituto Ricovero e Cura a Carattere Scientifico
(IRCCS), Via Cassia 1167, 00189 Rome, Italy. 92 Department of Medicine, Helsinki University Hospital, FI-00029
Helsinki, Finland. 59 Hammersmith Hospital, National Heart and Lung Institute, Imperial
College London, London W12 0NN, UK. 93 Department of Psychiatry, Center for Neurobehavioral Genetics, The
Jane and Terry Semel Institute for Neuroscience and Human Behavior,
David Geffen School of Medicine, University of California, Los Angeles,
California 90095, USA. 60 Gerontology Research Center, National Institute on Aging, 5600
Nathan Shock Drive, Baltimore, Maryland 21224, USA. 94 Clinical Research Branch, National Institute on Aging, National
Institutes of Health, Baltimore, Maryland 21225, USA. 61 Department of Health Research and Policy, Stanford University,
Stanford, California 94305, USA. Adiponectin: Multiethnic Genome-Wide Meta-Analysis 62 Department of Clinical Chemistry, University of Oulu, FI-90220
Oulu, Finland. 95 Medizinische Klinik II, Universita¨t zu Lu¨beck, 23538 Lu¨beck,
Germany. 96 MRC-HPA Centre for Environment and Health, Imperial College
London, London W2 1PG, UK. 63 The John & Jennifer Ruddy Canadian Cardiovascular Genetics
Centre, University of Ottawa, Ottawa K1Y 4W7, Canada. 97 BHF Glasgow Cardiovascular Research Centre, University of
Glasgow, 126 University Place, Glasgow G12 8TA, UK. y
64 Departments of Medicine, Epidemiology, and Health Services, University of Washington, Group Health Research Institute, Group Health
Cooperative, Seattle, Washington 98101, USA. g
,
y
,
g
,
98 Division of Cardiovascular Epidemiology, Institute of Environmental g
y
g
98 Division of Cardiovascular Epidemiology, Institute of Environmental
Medicine Karolinska Institutet SE-17177 Stockholm Sweden 98 Division of Cardiovascular Epidemiology, Institute of Environ
Medicine, Karolinska Institutet, SE-17177 Stockholm, Sweden. p
g
65 Institute of Genetic Medicine, European Academy Bozen/Bolzano
(EURAC), Viale Druso 1, 39100 Bolzano, Italy – affiliated institute of the
University of Lu¨beck, Germany. p
gy
Medicine, Karolinska Institutet, SE-17177 Stockholm, Sweden. 99 National Human Genome Research Institute, National Institutes of
Health, Bethesda, Maryland, 20892, USA. University of Lu¨beck, Germany. 66 Department of Medical Epidemiology and Biostatistics, Karolinska
Institutet, SE-17177 Stockholm, Sweden. 66 Department of Medical Epidemiology and Biostatistics, Karolinska
Institutet, SE-17177 Stockholm, Sweden. 100 Departments of Vascular Medicine & Cardiology, Academic
Medical Centre, 1105 AZ Amsterdam, The Netherlands. 101 Department of Physiology and Biophysics, University of Southern
California, Los Angeles, California 90033, USA. 67 Amgen, Thousand Oaks, California 91320, USA. 67 Amgen, Thousand Oaks, California 91320, USA. 68 Genetic and Molecular Epidemiology Laboratory, McMaster
University, Hamilton, Ontario L8N3Z5, Canada. 102 Geriatric Unit, Azienda Sanitaria Firenze (ASF), 50125 Florence,
Italy. 69 Department of Clinical Genetics, Erasmus University Medical
Center, 3000 CA Rotterdam, The Netherlands. 103 Department of Medicine, Baylor College of Medicine, Houston,
Texas 77030, USA. 70 Institut fur Humangenetik, Helmholtz Zentrum Munchen, Deutsches
Forschungszentrum fur Umwelt und Gesundheit, 85764 Neuherberg,
Germany. 104 Department of Medicine, Stanford University School of Medicine,
Stanford, California 94305, USA. 71 Institute of Human Genetics, Klinikum rechts der Isar, Technische
Universita¨t Mu¨nchen, 81675 Muenchen, Germany. 105 Singapore Eye Research Institute, National University of Singapore,
Singapore 168751, Republic of Singapore. g p
,
p
g p
106 Departments of Medicine/Epidemiology and Public Health, Yong 72 Wellcome Trust Centre for Human Genetics, University of Oxford,
Roosevelt Drive, Oxford OX3 7BN, UK. Adiponectin: Multiethnic Genome-Wide Meta-Analysis 39 Division of Genetic Epidemiology, Department of Medical Genetics,
Molecular and Clinical Pharmacology, Innsbruck Medical University,
Schoepfstrasse 41, A-6020 Innsbruck, Austria. 4 Department of Medicine, Harvard Medical School, Boston,
Massachusetts 02115, USA. 40 Institut fu¨r Medizinische Biometrie und Statistik, Universita¨t zu
Lu¨beck, 23562 Lu¨beck, Germany. 5 Broad Institute, Cambridge, Massachusetts 02142, USA. 6 Johns Hopkins University School of Medicine, Baltimore, Maryland
21287, USA. 41 MRC Human Genetics Unit, Institute of Genetics and Molecular
Medicine, Edinburgh EH4 2XU, UK. 7 Icelandic Heart Association, Heart Preventive Clinic and Research
Institute, 201 Kopavogur, Iceland. 42 Netherlands Genomics Initiative (NGI)-sponsored Netherlands
Consortium for Healthy Aging (NCHA) and Center of Medical Systems
Biology (CMSB), 2300 RC Leiden, The Netherlands. 8 University of Iceland, 101 Reykjavik, Iceland. 9 Cardiovascular Institute, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104, USA. 43 Centre for Population Health Sciences, University of Edinburgh,
Edinburgh EH8 9AG, UK. 10 Institute for Translational Medicine and Therapeutics, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania, 19104,
USA. 44 Department of Biological Psychology, VU University Amsterdam
Van der Boechorststraat 1, 1081 BT Amsterdam, The Netherlands. 45 Institute of Epidemiology, Helmholtz Zentrum Munchen – German
Research Center for Environmental Health, 85764 Neuherberg, Germany. 11 Division of Molecular Medicine, Department of Medicine, Columbia
University, New York, New York 10032, USA. 46 Genetic Epidemiology Unit, Queensland Institute of Medical
Research, PO Royal Brisbane Hospital, Queensland 4029, Australia. 12 Institute for Molecular Medicine Finland FIMM, University of
Helsinki, FI-00014 Helsinki, Finland. 47 Department of Internal Medicine, Centre Hospitalier Universitaire
Vaudois, 1011 Lausanne, Switzerland. 13 National Institute for Health and Welfare, P.O. Box 104, FI-00251
Helsinki, Finland. 14 Division of Preventive Medicine, Brigham and Women’s Hospital,
Boston Massachusetts 02215, USA. 48 Department of Internal Medicine, Erasmus University Medical
Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands. PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 PLoS Genetics | www.plosgenetics.org 20 Adiponectin: Multiethnic Genome-Wide Meta-Analysis References 1. Hivert MF, Sullivan LM, Fox CS, Nathan DM, D’Agostino RB, Sr, et al. (2008)
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495–504. 28. Rung J, Cauchi S, Albrechtsen A, Shen L, Rocheleau G, et al. Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis Borja, LS Adair, M Haun. Analyzed the data: Z Dastani, N Timpson, T
Tanaka, KA Kapur, R Semple, X Yuan, P Henneman, A Isaacs, J Dupuis,
J Grimsby, AK Manning, CT Liu, JRB Perry, AR Wood, F Kronenberg,
IM Heid, TW Winkler, C Langenberg, C Fuchsberger, J Brody, L-P
Lyytika¨inen, RA Scott, Y Liu, M Garcia, L Qi, FB Hu, M Beekman, I
Meulenbelt, H-W Uh, JS Pankow, S Kanoni, A Bidulescu, SG Buxbaum,
Y Wu, AP Morris, K Small, TM Teslovich, BA Oostra, AR Wood. Contributed reagents/materials/analysis tools: DM Evans, B St. Pourcain,
N Sattar, CMT Greenwood, TD Spector, M Ladouceur, CV Dedoussis,
JB Meigs, F Kronenberg, IM Heid, TW Winkler, C Langenberg, AA
Hicks, PP Pramstaller, BM Psaty, I Chen, RP Tracy, A Jula, B-M Loo, PE
Slagboom, S Bandinelli, I Meulenbelt, H-W Uh, M Kloppenburg, DJ
Couper, H-E Wichmann, B Paulweber, GV Dedoussis, P Deloukas, S
Redline, T Johnson, BA Oostra, GD Smith. Wrote the paper: Z Dastani,
JB Richards, CMT Greenwood, N Timpson, DM Waterworth, VE
Mooser, CV Dedoussis, A Isaacs, JB Meigs, JC Florez, J Dupuis, JRB
Perry, M-F Hivert, F Kronenberg, IM Heid, T Lehtima¨ki, M Perola, RA
Scott, C Langenberg, RJF Loos, T Harris, DJ Couper, CM Ballantyne, BB
Duncan, H-E Wichmann, KL Mohlke, AP Morris, K Small, R Semple. Undertook meta-analysis: Z Dastani, J Dupuis. Did multi-ethnic analysis:
AP Morris. Did expression analysis: K Small. Wrote the first draft of the
manuscript: Z Dastani. 115 Sage Bionetworks, Seattle, Washington 98109, USA. 116 Division of Community Health Sciences, St George’s University of
London, London SW17 0RE, UK. 117 Department of Cardiovascular Sciences, University of Leicester,
NIHR Biomedical Research Unit in Cardiovascular Disease, Glenfield
Hospital, Leicester LE3 9QP, UK. Author Contributions Conceived and designed the experiments: Z Dastani, JB Richards, DM
Waterworth, VE Mooser, C van Duijn, A Isaacs, JB Meigs, JC Florez, M-F
Hivert, F Kronenberg, IM Heid, PP Pramstaller, JR Kizer, DS Siscovick,
BM Psaty, O Raitakari, T Lehtima¨ki, JG Eriksson, M Perola, V Salomaa,
RJF Loos, NJ Wareham, T Harris, L Qi, FB Hu, I Meulenbelt, M
Kloppenburg, CM Ballantyne, H-E Wichmann, B Paulweber, L
Kendenko, JG Wilson, A Bidulescu, S Musani, SG Buxbaum, S Redline,
MA Allison, KL Mohlke, AP Morris, K Small. Performed the experiments:
L Ferrucci, JM Egan, OD Carlson, P Vollenweider, C Lamina, NJ
Wareham, J Chambers, J Kooner, R Frants, K Willems-vanDijk, SM
Willems, TM Frayling, S Bo¨hringer, F Kronenberg, AA Hicks, BM Psaty, I
Chen, RP Tracy, M Ka¨ho¨nen, J Viikari, K Kristiansson, M-L Nuotio, K
Lohman, A Kanaya, PE Slagboom, M Beekman, D van Heemst, I
Meulenbelt, M Kloppenburg, CM Ballantyne, N Klopp, S Coassin, E
Katsareli, A Bidulescu, MA Allison, LJ Rasmussen-Torvik, X Guo, JB Meulenbelt, M Kloppenburg, CM Ballantyne, N Klopp, S Coassin, E
Katsareli, A Bidulescu, MA Allison, LJ Rasmussen-Torvik, X Guo, JB Katsareli, A Bidulescu, MA Allison, LJ Rasmussen-Torvik, X Guo, JB Katsareli, A Bidulescu, MA Allison, LJ Rasmussen-Torvik, X Guo, JB Adiponectin: Multiethnic Genome-Wide Meta-Analysis Loo Lin School of Medicine, National University of Singapore, Singapore
117597, Republic of Singapore. 73 Oxford Centre for Diabetes, Endocrinology and Medicine,
University of Oxford, Churchill Hospital, Oxford OX3 7LJ, UK. 107 Office of Population Studies Foundation, University of San Carlos,
Cebu City 6000, Philippines. 74 Oxford NIHR Biomedical Research Centre, Churchill Hospital,
Oxford OX3 7LJ, UK. 108 Department of Anthropology, Northwestern University, Evanston,
Illinois 60208, USA. 75 Avon Longitudinal Study of Parents and Children, University of
Bristol, Bristol BS8 2BN, UK. 109 Department of Nutrition, Carolina Population Center, University of 109 Department of Nutrition, Carolina Population Center, University of
North Carolina, Chapel Hill, North Carolina 27516, USA. 109 Department of Nutrition, Carolina Population Center, University of
North Carolina, Chapel Hill, North Carolina 27516, USA. 76 Institute of Applied Genomics, via Linussio 51, 33100 Udine, Italy. 110 Department of Medicine, University of Mississippi Medical Center,
Jackson, Mississippi 39126, USA. 77 Cardiovascular Epidemiology and Genetics, Institut Municipal
d’Investigacio Medica, 08003 Barcelona, Spain. 111 Children’s Hospital Oakland Research Institute, Oakland, Califor-
nia 94609, USA. 78 Transplantation Laboratory, Haartman Institute, University of
Helsinki, FI-00014 Helsinki, Finland. 79 Montreal Heart Institute (Research Center), Universite´ de Montre´al,
Montre´al, Que´bec H1T 1C8, Canada. 112 Department of Cardiovascular Epidemiology and Population
Genetics, Centro Nacional de Investigaciones Cardiovasculares, 28029
Madrid, Spain. 80 Laboratory of Epidemiology, Demography, and Biometry, National
Institute on Aging, National Institutes of Health, Bethesda, Maryland
20892, USA. 113 Nutrition and Genomics Laboratory, Jean Mayer United States
Department of Agriculture Human Nutrition Research Center on Aging at
Tufts University, Boston, Massachusetts 02111, USA. 81 Science Center, Tampere University Hospital, FI-33521 Tampere,
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William Harvey Research Institute, Barts and The London School of
Medicine and Dentistry, Queen Mary University of London, Charterhouse
Square, London EC1M 6BQ, UK. 82 Institute of Regional Health Research and the Danish Twin Registry,
Institute of Public Health, University of Southern Denmark, JBWinsløws
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Confirmation of multiple risk Loci and genetic impacts by a genome-wide
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bacteria-binding and angiogenesis-modulating activities. J Biol Chem 277:
34264–34270. Framingham Offspring Study. Diabetes 57: 3353–3359. 14. Ling H, Waterworth DM, Stirnadel HA, Pollin TI, Barter PJ, et al. (2009)
Genome-wide Linkage and Association Analyses to Identify Genes Influencing
Adiponectin Levels: The GEMS Study. Obesity (Silver Spring). 31. Gray SL, Vidal-Puig AJ (2007) Adipose tissue expandability in the maintenance
of metabolic homeostasis. Nutr Rev 65: S7–12. 15. Heid IM, Henneman P, Hicks A, Coassin S, Winkler T, et al. (2010) Clear
detection of ADIPOQ locus as the major gene for plasma adiponectin: results of
genome-wide association analyses including 4659 European individuals. Atherosclerosis 208: 412–420. 32. Eremina V, Baelde HJ, Quaggin SE (2007) Role of the VEGF–a signaling
pathway in the glomerulus: evidence for crosstalk between components of the
glomerular filtration barrier. Nephron Physiol 106: p32–37. 16. Richards JB, Waterworth D, O’Rahilly S, Hivert MF, Loos RJ, et al. (2009) A
genome-wide association study reveals variants in ARL15 that influence
adiponectin levels. PLoS Genet 5: e1000768. doi:10.1371/journal.pgen.1000768. 33. Buraczynska M, Ksiazek P, Baranowicz-Gaszczyk I, Jozwiak L (2007)
Association of the VEGF gene polymorphism with diabetic retinopathy in type
2 diabetes patients. Nephrol Dial Transplant 22: 827–832. PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 March 2012 | Volume 8 | Issue 3 | e1002607 22 Adiponectin: Multiethnic Genome-Wide Meta-Analysis Adiponectin: Multiethnic Genome-Wide Meta-Analysis (2008)
Concept, design and implementation of a cardiovascular gene-centric 50 k SNP
array for large-scale genomic association studies. PLoS ONE 3: e3583. doi:10.1371/journal.pone.0003583. 41. Park MH, Kim N, Lee JY, Park HY (2011) Genetic loci associated with lipid
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inconsistency in meta-analyses. Bmj 327: 557–560. 43. Willer CJ, Sanna S, Jackson AU, Scuteri A, Bonnycastle LL, et al. (2008) Newly
identified loci that influence lipid concentrations and risk of coronary artery
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Fibrinogen beta variants confer protection against coronary artery disease in a
Greek case-control study. BMC Med Genet 11: 28. 44. Ivanov D, Philippova M, Antropova J, Gubaeva F, Iljinskaya O, et al. (2001)
Expression of cell adhesion molecule T-cadherin in the human vasculature. Histochemistry and cell biology 115: 231–242. 56. Storey JD, Tibshirani R (2003) Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 100: 9440–9445. PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 PLoS Genetics | www.plosgenetics.org 23
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Metal Nanoparticles and Nano-Filters for the Disposal of Hospital Waste: A Review
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Nano biomedicine and engineering
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Metal Nanoparticles and Nano-Filters for the
Disposal of Hospital Waste: A Review Ali Kaswer Musa Al-Toriahi1, Ebaa Adnan Azooz2 , Emad Abbas Jaffar Al-Mulla3
1 The Gifted Students’ School in Najaf, The General Directorate of Education Al-Najaf, Ministry of Education, Najaf, Iraq
2 Medical Laboratory Technology Department, College of Medical Technology, The Islamic University, Najaf, Iraq
3 Pathological Analysis Techniques, Faculty of Health and Medical Techniques, Al-Furat Al-Awsat Technical University, Najaf, Iraq Corresponding author. E-mail: ebaaadnan665@gmail.com p
p
Citation: A.K.M. Al-Toriahi, E.A. Azooz, E.A.J. Al-Mulla. Metal nanoparticles and nano-filters for the disposal of hospital waste: A review. Nano
Biomedicine and Engineering, 2023, 15(2): 179−190. Citation: A.K.M. Al-Toriahi, E.A. Azooz, E.A.J. Al-Mulla. Metal nanoparticles and nano-filters for the disposal of hospital waste
Biomedicine and Engineering, 2023, 15(2): 179−190. Abstract The waste of multiple residually harmful hospital pharmaceutical materials such as antibiotics,
chemicals, hormonal waste, and residual particles from different types of medicine used in
anticonvulsants and antipyretics is one of the most serious health issues confronting health workers
today. During the past decades, many efforts were made to remove the effects of this environmental
pollution. Efficient, stable, fast, and environmentally friendly methods use nanoparticles such as
nanomaterials, carbon nanotubes, and nanofilters to reduce the toxicity and side effects of the waste
even if they are present at a low concentration. This review describes the use of the nanomaterials, the
effects of their concentration, the investigation of their application, and the effectiveness on hospital
waste removal. Keywords: nanoparticles; hospital wastewater; pharmaceuticals; carbon nanotube; nano-filters 179 179 Nano Biomed. Eng., 2023, 15(2) Nano Biomed Eng Received: Nov. 16, 2022; Revised: Jan. 6, 2023; Accepted: Apr. 25, 2023
Citation: A.K.M. Al-Toriahi, E.A. Azooz, E.A.J. Al-Mulla. Metal nanoparticles and nano-filters for the disposal of hospital waste: A review. Nano
Biomedicine and Engineering, 2023, 15(2): 179−190.
http://doi.org/10.26599/NBE.2023.9290017 https://www.sciopen.com/journal/2150-5578 Introduction on the size of the hospital and the season. The smaller
hospitals produced higher average amounts than the
larger hospitals, and in the winter months, levels were
more elevated than in the summer [5]. The aim and
prospects of the project for using nanostructures in
medical separation of hospital wastewater treatment
have increased, but the difficulty has increased for the
following reasons: In addition to multiple chemically harmful substances
and microbiological contaminants that can reach
drinking water and the ground, the presence of
pharmaceuticals and drug components is considered
one of the biggest environmental challenges [1, 2]. The most important point for assurance of safe water
is to understand the nature, composition, source of the
contamination, and the way these substances reach
the water [3]. Hospital waste and its pharmaceutical
input are considered as the main sources of harmful
effects on the environment [4]. (1) A hospital’s main waste, including hazardous
materials, is discharged to a general wastewater
treatment facility [5, 6]. (1) A hospital’s main waste, including hazardous
materials, is discharged to a general wastewater
treatment facility [5, 6]. (2) The majority of hospital waste contamination is
resistant to standard treatment [7, 8]. In
their
work,
researchers
state
that
the
characteristics of hospital waste and its effects depend Hospitals are considered the main source of many https://www.sciopen.com/journal/2150-5578 https://www.sciopen.com/journal/2150-5578 Nano Biomed. Eng., 2023, 15(2) 180 microbiological pathogens and contrast media used in
iodinated X-rays [9]. Hospital waste contains a
complex
mixture
of
microorganisms
and
pharmaceutical materials that are typically discharged
without treatment. The increased use of antibiotics
and antimicrobics around the world is exacerbating
the problem [10]. In the case of pharmaceuticals,
significant quantities of various substances are
utilized all over the world. Figure 1 shows the most
important
routes
suggested
for
pharmaceutical
contamination of the aquatic environment. 2×106
total production of around
t [14]. https://www.sciopen.com/journal/2150-5578 2×106
total production of around
t [14]. A diverse mixture of active medicinal compounds
and bacteria can be found in hospital effluent. This
waste is typically sent to municipal effluent treatment
plants (WWTPs) without any prior handling [15]. Table 1 lists some of the most popular pharmaceutical
compounds found in hospital wastewater. Figure 2
depicts the amount (t/year) of the sulphamethoxazole
antibiotic’s sales over the course of a year and across
several countries. Most of the waste developed today evolves during
the passing of them through different substances and
filtering activities to reduce and remove pathogens,
nitrogen, carbon, organic material, and other
substances
in
order
to
reduce
its
harmful
effects [16]. But even after full treatment, their micro-
pollutants can still pass and produce harmful effects
on the environment and human health [16]. It is
important and necessary to introduce other more
effective and advanced technologies to improve 2.3×104
In recent decades, these substance sales have been
gradually growing. In France, for instance, yearly
sulphamethoxazole
(an
antibiotic)
using
was
22.4 t/year, 47 t/year in Germany, and 12.7 t/year in
Spain. Each year, roughly
t of antibiotics are
utilized in USA [11–13]. The medical industry in
China is the world’s largest. It produces more than a
thousand different kinds of pharmaceuticals. The
majority of the components are made in China, with a Wastewater treatment
Aquaculture treatment
Animals treatment
Incorrct removal of unutilized medicines
Spreading manure and slurry
Manure and slurry storage
Livestock treatment
Drugs
Human treatment
Manufacturing
methods
Water
Fig 1 Pharmaceutical contamination of the aquatic environment Table 1 Examples of common and frequent pharmaceutical materials detected in hospital wastewater
Pharmaceutical hospital wastewater
Examples
Antibiotics
Fluoroquinolones, sulfonamides, and penicillin group
Analgesic and antipyretics
Diclofenac, ibuprofen, and naproxen
CNS drugs effect
Caffeine
Cardiovascular (beta blockers and others )
Propranolol, atenolol, and metoprolol
Endocrinological drug
Estoile, estrone, and ethinylestradiol
Anticancer
Tamoxifen and methotrexate cyclophosphamide
Wastewater treatment
Aquaculture treatment
Animals treatment
Incorrct removal of unutilized medicines
Spreading manure and slurry
Manure and slurry storage
Livestock treatment
Drugs
Human treatment
Manufacturing
methods
Water
Fig. 1 Pharmaceutical contamination of the aquatic environment. wastewater treatment [17]. Generally speaking, wastewater treatment procedures
have emphasized the removal of pathogens, organic
carbon, nitrogen, and other substances that may
otherwise allow hazardous bacteria to proliferate and
infiltrate waterways [18]. Previous studies have found
that minor levels of chemicals, such as pharmaceuticals
and hormonal disruptors, are present in wastewater and
persist even after cleaning [19]. The issue is that these
micro-pollutant substances are harmful to humans,
aquatic species, and their environments. Because
current water and wastewater treatment methods are
unable to provide a dependable barrier against some
resistant medicines, innovative treatment technologies
must be introduced to detect and predict persistent
pollutants
[20]. Chemical
oxidation
employing
coagulation, ozone, and ozone/hydrogen peroxide [21,
22], biological treatment such as the activated sludge
reactor (SBR) method, membrane filtration such as
reverse osmosis [23, 24], and adsorption by activated
carbon have all been examined in the latest years for
this aim. The aim of this review is to highlight the
applications of metal nanoparticles (NPs), nanofilters,
and the application of carbon nanotubes (CNTs) in
removing hospital pharmaceutical particles from
waste. This study also focuses on the use of
nanoparticles in photocatalysis as a cost-effective
approach. The nanoparticles have a huge potential in
mineralizing pharmaceutical substances in aqueous
solutions at a low cost with simple handling and great
efficacy. Moreover, because nanoparticles are easily
recycled, they can absorb pharmaceutical by-products
and waste at a low cost. With more research being
done in this area, the value of pharmaceutical wastes
and by-products will continue to rise as we move
away from traditional methodologies and toward
nanoscience. The
use
of
nanoparticles
in
pharmaceutical wastewater treatment is reviewed,
with a focus on recovery, energy generation,
limitation, and its function in the circular economy,
which promotes long-term development. The ozone and ozone/hydrogen peroxide methods
are both energy and material costly [25] and are only
suited for treating generally clean surface water and
groundwater with minimal background pollution such
as organic matter (OM). Wastes generated during the
physical treatment operations, such as membrane
retentate and spent activated carbon, must also be
disposed of. Furthermore, activated carbon adsorption
has a narrow ability to remove polar organic
molecules due to its removal mechanism (i.e.,
hydrophobic interactions), especially in the presence
of competitive OM [26], despite the fact that many
pharmaceuticals and metabolic products are polar
materials. 2×106
total production of around
t [14]. https://www.sciopen.com/journal/2150-5578 Manufacturing
methods Drugs Livestock treatment Livestock treatment Table 1 Examples of common and frequent pharmaceutical materials detected in hospital wastewater
Pharmaceutical hospital wastewater
Examples
Antibiotics
Fluoroquinolones, sulfonamides, and penicillin group
Analgesic and antipyretics
Diclofenac, ibuprofen, and naproxen
CNS drugs effect
Caffeine
Cardiovascular (beta blockers and others )
Propranolol, atenolol, and metoprolol
Endocrinological drug
Estoile, estrone, and ethinylestradiol
Anticancer
Tamoxifen and methotrexate cyclophosphamide Table 1 Examples of common and frequent pharmaceutical materials detected in hospital wastewater
Pharmaceutical hospital wastewater
Examples
Antibiotics
Fluoroquinolones, sulfonamides, and penicillin group
Analgesic and antipyretics
Diclofenac, ibuprofen, and naproxen
CNS drugs effect
Caffeine
Cardiovascular (beta blockers and others )
Propranolol, atenolol, and metoprolol
Endocrinological drug
Estoile, estrone, and ethinylestradiol
Anticancer
Tamoxifen and methotrexate cyclophosphamide mples of common and frequent pharmaceutical materials detected in hospital wastewater https://www.sciopen.com/journal/2150-5578 181 Nano Biomed. Eng., 2023, 15(2) 0
10
20
30
40
50
60
70
80
90
100
Amount of sulphamethoxazole
antibiotic sold (t/year )
Countries
France
Germany
Spain
USA
Japan
China
Brazil
Fig. 2 Annual tonnage of sulphamethoxazole sold in seven
countries. that have a size scale of less than 100 nm. This
process takes advantages of its characteristics, which
include unusual and scarce phenomena that depend
on scales (length of particles) and high activity [24,
25]. Pharmaceutical material removal by using
nanomaterials has become an interesting area of
research. Its high adsorptive efficiency is due to the
development of specific functional groups [26, 27]. Even though the knowledge about the use of
nanomaterials to improve environmental fat and
toxicity is still limited, it is still considered the most
effective method for water purification, and research
has found it to have multiple advantages in
comparison with others [28, 29]. According to the
above, the development of nanotechnology has
become an interesting area for research and it adds
more effective methods for the treatment of hospital
waste and the removal of pharmaceuticals, organic
materials, and microbiological contamination from
it [30–35]. Fig. 2 Annual tonnage of sulphamethoxazole sold in seven
countries. Metal nanoparticles Attractive nanoparticles have a large surface area to
enhance their activity and can be functionalized with
various chemical groups in order to produce high- Nanotechnology is a process dealing with particles Nanotechnology is a process dealing with particles https://www.sciopen.com/journal/2150-5578 182 Nano Biomed. Eng., 2023, 15(2) capacity substances and selective sorbent materials
with
unique
properties
for
pharmaceutical
removal [30]. They were studied by using electron
microscopy both for transition (TEM) and scanning
(SEM), or atomic force microscopy (AFM) [31]. They act as a photocatalytic/sorbent phenomenon in
the adsorption and degradation of pharmaceutical
materials through advanced oxidation processes
(AOPs) [32]. Process of photo-degradation is an
effective metal-nanoparticle method that increases the
rate and speed of the degradation process [33]. Recent research proposes the use of two or more
appropriate elements on nanocrystalline TiO2 and UV
length to improve photocatalytic effect [41–43]. Ag/TiO2 nanoparticles were prepared by the chemical
reduction method. Photocatalytic degradation was
studied under UV irradiation and found to be
effective [44]. One of the disadvantages of the above
method is that the production particles dispersed in
the waste and lost with difficulty recollected [45]. One method to improve and reduce this disadvantage
is photo active of TiO2 particles coated by super para
magnetic material (magnetic core) with no remnant
magnetism effect for early separation of particles [45]. In general, this method is considered low cost, strong
adoptive capacity, and early separation, and enhances
stability for hospital pharmaceutical wastewater
separation [46]. Due to their energy gap being close
to that of TiO2NPs, outstanding bulk electron
mobility, a higher number of active locations with
increased reactivity, and the production of the most
active and non-selective hydroxyl radicals (·OH),
zinc oxide nanoparticles (ZnONPs) have been
employed as photocatalyst. However, because of the
large bandgap energy (3.2 eV) of the solar light (high
intensity UV radiation; 380 nm), only a small
percentage (5%) of it can be absorbed by the
ZnONPs. The photocatalytic effectiveness of the
ZnONPs
is
severely
hampered
by
the
high
recombination of photo induced charge carriers
during photocatalytic processes. Due to the significant
improvement in their optical and catalytic capabilities,
noble (Ag, Au, and Pt) metal nanoparticle-modified
ZnO semiconductor photocatalysts in particular have
drawn enormous interest [47, 48]. More studies are
explained in Table 2. AOPs are one of the most common methods for
destroying residual medicines. This method can
produce a nearly complete degradation. https://www.sciopen.com/journal/2150-5578 Metal nanoparticles There are
numerous studies on the effectiveness of AOPs for
eliminating residual medicines [34–37]. In the photo
degradation process, photo catalyst semiconductors
such as TiO2, ZnS, ZnO, CdS, Fe2O3, and others are
effective metallic nanoparticles for raising the
degradation rate [38–40]. The benefits of an
appropriate
photocatalyst
include
a
high
photocatalytic activity, resistance to photo corrosion,
cheapness, non-toxicity, and a good band gap
energy [38]. TiO2 has been a popular AOP for heterogeneous
semiconductor photocatalysis in recent years. When
the semiconductor is subjected to ultraviolet (UV)
irradiation, hydroxyl radicals (·OH) and/or valence
band (Vb) holes (h+) are produced, resulting in the
full oxidation of the different pollutants [34]. The
catalytic concentration is an important element in
determining the high efficiency of photocatalysis,
which commonly utilizes nanoparticle suspensions. The following cofactors can enhance functions [38]: (1) Light wave length and intensity. (1) Light wave length and intensity. Carbon nano-tubes A great advance and potential effect have been added
to medical, environmental, and drug applications. Since the discovery of CNTs in 1991, CNTs have
graphite sheets with a cylindrical shape and a very
high (van de Waals) index [49]. CNTs’ characteristics
and
properties
make
them
super-hydrophobic
materials that strongly deal with and interact with
pollutants, especially aromatic ones. Their action is
through
p–p
coupling
and
stacking
process
interactions. This aromatic pollutant substance
includes nitro aromatics, hydroxyl substitutes, and
amino
aromatic
compounds
[50]. Several
conventional and advanced techniques, such as (2) The pH of the solution determines the ionic
position of the catalyst surface, which has an impact
on the amount of organic adsorption and degradation. (3) Addition of H2O2 as an oxidant increases and
promotes reaction. Various metal atoms can improve separation and
trapping of photo electrons and facilitate the photo
oxidation process, e.g., silver, platinum, palladium,
rhodium, gold, and others. Sliver is considered the
most suitable material for usage in different industrial
productions because of its easy abundance and lower
cost in comparison with others [39, 40]. Nano Biomed. Eng., 2023, 15(2) 183 electrochemical treatments, filtration, precipitation,
membrane separation, photocatalysis, ion-exchange,
reverse osmosis, and adsorption, have been used for
the treatment of antibiotics in wastewater [51]. However, they still face challenges of cost-
effectiveness, environmental friendliness, and process
efficiency, from material preparation to process
optimization. Graphene and its derivatives have
impacted wastewater treatment and have been utilized
in photocatalysis, adsorption, or as an effective
electrode in various treatment technologies and
applications. Adsorption has numerous advantages,
such as its high efficacy, low cost, and ecological
viability to remove organic contaminants from
water [52–55]. Graphene is a planar, single-atom-
thick
sheet,
and
two-dimensionally
structured
material composed of tightly packed sp2-bonded
carbon atoms in a honeycomb crystal lattice with a
distinct
charge
mobility
carrier,
a
broad
electrochemical
spectrum,
and
physicochemical
properties. As a result of its outstanding optical,
thermal, electrical, and mechanical properties as well
as its high specific surface area, graphene has
emerged as a revolutionary material with a wide
range of applications, including its use as innovative
adsorbents for water treatment [56–60]. It is an
excellent adsorbent for removing a wide range of
inorganic and organic pollutants because of its high
surface area, abundance of active sites, and excellent
delocalized electron systems. Despite the significant
progress made in the development and application of
grapheme-based
adsorbents,
some
inherent
disadvantages remain [61–65]. Carbon nano-tubes nanostructure sponge, also called nana sponge, has
more effective materials like iron and sulfur, making
it exceedingly effective in intake and sucking
contaminated
with
pharmaceutical
substances,
pesticides, and oil-fertilizers [54–56]. Recently, nanotubes have proven to be effective
against both biological and chemical contaminants
and have the ability to remove contaminants with
heavy metals such as Zn(II), Pb(II), and Cr(III) [57–60]. Also, they are effective for removing aromatic
hydrocarbons from the organic continent and a wide
range of biological materials like viruses, bacteria,
and
their
toxins
related
to
unique
surface
functionalization
and
their
physical
cytotoxic
properties
[58,
61–65]. The
effectiveness
of
nanotubes for the removal of wastewater treatment
can also be improved by the addition of UV light with
improved nanocomposites. Improving photocatalytic
activity is to deal with pollutants. But the cost-
effective is one important disadvantage of this
procedure, but still, there’s a belief that if production
is scaled up, its cost will decrease markedly [66–71]. On the other hand, modified multiwall carbon
nanotubes (MWCNTs) were used to remove dyes in 5
min from wastewater and support a high-flux cyclodextrin/
trimesoyl chloride (CD/TMC) polyester membrane. The results appear as adsorption capacity (AC)
for Brilliant Green (97.41%) and Congo Red
(96.39%) [72–75]. Table 2 explains many medicines
removed by CNTs. https://www.sciopen.com/journal/2150-5578 Nano-filters 2014
[78]
pH 2, 8 h, 45 °C, AC 45.2%
2014
[79]
pH 5.7, 12 h, 23.5 °C, AC 441.3 mg/g
2011
[80]
pH 3, 90 min, AC 93.8%, ultrasonic waves
2020
[81]
pH 7, AC 71.4 mg/g, magnetic carbon for adsorption
of antibiotic prepared from PET
2018
[82]
pH 2, 70 min, 25 °C, AC 1667, 1428 mg/g
2018
[83]
pH 4, 25 °C, AC 97.8%, ultrasonic waves
2020
[86]
pH 5, 105 min, AC 92.8%
2020
[87]
pH 3, 5 h, AC 100%
2019
[88]
pH 7, 30 min, AC 93.8%, immobilized on the surface
of stones
2019
[89]
90 min, AC 100%, graphitized mesoporous carbon-
Tatiana nanocomposite
2018
[90]
pH 6.5, 20 min, 25 °C , AC 87.7 mg/g
2018
[91]
50 °C, 100 min, AC 90. 3%
2018
[92]
pH 9, 20 min, 25 °C, AC 97%
2018
[93]
pH 3.14, 90 min, 25 °C, AC 97.1%. 2018
[94]
pH 9, 20 min, 25 °C, AC 85%
2018
[95]
pH 6, 90 min, AC 90%
2018
[96]
pH 5, 120 min, 25 °C, AC 92%
2020
[97]
90 min, AC 99%
2020
[98]
(mole ratio of Fe/Zn 1:1) loading on hydroxyl ethyl
cellulose-graphene oxide, pH 3, AC 95%
2023
[99]
AC 80%, H2O2 generated under visible light
irradiation
2022
[100]
120 min, AC 99.6%
2020
[101]
pH 7.9, water recovery 75%, coupled with AOPs
2014
[102]
pH 3.1, maximum AC 310 mg/g, MWCNTs as
adsorbent
2022
[103]
Nanoparticles were prepared by solvothermal method. 89% f
fti f
di
(CFS)
i
li
d i
2023
[104] Table 2 Overview of degradation processes used in nanomaterials-assisted pharmaceutical treatment
Pharmaceutical
particles
Nanomaterials
Conditions and results
Year
References
Amoxicillin
TiO2/ceramic membrane reactor
pH 6.5, 0 min, AC 83.3%
2019
[68]
Nano-Fe3O4
pH 3, 0 min, AC 98.2%
2018
[69]
TiO2/UV-C nano
pH 11, 120 min, AC 99.1%, photocatalytic method
doped with Fe(III)
2018
[70]
TiO2/polyimide sheets
12 h, 37 °C, AC 100%, photocatalytic method
2017
[71]
C3N4@MnFe2O4 graphene nanocomposites
30 min, AC 70%, photocatalytic method
2017
[72]
Bimetallic nanoparticles CuFe2O4
pH 4, 30 min, 20 °C, AC 99.2%
2017
[73]
NZVI
pH 3, 95 min, AC 100%
2017
[74]
ZnO NPs
pH 10, 6 h, AC 62.8%
2016
[75]
Fe/Ni NPs
pH 2, 76 min, AC 85%, bimetallic stabilized with
chitosan
2016
[77]
Fe/Ni NPs
pH 4, 60 min, 25 °C, AC 84%, clay supported
bimetallic. Nano-filters Nano filters have been widely used, increasing
rapidly in the last decade (Fig. 3). It is considered the
most recent pressure drive in the membrane
separation process to have been developed, and
its application in different parts is increasing
nowadays [76]. Nano-filters are used in a variety of
industrial applications to remove a wide range of
waste materials, such as pharmaceutical substances
that remove hardness from water and also remove
arsenic heavy metal ions from the water [77–79]. The
characteristics of the nano-filter lie in between
reverse osmosis (RO) and ultrafiltration (UF). Most
recent studies found that the application of nano-
filters to remove pharmaceutical materials and toxic
substances from hospital waste reached 90%
effectiveness [65, 80]. Despite having the same basic form and graphite
sheets, CNTs differ in layer number, helicity
thickness, and length [66–70]. Also, their electrical
properties differ depending on their variation, which
acts either as a metal or a semiconductor [52]. CNTs
with diameters ranging from 1 to 50 mm and lengths
ranging from several microns to centimeters will
become much longer as technology develops and
advances. Due to their structures, CNTs can be
classified into single-wall nano-tube (SWNT), double-
wall nanotube (DWNT), and multi-wall nano-tubes
(MWNT) [48]. According to high chemical and mechanical
properties and their large specific areas, CNTs are
considered one of the effected methods for
wastewater treatment. Because of their magnetic
properties, nanotubes are easier to clean up. CNTs Other studies found that application of inorganic https://www.sciopen.com/journal/2150-5578 Nano Biomed. Eng., 2023, 15(2) 184 es used in nanomaterials-assisted pharmaceutical treatment
Conditions and results
Year
References
pH 6.5, 0 min, AC 83.3%
2019
[68]
pH 3, 0 min, AC 98.2%
2018
[69]
pH 11, 120 min, AC 99.1%, photocatalytic method
doped with Fe(III)
2018
[70]
12 h, 37 °C, AC 100%, photocatalytic method
2017
[71]
30 min, AC 70%, photocatalytic method
2017
[72]
pH 4, 30 min, 20 °C, AC 99.2%
2017
[73]
pH 3, 95 min, AC 100%
2017
[74]
pH 10, 6 h, AC 62.8%
2016
[75]
pH 2, 76 min, AC 85%, bimetallic stabilized with
chitosan
2016
[77]
pH 4, 60 min, 25 °C, AC 84%, clay supported
bimetallic. Nano-filters 2014
[78]
Carbon NPs
pH 2, 8 h, 45 °C, AC 45.2%
2014
[79]
Carbon/TiO2 NPs
pH 5.7, 12 h, 23.5 °C, AC 441.3 mg/g
2011
[80]
Cephalexin
H2O2/NiO NPs
pH 3, 90 min, AC 93.8%, ultrasonic waves
2020
[81]
Ferromagnetic Fe-O NPs
pH 7, AC 71.4 mg/g, magnetic carbon for adsorption
of antibiotic prepared from PET
2018
[82]
Nano-ZVI from nettle and thyme extracts
pH 2, 70 min, 25 °C, AC 1667, 1428 mg/g
2018
[83]
Ciprofloxacin
α-Al2O3 NPs/polyanion
pH 4, 25 °C, AC 97.8%, ultrasonic waves
2020
[86]
TiO2 NPs
pH 5, 105 min, AC 92.8%
2020
[87]
Mesoporous MnxCo3−x O4 NPs
pH 3, 5 h, AC 100%
2019
[88]
ZnO NPs
pH 7, 30 min, AC 93.8%, immobilized on the surface
of stones
2019
[89]
GMC-TiO2
90 min, AC 100%, graphitized mesoporous carbon-
Tatiana nanocomposite
2018
[90]
CNT/V2O5-ZnO NPs
pH 6.5, 20 min, 25 °C , AC 87.7 mg/g
2018
[91]
Ceftriaxone
RuO2-TiO2/G-nano
50 °C, 100 min, AC 90. 3%
2018
[92]
NZVC
pH 9, 20 min, 25 °C, AC 97%
2018
[93]
Fe3O4 NPs/ PAC
pH 3.14, 90 min, 25 °C, AC 97.1%. 2018
[94]
Cefadroxil
NZVC
pH 9, 20 min, 25 °C, AC 85%
2018
[95]
Cefixime
TiO2 NPs
pH 6, 90 min, AC 90%
2018
[96]
Rifampicin
Iron NPs
pH 5, 120 min, 25 °C, AC 92%
2020
[97]
Tetracycline
NiFe NPs
90 min, AC 99%
2020
[98]
bimetallic Fe/Zn NPs
(mole ratio of Fe/Zn 1:1) loading on hydroxyl ethyl
cellulose-graphene oxide, pH 3, AC 95%
2023
[99]
CdS/(rGO)/ZnFe2O4
AC 80%, H2O2 generated under visible light
irradiation
2022
[100]
Anticancer
ZnO NPs/GONS
120 min, AC 99.6%
2020
[101]
Four antibiotics
NF/O3
pH 7.9, water recovery 75%, coupled with AOPs
2014
[102]
17b-estradiol
MWCNTs
pH 3.1, maximum AC 310 mg/g, MWCNTs as
adsorbent
2022
[103]
Ceftiofur sodium
Ag-ZnO nanorods
Nanoparticles were prepared by solvothermal method. https://www.sciopen.com/journal/2150-5578 Nano-filters NF is a temperature-sensitive process
that depends on organic substances naturally present
in the water matrix, the concentration of dissolved
salts, and feed pump pressure (this parameter also has
a significant impact on the hydraulic flux balance
between steps and the solubility of the dissolved salts
in the effluent). When these chemicals are present in
large concentrations, the porous structure may
deteriorate or become foul [109, 110]. Dyes can also
be removed by nanofiltration using 2-aminophenol-4-
sulfonic acid or piperazine. The time required was
120 h, and the result indicates that the AC was
93.6%–99.7% [102]. Table 2 contains many examples
of nanofilters using to separate wastes. pharmaceutical molecules from wastewater sources
has the following two major challenges: an adverse effect on feed bump pressure and the
presence of highly concentrated organic material
such
as
salts
caused
membrane
structure
deterioration [100–105]. These methods create a fresh
solid residue (membrane), similar to adsorption,
where the pollutant is focused. These methods have
mainly been combined with other approaches up until
now [106–108]. NF is a temperature-sensitive process
that depends on organic substances naturally present
in the water matrix, the concentration of dissolved
salts, and feed pump pressure (this parameter also has
a significant impact on the hydraulic flux balance
between steps and the solubility of the dissolved salts
in the effluent). When these chemicals are present in
large concentrations, the porous structure may
deteriorate or become foul [109, 110]. Dyes can also
be removed by nanofiltration using 2-aminophenol-4-
sulfonic acid or piperazine. The time required was
120 h, and the result indicates that the AC was
93.6%–99.7% [102]. Table 2 contains many examples
of nanofilters using to separate wastes. (1)
The
determination
of
the
material’s
effectiveness and performance in both systems, the
evaluation of removal efficiency, and the usability of
various approaches to validating nanomaterials using
enabling active sensors. (1)
The
determination
of
the
material’s
effectiveness and performance in both systems, the
evaluation of removal efficiency, and the usability of
various approaches to validating nanomaterials using
enabling active sensors. (2) The mentioned research was conducted in
laboratories for a short period of time, and the long-
term capabilities of these nanoparticles are still in
doubt. Inspite of nanomaterials’ high removal efficiency,
the adaptability of these technologies to pilot research
is completely based on the possible risk and cost-
effectiveness. Nano-filters Except for nanoscale ion-oxide, nano
TiO2, and polymeric nanofibers, nanomaterials are
now prohibitively expensive. However, by reusing
and maintaining the nanomaterials, this can be
enhanced. The surface morphology of nanomaterials
enhances the activity and sensitivity of the
nanostructures, resulting in the removal of medicinal
substances
[111–113]. Chemical
modification
improves photocatalytic activity by combining the
nanoparticles with a catalytic metal, boosting
nanoparticle
efficiency
and
affinity
for
the
contaminant. The use of modified materials to remove
antibiotics from wastewater is modest, and various
techniques to overcome the involvement of other
contaminants, such as complex organic compounds,
heavy metal ions, colors, and viruses, should be
explored
[114]. Despite
the
fact
that
these
functionalized nanoparticles are made in a cost- Nano-filters 89% of ceftiofur sodium (CFS) was mineralized in
f A
Z O/PMS
6 h ill
i
i
2023
[104] Table 2 Overview of degradation proces
Pharmaceutical
particles
Nanomaterials
Amoxicillin
TiO2/ceramic membrane reactor
Nano-Fe3O4
TiO2/UV-C nano
TiO2/polyimide sheets
C3N4@MnFe2O4 graphene nanocomposites
Bimetallic nanoparticles CuFe2O4
NZVI
ZnO NPs
Fe/Ni NPs
Fe/Ni NPs
Carbon NPs
Carbon/TiO2 NPs
Cephalexin
H2O2/NiO NPs
Ferromagnetic Fe-O NPs
Nano-ZVI from nettle and thyme extracts
Ciprofloxacin
α-Al2O3 NPs/polyanion
TiO2 NPs
Mesoporous MnxCo3−x O4 NPs
ZnO NPs
GMC-TiO2
CNT/V2O5-ZnO NPs
Ceftriaxone
RuO2-TiO2/G-nano
NZVC
Fe3O4 NPs/ PAC
Cefadroxil
NZVC
Cefixime
TiO2 NPs
Rifampicin
Iron NPs
Tetracycline
NiFe NPs
bimetallic Fe/Zn NPs
CdS/(rGO)/ZnFe2O4
Anticancer
ZnO NPs/GONS
Four antibiotics
NF/O3
17b-estradiol
MWCNTs
Ceftiofur sodium
Ag-ZnO nanorods pH 5, 105 min, AC 92.8%
2020 pH 3, 5 h, AC 100%
2019 50 °C, 100 min, AC 90. 3%
2018 pH 9, 20 min, 25 °C, AC 85%
2018
[95] 90 min, AC 99%
2020
[98]
(mole ratio of Fe/Zn 1:1) loading on hydroxyl ethyl
cellulose-graphene oxide, pH 3, AC 95%
2023
[99]
AC 80%, H2O2 generated under visible light
irradiation
2022
[100]
120 min, AC 99.6%
2020
[101]
pH 7.9, water recovery 75%, coupled with AOPs
2014
[102]
pH 3.1, maximum AC 310 mg/g, MWCNTs as
adsorbent
2022
[103]
Nanoparticles were prepared by solvothermal method. 89% of ceftiofur sodium (CFS) was mineralized in
presence of Ag-ZnO/PMS at 6 h illumination
2023
[104] additives such as NaCl, NaHCO3, and NH4HCO3
increased the absorptive capacity of the nano- additives such as NaCl, NaHCO3, and NH4HCO3
increased the absorptive capacity of the nano- filter [81–85]. One of their disadvantages is a
temperature-sensitive process which significantly has https://www.sciopen.com/journal/2150-5578 185 Nano Biomed. Eng., 2023, 15(2) 1-Size exclusion
2-Donnan effect (charge balance)
3-Electrostatics interactions
Water molecules
Nonfilter
Medicines
Salts molecules
Fig. 3 Nonfilter mechanisms for removing medicines and salts. Medicines
Salts molecules Nonfilter Water molecules Fig. 3 Nonfilter mechanisms for removing medicines and salts. an adverse effect on feed bump pressure and the
presence of highly concentrated organic material
such
as
salts
caused
membrane
structure
deterioration [100–105]. These methods create a fresh
solid residue (membrane), similar to adsorption,
where the pollutant is focused. These methods have
mainly been combined with other approaches up until
now [106–108]. https://www.sciopen.com/journal/2150-5578 Limitation Pharmaceutical chemicals can be removed from
wastewater using a variety of nanotechnology-based
techniques. However, they show promise in lab
research, and their readiness in pilot-scale studies
differs significantly. Cost-effectiveness, technological
difficulties, potential human, environmental harm,
and other issues face their commercialization and
transition
to
pilot
projects. The
pilot-scale
deployment of nanoparticles for the elimination of https://www.sciopen.com/journal/2150-5578 https://www.sciopen.com/journal/2150-5578 Nano Biomed. Eng., 2023, 15(2) 186 2023, 51: 103440. https://doi.org/10.1016/j.jwpe.2022. 103440 2023, 51: 103440. https://doi.org/10.1016/j.jwpe.2022. 103440 effective as well as simple manner, large-scale
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Study of the parameters for a parallel hybrid electric vehicle
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* Corresponding author: zarni.znl15@gmail.com Study of the parameters for a parallel hybrid
electric vehicle
Zar Ni Lin1*, V. Sidorov1
1 Wheeled Vehicles and Applied Mechanics, Bauman Moscow State Technical University, 248000,
Kaluga Branch, Bazhenova st., 2, Kaluga, Russia Abstract. At present, the expansion in vehicle population has contributed
to the increase in carbon emissions and has led to an even greater increase
in the need for fuel. In this way, the hybrid vehicle (HEV) and the electric
vehicle (EV) are being introduced to reduce fuel consumption and exhaust
gas emission instead of conventional vehicles. The study of parameters for
the hybrid electric vehicle is important to design the powertrain of the
vehicle. The parameters of hybrid vehicles consist of the rate of internal
combustion engine (ICE) power, transmission gear ratios, motor maximum
torque and power, capacity and power of battery. The vehicle model with
the regenerative model is built to calculate the regenerative energy for
driving on a road condition. The design purposes to match the vehicle with
the specified performances, like as speed and acceleration. Many
parameters are chosen from analysis of this results. And then, as a result,
fuel consumption of test vehicle model reduces 15% than the conventional
vehicle in this study. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative
Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 402, 04014 (2023)
TransSiberia 2023 E3S Web of Conferences 402, 04014 (2023)
TransSiberia 2023 https://doi.org/10.1051/e3sconf/202340204014 Study of the parameters for a parallel hybrid
electric vehicle
Zar Ni Lin1*, V. Sidorov1
1 Wheeled Vehicles and Applied Mechanics, Bauman Moscow State Technical University, 248000,
Kaluga Branch, Bazhenova st., 2, Kaluga, Russia 1 Introduction With global gas emissions and rising fuel prices in the world, automotive manufacturers are
searching to reduce exhaust emission and fuel consumption, and with these vehicles like
this, more efficient powertrain vehicles are needed to produce [1]. The hybrid vehicle is one
of the choices for producing this vehicle because its internal combustion engine with the
electric motor and alternative powertrain can able to reduce exhaust emission and fuel
consumption. Many automakers and powertrains producers are engaged in projects to
develop clearer and more economical hybrid vehicles to meet the requirements [2]. When the vehicles drive in the city, they often move at low speed and need to
accelerate rapidly. The average speed of vehicle is less than 35 km/hr and the ranges
between two bus stops also are less than 500 meters and the maximum speed of city vans is
mostly less than 50 km/hr. Generally, the system of hybrid vehicle should be able to
emulate the engine driving at low speed, to recover brake energy, and to stop the engine
when the vehicle is in bus stops and waiting for traffic lights [3, 4]. * Corresponding author: zarni.znl15@gmail.com E3S Web of Conferences 402, 04014 (2023)
TransSiberia 2023 https://doi.org/10.1051/e3sconf/202340204014 (i) In EV function, the motor is only used to drive the vehicle from 0 to a certain speed, if
then the function is to avoid high emission and high fuel consumption of engine and clutch-
slipping at the start process. (i) In EV function, the motor is only used to drive the vehicle from 0 to a certain speed, if
then the function is to avoid high emission and high fuel consumption of engine and clutch-
slipping at the start process. (i) In EV function, the motor is only used to drive the vehicle from 0 to a certain speed, if
then the function is to avoid high emission and high fuel consumption of engine and clutch-
slipping at the start process. (ii) If the brake regeneration is larger, the fuel consumption can be more reduced. To
increase the brake regeneration, the engine must be disengaged from the powertrain to
avoid its friction. (iii) The Stop & Go function enables the engine to shut down when the vehicle is waiting (ii) If the brake regeneration is larger, the fuel consumption can be more reduced. To
increase the brake regeneration, the engine must be disengaged from the powertrain to
avoid its friction. (iii) The Stop & Go function enables the engine to shut down when the vehicle is waiting
and the engine is idling. This function in city conditions is important because there are too
many traffic problems. For the parallel hybrid vehicle, the four schemes are shown in (Fig. 1). In scheme (a) the
motor cannot recover the maximum regenerative energy when the vehicle is braking
because its clutch cannot be disengaged for the motor. The powertrain systems of the
scheme (b) and (c) are complex and difficult to realize in the vehicle. The mechanical
construction in the scheme (d) is not difficult for the vehicle. According to this analysis, the
powertrain system in the scheme (d) might be suitable for vehicles. Fig. 1. Configurations of parallel hybrid vehicle (E - engine, C - clutch, T - transmission, M – electric
motor and W - wheel). Fig. 1. Configurations of parallel hybrid vehicle (E - engine, C - clutch, T - transmission, M – electric
motor and W - wheel). * Corresponding author: zarni.znl15@gmail.com The complex mechanical construction for the series-parallel hybrid vehicle [5, 6] has
prevented it from being used as its main hybrid topology. The configuration as shown in
(Fig. 2) is an improvement for the hybrid series vehicle which installs a clutch between the
generator and the motor. Therefore, the vehicle can be driven by the engine in a proper
speed range and the overall efficiency can be improved compared with the pure series
hybrid because the energy conversion process is avoided when the engine drives. There are
also several applications for the series-parallel hybrid vehicle. pp
p
y
Fig. 2. Configurations of the series-parallel hybrid vehicle (E - engine, C - clutch, G - generator, M -
electric motor, R - final reducer and W - wheel) Fig. 2. Configurations of the series-parallel hybrid vehicle (E - engine, C - clutch, G - generator, M -
electric motor, R - final reducer and W - wheel) In the past, the focus has often been on developing control strategies for hybrid design
[8, 9]. In fact, hybrid powertrain parameters include not only motor and battery parameters,
such as engine and motor power rating and maximum torque of electric motors but also
gear ratio of the transmission and final reduction. According to the vehicle dynamics
theory, the gear ratio is related to the optimum performance of a conventional vehicle, but 2 2 E3S Web of Conferences 402, 04014 (2023)
TransSiberia 2023 https://doi.org/10.1051/e3sconf/202340204014 also affects the regenerative power. The parameters related to the motor and battery should
be solved by the hybrid vehicle model. This study tries to prove a matching strategy or methodology for parallel hybrid vehicle
which can solve the matching problem. The city van is chosen for the prototype vehicle
because many city vans for transport are used a lot in cities. The requirements and
limitations of this vehicle are introduced with a detailed illustration of hybrid models. The
parameters of the matching strategy are then described to analyse the influence of each
parameter and the control strategy for the hybrid system used in this paper is briefly
introduced. 2 Operation modes of hybrid vehicle system The operation modes of the parallel hybrid vehicle are described below: (i)
EV: normally the conventional vehicle has clutch-slipping effect when it
accelerates from 0 to a certain speed, but hybrid vehicle is only driven by the electric motor
to a certain speed for avoiding slipping effect. p
g
pp g
(ii)
Boost: when the gas pedal is fully pressed by the driver, the motor will assist the
engine to reduce engine load and keep it running at low load engine speed to improve the
fuel economy. (ii)
Boost: when the gas pedal is fully pressed by the driver, the motor will assist the
engine to reduce engine load and keep it running at low load engine speed to improve the
fuel economy. y
(iii)
Pure engine drive: the gearbox often shifts to high gear for medium and high
speed and the effective way is to drive the engine directly. In this case, the motor will not
work even if battery energy is high enough. However, if the battery energy is too low, the
engine should provide to charge it in this case. g
p
g
(iv)
Regeneration: when the driver presses brake pedal, the motor will produce the
negative torque to charge the battery. The greater of the motor power is, the more
regenerative energy is recovered. (iv)
Regeneration: when the driver presses brake pedal, the motor will produce the
negative torque to charge the battery. The greater of the motor power is, the more
regenerative energy is recovered. Fig. 3. Operation modes of the hybrid vehicle system
A typical driving pattern of the hybrid vehicle system is shown in (Fig. 3). In real road, the
distance of EV mode can be very long in heavy traffic conditions and mode of the pure
engine drive can be short because the maximum speed in the urban cycle is often lower
than 50 km/h. The electric motor consumes the battery energy in EV and boost modes and
this energy is replenished by the regeneration; however, it might cause over discharge of
the battery at low velocity as well as small regeneration conditions. The control strategy of
the hybrid system should be to avoid using the engine to charge the battery directly due to
low efficiency. This means that the engine of a hybrid vehicle should be downsized to
avoid excessive fuel consumption to charge the battery in boost mode. 3.1 Engine Capacity The power requirement of the hybrid vehicle can be calculated by
𝐶𝐴
2 𝑃𝑟𝑜𝑎𝑑= 𝑢𝑎
3.6𝜂[𝑀𝑔(𝑓0 + 𝑓1𝑢𝑎)𝑐𝑜𝑠𝜃+ 𝑀𝑔𝑠𝑖𝑛𝜃+ 𝐶𝐷𝐴𝑢𝑎
2
21.15 ], (1) 𝑃𝑟𝑜𝑎𝑑= 𝑢𝑎
3.6𝜂[𝑀𝑔(𝑓0 + 𝑓1𝑢𝑎)𝑐𝑜𝑠𝜃+ 𝑀𝑔𝑠𝑖𝑛𝜃+ 𝐶𝐷𝐴𝑢𝑎
2
21.15 ], (1)
where 𝑢𝑎 - car velocity, km/h; 𝑀 - total mass of car, kg which is the sum of curb mass and
cargo mass; 𝜃 - percentage of slope; 𝑔 - acceleration due to gravity, 9.81 kg⋅m/s; 𝐴 – car
front area; 𝐶D – wind coefficient; 𝑓0 and f1 - zero and first coefficients of road rolling
resistance. The values of the parameters used in equation (1) are shown in (Table 1). 𝑃𝑟𝑜𝑎𝑑= 𝑢𝑎
3.6𝜂[𝑀𝑔(𝑓0 + 𝑓1𝑢𝑎)𝑐𝑜𝑠𝜃+ 𝑀𝑔𝑠𝑖𝑛𝜃+ 𝐶𝐷𝐴𝑢𝑎
2
21.15 ], (1)
where 𝑢𝑎 - car velocity, km/h; 𝑀 - total mass of car, kg which is the sum of curb mass and
cargo mass; 𝜃 - percentage of slope; 𝑔 - acceleration due to gravity, 9.81 kg⋅m/s; 𝐴 – car
front area; 𝐶D – wind coefficient; 𝑓0 and f1 - zero and first coefficients of road rolling
resistance. The values of the parameters used in equation (1) are shown in (Table 1). (1) The text of your paper should be formatted as follows: The text of your paper should be formatted as follows: - 10-point Times, Times Roman or Times New Roman. - 10-point Times, Times Roman or Times New Roman. - The text should be set to single line spacing. - The text should be set to single line spacing. 3 Engine Matching Usually, the engine power capacity should meet the power requirement in the maximum
velocity. The dynamics factor in the high gear should be greater than 0.04 to overcome road
unevenness while driving at high speeds. 2 Operation modes of hybrid vehicle system However, if this
situation happens, the engine will be able to drive the vehicle and charge the battery at the
same time. Therefore, the purpose of parameter matching is described as follows: Fig. 3. Operation modes of the hybrid vehicle system Fig. 3. Operation modes of the hybrid vehicle system A typical driving pattern of the hybrid vehicle system is shown in (Fig. 3). In real road, the
distance of EV mode can be very long in heavy traffic conditions and mode of the pure
engine drive can be short because the maximum speed in the urban cycle is often lower
than 50 km/h. The electric motor consumes the battery energy in EV and boost modes and
this energy is replenished by the regeneration; however, it might cause over discharge of
the battery at low velocity as well as small regeneration conditions. The control strategy of
the hybrid system should be to avoid using the engine to charge the battery directly due to
low efficiency. This means that the engine of a hybrid vehicle should be downsized to
avoid excessive fuel consumption to charge the battery in boost mode. However, if this
situation happens, the engine will be able to drive the vehicle and charge the battery at the
same time. Therefore, the purpose of parameter matching is described as follows: 3 3 E3S Web of Conferences 402, 04014 (2023)
TransSiberia 2023 https://doi.org/10.1051/e3sconf/202340204014 (i)
The requirement of drivability in boosting and the potential regeneration energy
in braking are the main concern for motor design. (i)
The requirement of drivability in boosting and the potential regeneration energy
in braking are the main concern for motor design. g
g
(ii)
Engine power rating should meet the power requirements of pure engine drive
mode and achieve acceptable dynamics in acceleration without the assistance of the motor. (ii)
Engine power rating should meet the power requirements of pure engine drive
mode and achieve acceptable dynamics in acceleration without the assistance of the motor. 3.3 Engine torque capacity If the battery or motor fails, the dynamic characteristics of the mode of pure engine drive
should also be considered. The dynamics factor that indicates the acceleration ability and
slope-climbing ability is expressed in y
p
𝐷= 1
𝑚[(𝑇𝐼𝐶𝐸−𝑇𝐴𝑐𝑐)𝑖𝑔𝑖0𝑟𝜂−𝐶𝐷𝐴𝑢𝑎
2
21.15 ], (3) (3) where 𝑇ICE - maximum engine torque, 𝑇Acc - accessories torque, 𝑖𝑔 - gear ratio of the
transmission, 𝑖0 - ratio of final reducer, and 𝑟 - wheel radius. The average traction power is calculated by The average traction power is calculated by e average traction power is calculated by p
y
𝑃𝑒= 1
𝑇∫(𝑃𝑟𝑜𝑎𝑑+ 𝜎𝑀𝑢𝑎
12.96𝜂
𝑑𝑢𝑎
𝑑𝑡)𝑑𝑡, (2) (2) where 𝜎 - equivalent factor to transform the inertia to traction and other parameters are the
same as equation (1). The average traction power is highly related to the average velocity,
but it is much smaller than the engine power capacity selected in the previous section. Therefore, the engine power design is natural to meet the requirements of the urban cycle. where 𝜎 - equivalent factor to transform the inertia to traction and other parameters are the
same as equation (1). The average traction power is highly related to the average velocity,
but it is much smaller than the engine power capacity selected in the previous section. Therefore, the engine power design is natural to meet the requirements of the urban cycle. 4 Electric motor matching The main function of motor is to drive the vehicle to a certain speed and to assist the engine
when the gas pedal is pressed fully, and to generate electric power to charge the battery if
brake pedal is pressed. The maximum regeneration power is applied to recover the
electricity as much as possible when the vehicle is braking; thus the turning point and
maximal power are decided. Finally the electric power should be balanced for the
sustainable drive. 3.2 Average traction power The average traction power output of the engine will be greater or at least not smaller than
the road resistance when the vehicle drives in urban cycles which has many stop & go
situations. Otherwise, it cannot able to charge the battery in regeneration mode and
eventually, the charging drive will not be avoided. The average traction power output of the engine will be greater or at least not smaller than
the road resistance when the vehicle drives in urban cycles which has many stop & go
situations. Otherwise, it cannot able to charge the battery in regeneration mode and
eventually, the charging drive will not be avoided. Table 1. Parameters used for the prototype vehicle. Table 1. Parameters used for the prototype vehicle. Symbol
Value
Unit
Vehicle mass
3500
kg
Wind
coefficient
Cd
0.65
-
Front area
A
5.7
m2
Coefficient
of zero road
resistance
f0
0.0078
-
Coefficient
of first road
resistance
f1
0.000056
-
Acceleration
due to
gravity
g
9.81
ms-2
Powertrain
efficiency
η
0.9
- E3S Web of Conferences 402, 04014 (2023)
TransSiberia 2023 https://doi.org/10.1051/e3sconf/202340204014 TransSiberia 2023 5 Gear ratio of transmission According to (Fig. 1), the transmission affects the dynamics and fuel economy of
traditional vehicles, and it also relates to the hybrid vehicle because the route of power in
this parallel hybrid configuration is the same as the traditional vehicle [10]. However for the powertrain of hybrid vehicle as to the analysis before, the dynamics
would be improved greatly with the assist of the electric motor. Thus, the main factor in
gear ratio design is the only fuel consumption. Usually, the equivalent fuel consumption is
calculated by 𝑄𝑒=
𝑔𝑒𝑛𝑟
95490𝜌𝑢𝑎𝑖𝑔𝑖0
[𝑀𝑔(𝑓0 + 𝑓1𝑢𝑎) + 𝐶𝐷𝐴𝑢𝑎
2
21.15 ], (9) (9) it is used to evaluate the fuel economy of different gear ratios to compare the fuel economy
of different gear ratio groups. Where, 𝑔𝑒 - equivalent fuel consumption in g/(kw⋅h), 𝜌 -
density of diesel fuel in g/mm3. For the same diesel engine, 𝑔𝑒 is the same and 𝑄𝑒 is
influenced by 𝑖𝑔 and 𝑖0 greatly. The relationship of the speed 𝑛 and 𝑢𝑎 can be written by 𝑛
𝑢𝑎
= 250𝑖𝑔𝑖0
3𝜋𝑟
, (10) (10) 𝑎
and the equation can be rewritten as
𝑄𝑒= 𝐶𝑔𝑒𝑖𝑔
2𝑖𝑜
2
𝜌
[𝑀𝑔(𝑓0 + 𝑓1𝑢𝑎) + 𝐶𝐷𝐴𝑢𝑎
2
21.15 ]. (11) 𝑎
and the equation can be rewritten as
2 be rewritten as
𝑄𝑒= 𝐶𝑔𝑒𝑖𝑔
2𝑖𝑜
2
𝜌
[𝑀𝑔(𝑓0 + 𝑓1𝑢𝑎) + 𝐶𝐷𝐴𝑢𝑎
2
21.15 ]. (11) (11) From the above equation (11), for the vehicles, the first road resistance 𝑓1 is very small, so
it has been ignored in this equation; therefore equation (11) is rewritten as From the above equation (11), for the vehicles, the first road resistance 𝑓1 is very small, so
it has been ignored in this equation; therefore equation (11) is rewritten as
𝐶𝐴 q
q
(
)
𝑄𝑒= 𝑔𝑒
𝜌(𝐶1𝑖𝑔
2𝑖𝑜
2𝑀𝑔𝑓0 + 𝐶2
𝐶𝐷𝐴
21.15𝑛2), (12) (12) where 𝐶1 and 𝐶2 - constant values. From this equation it can be found that the smaller 𝑖𝑔 and
𝑖0 are, the lower 𝑄𝑒 we get for the same velocity. 21.15𝑚 where the propelling torque is the motor torque only compared with equation (8). Also as the EV mode will not be used too many in this parallel hybrid vehicle
configuration because the motor will only drive the vehicle to a certain speed until the
motor speed is greater than idle speed, the acceleration time is not mainly decided by the
motor but by the motor and engine together. 4.1 Evaluation of regeneration energy The electric motor will generate the maximum negative torque to recover the kinetic energy
during braking. The brake force is calculated by 𝑇𝑏𝑟𝑎𝑘𝑒= 𝑇𝑤ℎ𝑒𝑒𝑙+ 𝑇𝑚+ 𝑀𝑔(𝑓0 + 𝑓1𝑢𝑎) + 𝐶𝐷𝐴𝑢𝑎
2
21.15 , (4) (4) where 𝑇brake - total brake force, 𝑇wheel - mechanical brake force, and 𝑇𝑚 - negative torque
of motor. To achieve the maximum regenerative energy, 𝑇wheel is set to zero. Therefore, the brake force can be written by y
𝑇𝑏𝑟𝑎𝑘𝑒= 𝑀𝑔(𝑓0 + 𝑓1𝑢𝑎) + 𝐶𝐷𝐴𝑢𝑎
2
21.15 + 𝑇𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒. (5) (5) 2
The total electric energy can be calculated by The total electric energy can be calculated by 𝐸𝑚= ∫𝜂𝑚𝜂𝑏𝑇𝑚𝑖𝑔𝑖0𝑢𝑎
1145.88𝜋𝑟, (6) (6) and the efficiency of motor 𝜂𝑚 and battery 𝜂𝑏 is set to 90%. Thus the regeneration rate
𝜆regen, which is the total regenerative energy versus the motive from a certain velocity to
zero, is an indicator to value how the power and the basic point 𝑛𝑏 affect the brake
recovering, derived by 𝜆𝑟𝑒𝑔𝑒𝑛= 2𝐸𝑚
𝑀𝑢𝑎2. 𝜆𝑟𝑒𝑔𝑒𝑛= 2𝐸𝑚
𝑀𝑢𝑎2. (7) (7) 5 5 E3S Web of Conferences 402, 04014 (2023)
TransSiberia 2023 https://doi.org/10.1051/e3sconf/202340204014 6 Battery matching According to (Fig. 1), the transmission affects the dynamics and fuel economy of
traditional vehicles, and it also relates 4.2 Dynamics Evaluation The design of electric motor torque is to satisfy mode of the pure motor drive and to assist
the engine when the pedal is pressed fully. To overcome the slopes, the 𝐷 factor of the first
gear and second gear should be large enough. The 𝐷 factor is shown in g
g
𝐷= 21.15𝑇𝑚𝑜𝑡𝑜𝑟𝑖𝑔𝑖𝑜𝑟𝜂−𝐶𝐷𝐴𝑢𝑎
2
21.15𝑚
, (8) g
g
𝐷= 21.15𝑇𝑚𝑜𝑡𝑜𝑟𝑖𝑔𝑖𝑜𝑟𝜂−𝐶𝐷𝐴𝑢𝑎
2
21.15𝑚
, (8)
torque is the motor torque only compared with equation (8). (8) 6.2 Electric energy matching The requirement of electric energy will be confirmed by simulation equipment and test on
real road. For the hybrid vehicle, the typical European city cycle (EUDC) is used to
simulate the electric energy demand. The electric energy is consumed when the vehicle
accelerates at high speed. According to previous calculation, the total energy 𝐸BP of the
LMO battery pack is calculated as (17) 𝐸𝐵𝑃= 𝑉𝑐𝑒𝑙𝑙𝑛𝑠𝐶𝑛𝑝. 𝐸𝐵𝑃= 𝑉𝑐𝑒𝑙𝑙𝑛𝑠𝐶𝑛𝑝. (17) 𝑝
The SOC using range has been set from 30% to 70%. Thus, the energy of battery pack
can satisfy the energy requirement of hybrid electric vehicle. 6.1 Electric power matching The power selection of battery is determined by the maximum power of electric motor as
shown in 6 6 6 E3S Web of Conferences 402, 04014 (2023)
TransSiberia 2023 https://doi.org/10.1051/e3sconf/202340204014 𝑃𝑏= 𝑃𝑚
𝜂𝑏
, (13) 𝑃𝑏= 𝑃𝑚
𝜂𝑏
,
i
h
i
f (13) where 𝜂𝑏 – battery charging and discharging efficiency, 𝑃𝑏 - battery power and The lithium-
ion battery is widely used in hybrid electric vehicles due to its high power density (𝜌𝑃,
W/kg) [11]. In general, the charging 𝜌𝐶 and discharging 𝜌𝐷 power density of the battery
are different. In HEV application, the battery pack is often a set of many cells, and for the
first, several cells are paralleled together to form a cell module that has greater capacity,
and then the cell modules are connected in series to battery pack so as to meet the voltage
and energy requirement [12]. The number of cell modules 𝑛𝑠 is decided by the motor maximum voltage demand
𝑉max of the motor as shown in 𝑛𝑠≈
𝑉𝑚𝑎𝑥
𝑉𝑐𝑒𝑙𝑙𝑚𝑎𝑥
, (14) (14) 𝑚𝑎𝑥
where 𝑉cell max - maximum voltage of battery cell. The power demand of each cell 𝑃𝑏 can
be written as
𝑃 𝑃𝑐𝑒𝑙𝑙= 𝑃𝑏
𝑛𝑠
. (15)
The number of parallel cells 𝑛𝑝 in cell module can be calculated by
𝑛𝑝= 𝜌𝐸𝑃𝑐𝑒𝑙𝑙
𝜌𝐶𝐸𝑐𝑒𝑙𝑙
, (16)
𝑃
motor power 𝑃𝑐𝑒𝑙𝑙= 𝑃𝑏
𝑛𝑠
. (15) (15) 𝑛𝑠
The number of parallel cells 𝑛𝑝 in cell module can be calculated by
𝑛𝑝= 𝜌𝐸𝑃𝑐𝑒𝑙𝑙
𝜌𝐶𝐸𝑐𝑒𝑙𝑙
, (16)
𝑃
motor power 𝑠
The number of parallel cells 𝑛𝑝 in cell module can be calculated by allel cells 𝑛𝑝 in cell module can be calculated by
𝑛𝑝= 𝜌𝐸𝑃𝑐𝑒𝑙𝑙
𝜌𝐶𝐸𝑐𝑒𝑙𝑙
, (16) 𝑛𝑝= 𝜌𝐸𝑃𝑐𝑒𝑙𝑙
𝜌𝐶𝐸𝑐𝑒𝑙𝑙
, (16) (16) 𝑃𝑚 - motor power. p
where 𝐸cell - nominal energy of each cell. Since the charging power density and discharging
power density of the battery are different, the charging power density 𝜌𝐶 should be used in
this equation. 8 Conclusions The methodology of parameter design is effectively proven for the postposition parallel
hybrid powertrain. The keys to this methodology are summarized as follows. (i)
Power requirement determines the engine power rating when the vehicle is
driving at high speed on normal road, the highest gear ratio must correspond to the
maximum speed at high engine speed, and the dynamic requirement should be met by the
lowest gear ratio. In order to realize the low engine speed in the urban cycle, the 𝑛/𝑉
selection must be smaller. (i)
Power requirement determines the engine power rating when the vehicle is
driving at high speed on normal road, the highest gear ratio must correspond to the
maximum speed at high engine speed, and the dynamic requirement should be met by the
lowest gear ratio. In order to realize the low engine speed in the urban cycle, the 𝑛/𝑉
selection must be smaller. (ii)
The power and torque of the electric motor are related to the potential of
regenerative brake, and the proper regenerative rate should be achieved. (ii)
The power and torque of the electric motor are related to the potential of
regenerative brake, and the proper regenerative rate should be achieved. (iii)
The size of battery should satisfy the requirements of electric power and energy
during the specified driving cycle. (iii)
The size of battery should satisfy the requirements of electric power and energy
during the specified driving cycle. At last, this parameter matching methodology for a post-positioned parallel hybrid
configuration can be easily extended to other HEV types. At last, this parameter matching methodology for a post-positioned parallel hybrid
configuration can be easily extended to other HEV types. Fig. 4. Energy consumption using EUDC cycle. References
1. S.A. Skia, Basis of HEV (Beijing, China: Beijing Institute of Technology Press,
Translated by Chen Q and Sun F) (2001)
2. M. Nishihara, Operations Research, Letters 38(2), 87–93 (2010)
3. K.C. Agatay Bayindir, et.al., Energy Conversion and Management 52(2), 1305–1313
(2011) Fig. 4. Energy consumption using EUDC cycle. Fi
4 E
ti
i
EUDC
l Fig. 4. Energy consumption using EUDC cycle. 7 Comparison of experimental results For this study, a prototype car is built and then the experiment is conducted. According to
the European city cycle (EUDC), the test results of this experiment are shown in (Fig. 4)
and (Table 2). The fuel-saving is about 15% compared to the same size vehicle with the
conventional engine after adjusting fuel consumption for electric energy consumption. 7 E3S Web of Conferences 402, 04014 (2023)
TransSiberia 2023 https://doi.org/10.1051/e3sconf/202340204014 Table 2. Experimental results of fuel consumption. Fuel consumption
(L/100 km)
Battery energy
consumption (kW.h)
Conventional vehicle
9.8
-
Hybrid vehicle
8.4
10.87 Table 2. Experimental results of fuel consumption. References 1. S.A. Skia, Basis of HEV (Beijing, China: Beijing Institute of Technology Press,
Translated by Chen Q and Sun F) (2001) 1. S.A. Skia, Basis of HEV (Beijing, China: Beijing Institute of Technology Press,
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Immunoadsorption therapy in patients with multiple sclerosis with steroid-refractory optical neuritis
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Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 JOURNAL OF
NEUROINFLAMMATION Open Access Abstract Background: In multiple sclerosis relapses refractory to intravenous corticosteroid therapy, plasma exchange is
recommended. Immunoadsorption (IA) is regarded as an alternative therapy, but its efficacy and putative
mechanism of action still needs to be established. Methods: We prospectively treated 11 patients with multiple sclerosis who had optical neuritis and fulfilled the
indications for apheresis therapy (Trial registration DE/CA25/00007080-00). In total, five IA treatments were
performed using tryptophan-IA. Clinical activity (visual acuity, Expanded Disability Status Scale, Incapacity Status
Scale), laboratory values and visual evoked potentials were measured before, during and after IA, with a follow-up
of six months. Moreover, proteomic analyses were performed to analyze column-bound proteins as well as
corresponding changes in patients’ sera. Results: After the third IA, we detected an improvement of vision in eight of eleven patients, whom we termed
responders. Amongst these, the mean visual acuity improved from 0.15 ± 0.12 at baseline to 0.47 ± 0.32 after the
third IA (P = 0.0252) up to 0.89 ± 0.15 (P < 0.0001) at day 180 ± 10 after IA. Soluble interleukin-2 receptor decreased
in responders (P = 0.03), whereas in non-responders it did not. Proteomic analyses of proteins adsorbed to IA
columns revealed that several significant immunological proteins as well as central nervous system protein
fragments, including myelin basic protein, had been removed by IA. Conclusions: IA was effective in the treatment of corticosteroid-refractory optic neuritis. IA influenced the humoral
immune response. Strikingly, however, we found strong evidence that demyelination products and immunological
mediators were also cleared from plasma by IA. Keywords: Apheresis, Autoimmune diseases, Evoked potentials/visual, Immunoadsorption, Multiple sclerosis, Optic
neuritis Proteomics Keywords: Apheresis, Autoimmune diseases, Evoked potentials/visual, Immunoadsorption, Multiple sclerosis, Optic
neuritis, Proteomics Immunoadsorption therapy in patients with
multiple sclerosis with steroid-refractory optical
neuritis Michael J Koziolek1*, Desiree Tampe1, Matthias Bähr2, Hassan Dihazi1, Klaus Jung3, Dirk Fitzner2, Reinhard Klingel4,
Gerhard A Müller1 and Bernd Kitze2 © 2012 Koziolek 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. * Correspondence: mkoziolek@med.uni-goettingen.de
1Department of Nephrology and Rheumatology, Georg-August-University
Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany
Full list of author information is available at the end of the article Clinical chemistry
All l b Clinical chemistry
All laboratory parameters were measured by standard
methods. The complement components C3c and C4
as well as the immunoglobulins G (IgG), A (IgA) and
M (IgM) were measured by nephelometry (Behring
Nephelometer II Analyzer, Germany). Soluble interleukin-2
receptor (sIL-2R) was detected on an immulite system
(Siemens, Germany). The study protocol had been approved by the local
ethics committee prior to study initiation (no. 2/4/07)
and registered at the local government (no. DE/CA25/
00007080-00). All patients gave their written informed
consent before enrolment. Classification of side effects Side effects were defined as any unexpected or symptom-
atic event that had a possible, probable or definite causal
relationship with IA treatment [14]. They were classified
as mild, moderate or severe as described previously [15]
with small modifications. Briefly, mild side effects included
those of transient nature with little or no clinical signifi-
cance and without any temporary break of the procedure. Side effects that required medical intervention but were
not life-threatening were classified as moderate. Unstable
and life-threatening events requiring termination of the
procedure were classified as severe. Baseline and follow-up visits Baseline and follow-up visits part of European guidelines for the treatment of MS [7]. Clinical-pathological correlation analyses have shown that
all patients with pattern II pathology but none with
pattern I or pattern III experienced improvement in
neurological deficits after being treated with PE [8]. This
selective response suggests a removal of pathogenic
humoral and plasma factors by PE. All patients were followed up by a neurologist. Visits were
performed at baseline (visit 0), after each IA treatment
(visits 2 to 5) and after 30± 5 days (visit 6), 60 ± 10 days
(visit 7) and 180 ± 10 days (visit 8). The neurological find-
ings were assessed using the Expanded Disability Status
Scale (EDSS) and the Incapacity Status Scale (ISS) [11-13]. Changes of visual acuity were monitored using standar-
dized near vision types after correction of refractive error
at each visit and confirmed by an ophthalmologist before
and after IA, as well as at 180 ± 10 days. Visual evoked
potentials were determined at baseline, after the last IA
and 60 ± 10 days post-intervention with the Neuropack
M1 (Nihon Koden, Surbiton, UK). Immunoadsorption (IA) provides a more selective
approach and the potential for technical innovations in
therapeutic apheresis techniques, allowing the elimination
of pathogenic antibodies while sparing other plasma
proteins. With IA, relevant side effects of PE resulting
from protein substitution can be avoided [9]. We hypothe-
sized that IA is at least equally efficient compared to PE as
an escalation therapy for steroid-unresponsive relapses of
MS. Therefore, we performed a prospective trial to
compare IA treatment in 11 patients with MS with our
earlier patient population treated with PE for their MS
[10]. In addition, proteomic analyses of column-bound
proteins were performed as well as measurements of
corresponding changes in patients’ plasma samples. Methods We prospectively included 11 consecutive patients with
MS who had functionally disabling acute optical neuritis. Patients fulfilled the indications for apheresis treatment
due to this steroid-unresponsive MS relapse according
to German guidelines (http://www.dgn.org) [7]. Background however, at least part of the humoral response in MS is
derived systemically from the blood [3]. Previous studies have described four immunopathological
patterns of demyelination in early multiple sclerosis (MS)
lesions, with pattern II being characterized by antibody
and/or complement-associated demyelination [1]. Several
specific antibodies have been described and discussed
to contribute to the humoral autoimmune response in
MS [2]. Immunoglobulins are synthesized intrathecally; Therapeutic plasma exchange (PE) is based on the sep-
aration of plasma from cellular blood components, allow-
ing the removal of substances up to a molecular weight of
3 × 103 kDa. As shown in a randomized placebo controlled
cross-over study, PE was efficient for steroid - refractory
relapses in about 40% to 50% of cases of acute central
nervous system inflammatory demyelinating diseases [4]. The usefulness of PE has also been extended to severe
optic neuritis in patients with MS [5,6]. Thus, the use of
PE in steroid-refractory relapses has become an integral Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Page 2 of 10 Page 2 of 10 Identification of immunoadsorption column-binding
proteins by elution and proteomics IA was performed using the tryptophan-linked polyvinyl
alcohol adsorber TR-350, after membrane plasma separ-
ation with the polyethylene plasma separator OP-05 W
(Asahi Kasei Kuraray, Tokyo, Japan) in combination with
the Octo Nova extracorporeal circuit technology (SW
4.30.2, front 4.30.0) (Diamed Medizintechnik, Cologne,
Germany). The adsorber, plasma separator and tubing
system were for single use only. Combined anticoagulation,
with citrate and unfractionated heparin, was used for all
treatments. The treated plasma volume was 2,500 mL
plasma for all treatments of all patients. In total, five
sessions were performed in each patient on alternate days. In case of complications or decrease of fibrinogen below
100
mg/dL,
treatment-free
intervals
were
extended
individually. Internal jugular veins were used for central
vascular access with double lumen catheters in all patients. Immusorba TR-350 column-binding proteins were eluted
after the first IA treatment in five of eleven patients. Prior
to elution, the column was washed with PBS buffer. The
protein elution was carried out as following: PBS-washing
step was followed by a three-step elution protocol using
solution A (100 mM sodium acetate, 1 M NaCl, pH 5),
solution B (20 mM Tris–HCl, 1 M NaCl, pH 8.5) and
solution C (20% acetonitril in double diluted H2O). Proteins eluted from all three steps were pooled together
and aliquots of 10 mL were used for protein precipitation,
protein estimation and two-dimensional gel electrophoresis. Two-dimensional gel electrophoresis, protein visual-
ization and image analysis, in-gel digestion, mass spec-
trometry analysis of the digestion products and protein
identification using a database search were performed as Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Page 3 of 10 Page 3 of 10 to 0.6 or more was achieved in eight of eleven patients
(72.7%) undergoing IA treatment (responder group). Two patients did not respond to therapy at any time
point and one patient improved during IA therapy but
deteriorated shortly after the end of IA therapy, asso-
ciated with the incidence of a jugular venous thrombosis
on the same side (non-responder group). The relevant
data of patients’ characteristics are shown in Table 1. described in detail in our previous publication [16]. For
protein identification, qualitative criteria encompassed
optimized mass accuracy (<50 ppm), minimal mass de-
viation (in the millidalton range) and highest possible
probability score, which were assigned to each identified
protein. Western blot analysis Western blot analyses were performed according to
previously published data [17] with 20 μg of plasma
proteins. Equal loading was ascertained by Coomassie
staining. For immunodetection of proteins, the following
antibodies were used: rabbit polyclonal to human CD5
ligand (CD5L) and mouse monoclonal to human myelin
basic protein (both Abcam, Cambridge, UK); and horse-
radish peroxidase-linked donkey anti-rabbit antibody
(Amersham Biosciences, Freiburg, Germany). Results are
expressed as mean ± SD. Statistics
Vi i
d Visit and response effects were studied by two-way
repeated
measures
analysis
of
variance
using
the
mixed procedure for the software SAS (version 9.1,
SAS Institute). In this global analysis, P < 0.05 indi-
cated a significant effect. In the case of a significant
effect at a particular visit, subsequent pairwise com-
parisons
to baseline
values
were performed using
one-way repeated measures analysis of variance to de-
tect the point in time when the effect occurred. These
comparisons were performed at Bonferroni-adjusted sig-
nificance levels (related P-values are labeled with either
Bonf. sig. (significant) or with Bonf. n.s. (non-significant)). Relief of visual impairment inversely correlated with
the time interval between the start of symptoms and
the beginning of IA therapy (r = -0.539) with a trend
(P = 0.08) favoring positive clinical response in early
opposed to late IA initiation. Visual evoked potentials Before IA treatment, visual evoked potentials could not
be identified in six of our patients with MS due to the
severity of the optical neuritis. However, after treatment
in four of these participants, potentials recovered at day
60. In five patients, the visual evoked potential ampli-
tudes as well as latencies before and after IA could be
compared. The mean amplitude was 2.31 ± 2.57 mV at
baseline,
3.34 ± 2.60
mV
after
the
last
IA,
and
5.69 ± 1.76 mV at day 60 (P = 0.37). Latencies did not
show recovery in those five patients from baseline
(107.6 ± 21.7 ms) until day 60 ± 10 (112.3 ± 17.1 ms). Smoothing splines with three degrees of freedom were
fitted to mean values of each visit to illustrate the trends
and the potential interactions between visits and response
effects. Fitting was performed using the free software R
(version 2.8, http://www.r-project.org). Comparative statistical analyses of changes within-sub-
ject and within-treatment were performed using t-tests for
paired samples in case of normal data and Mann–Whitney
U-tests for non-normal data. Normality was checked by
quantile-quantile plots. Again, test results with P < 0.05
were considered significant. Expanded disability status scale and incapacity status scale
The EDSS continuously improved in the responder
group starting from 4.06 ± 1.82 at baseline to 3.44 ± 2.38
after the last IA to a minimum of 2.81 ± 2.72 at day
60 ± 10 (P = 0.58), in contrast to the non-responder
group who showed no changes. However, EDSS is clearly
dominated by ambulation [12] and rather insensitive for
visual dysfunction [13]. In parallel, ISS ameliorated in
the responder group from 8.00 ± 9.02 at baseline with a
continuous decrease to a minimum of 5.75 ± 6.78 at day
180 ± 10 (P = 0.04), in contrast to no significant changes
in the non-responder group. Visual acuity Mean visual acuity of all patients significantly improved
with a baseline value of 0.12 ± 0.12 before the start of IA,
0.36 ± 0.33 (P = 0.0234) after the third IA and 0.72 ± 0.38
(day 180 ± 10 after IA; P < 0.001). The effect was even
more pronounced in the responder group. Mean visual
acuity before start of IA was 0.15 ± 0.12 with an increase
after the third IA to 0.47 ± 0.32 (P = 0.0252 Bonf. n.s.)
and even more up to 0.89 ± 0.15 (P < 0.0001 Bonf. sig.)
at day 180 ± 10 after IA. By contrast, in the non-re-
sponder group, no significant changes of visual acuity
were detectable. Results are summarized in Figure 1. Identification of immunoadsorption column-binding
proteins by elution and proteomics Proteins identified by mass spectrometry are
described qualitatively. Side effects hemoglobin, hematocrit, thrombocytes, sodium, calcium,
creatinine, aspartate aminotransferase, alanine aminotrans-
ferase, alkaline phosphatase, gamma-glutamyltransferase,
creatine kinase and C-reactive protein. Significant changes
were detected for fibrinogen, leukocytes and total protein. Increased leukocyte counts at baseline were likely caused
by corticosteroid therapy before starting IA. In total, 55 IA treatments were analyzed. Mild side effects
were often reported and caused slight discomfort to the
patients, such as coldness, cough, dizziness, headache,
temporary hypertension, lacrimation, muscular cramps,
nausea,
palpations,
pruritus,
paresthesia,
rhinorrhea,
sweating, tinnitus or vomiting. They were transient and
could be easily managed without therapy. Immunoglobulins IgA, IgG and IgM as well as
complement components C3c and C4 significantly
decreased during IA therapy (P < 0.01). After the end of
IA, however, these parameters gradually recovered and
increased beyond baseline values (as shown for IgG and
C3c in Figure 2A, B). We found a significant decrease of
sIL-2R at a rate of -35.4 ± 20.7% (P = 0.0054) after just one
IA session in our patients with MS. Furthermore, time-
courses of sIL-2R in responders and non-responders
showed a significant difference (P = 0.03; Figure 2C). Sam-
ple dilution in the patients’ circulation due to IA was
excluded by laboratory follow-ups of hematocrit and albu-
min in serum. Ten moderate side effects were recorded during the
IA phase. These included vascular access problems such
as central venous catheter infection and jugular venous
thrombosis as well as therapy-associated events that
included chest pain, dyspnoe, transient hypotension and
urticaria, which could be easily managed by approved
medical interventions. Two patients developed febrile
infections (common cold and phlebitis of a peripheral
vein) within 24 h of IA, which were possibly related to
short-term immune dysfunction caused by corticosteroid
therapy and IA. No severe side effects occurred. Results
are summarized in Table 2. Glatiramer acetate Descriptive values are shown either as absolute numbers (frequencies) or mean ± standard deviation (minimum –maximum), except for baseline EDSS and relapse
rates which are shown as medians and ranges. Results IA treatments were started after a mean time of 26.6 days
after the initial symptoms and 10.8 days after the start of
corticosteroid therapy. Each patient received at least two
courses of corticosteroid therapy prior to IA therapy,
with a mean cumulative dose of 10.9 g prednisolone
equivalent but without a significant improvement, thus
fulfilling the indication of therapeutic apheresis as
adjunct treatment. An improvement of visual acuity up Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Page 4 of 10 Table 1 Clinical baseline characteristics of the participants
Parameter
Total (n = 11)
Responder (n = 8)
Non-responder (n = 3)
Gender
Female
7 (64%)
5 (62%)
2 (67%)
Male
4 (36%)
3 (38%)
1 (33%)
Age
33.6 ± 10.5
29.6 ± 9.2
44.3 ± 8.0
(19.0 to 55.0)
(19.0 to 47.0)
(39.0 to 55.0)
Body mass index
25.5 ± 3.0
22.9 ± 2.6
26.2 ± 4.0
(20.1 to 30.7)
(22.0 to 30.7)
(20.1 to 25.7)
Relapse rates in the past 12 months (median and range)
2
2
1
(1 to 4)
(1 to 4)
(1 to 2)
Baseline Expanded Disability Status Scale
4
3.5
4
(2 to 7.5)
(2 to 7.5)
(3 to 4)
Days after onset of relapse
26.6 ± 14.6
29.3 ± 15.6
19.3 ± 10.1
(10.0 to 60.0)
(10.0 to 60.0)
(10.0 to 30.0)
Days after initiation of corticosteroid therapy
10.8 ± 6.9
12.3 ± 6.6
7.0 ± 7.2
(1.0 to 20.0)
(3.0 to 20.0)
(1.0 to 15.0)
Cumulative dose of prednisolone equivalents (g)
9.5 ± 3.7
8.6 ± 3.7
12.0 ± 2.6
(4.0 to 15.0)
(4.0 to 15.0)
(10.0 to 15.0)
Duration of disease (years)
3.5 ± 4.9
5.7 ± 2.9
2.7 ± 9.0
(0.0 to 16.0)
(0 to 7.0)
(0.0 to 16.0)
Initial disease modifying therapy
3 (27.3%)
2 (25%)
1 (33.3%)
Interferon beta
2 (18.2%)
2 (25%)
0 (0%)
Glatiramer acetate
1 (9.1%)
0 (0%)
1 (33.3%)
Descriptive values are shown either as absolute numbers (frequencies) or mean ± standard deviation (minimum –maximum), except for baseline EDSS and relapse
rates which are shown as medians and ranges. Table 1 Clinical baseline characteristics of the participants Total (n = 11)
Responder (n = 8)
Non-responder (n = 3) Initial disease modifying therapy Interferon beta Proteomic data Our proteomic investigation allowed the identification of
41 proteins with a peptide mass fingerprinting-score In routine analyses, no significant changes were seen for
the
following
values:
partial
thromboplastin
time, Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Page 5 of 10 Page 5 of 10 sign. sign. sign. sign. Figure 1 Time-course of visual acuities of the affected eyes assessed by standard charts in 11 patients with multiple sclerosis treated
by immunoadsorption. Visual acuities were shown as box plots (outliers marked as X) at baseline (visit 0), after three IA therapies (visit 3), after
five IA therapies (visit 5), after 30 days (visit 6), after 60 days (visit 7), and after 180 days (visit 8). Three patients did not show long-term
improvement of visual acuity, they were termed non-responders in this study (shown as filled circles, partially overlapping each other). AE: affected eyes; sign.: significant (pairwise comparison to baseline). sign. sign. sign. sign. Figure 1 Time-course of visual acuities of the affected eyes assessed by standard charts in 11 patients with multiple sclerosis treated
by immunoadsorption. Visual acuities were shown as box plots (outliers marked as X) at baseline (visit 0), after three IA therapies (visit 3), after
five IA therapies (visit 5), after 30 days (visit 6), after 60 days (visit 7), and after 180 days (visit 8). Three patients did not show long-term
improvement of visual acuity, they were termed non-responders in this study (shown as filled circles, partially overlapping each other). AE: affected eyes; sign.: significant (pairwise comparison to baseline). Figure 1 Time-course of visual acuities of the affected eyes assessed by standard charts in 11 patients with multiple sclerosis treated
by immunoadsorption. Visual acuities were shown as box plots (outliers marked as X) at baseline (visit 0), after three IA therapies (visit 3), after
five IA therapies (visit 5), after 30 days (visit 6), after 60 days (visit 7), and after 180 days (visit 8). Three patients did not show long-term
improvement of visual acuity, they were termed non-responders in this study (shown as filled circles, partially overlapping each other). AE: affected eyes; sign.: significant (pairwise comparison to baseline). >65 in eluates of IA columns. We could identify apoli-
poproteins, hemorheologically relevant proteins, im-
munologically relevant proteins, myelin-related proteins
and others. A representative two-dimensional gel is shown in Figure 3A and results are summarized in
Table 3. Proteomic data Amongst all identified proteins, soluble CD5L and the
myelin basic protein (MBP) fragment were quantitatively
analyzed in patients’ sera by western blots using specific
antibodies (Figure 3B). The baseline value was set to
100% and follow-up values were given in percent of
baseline. Plasma MBP levels significantly decreased
during the first IA session to 46.4 ± 15.0% (P < 0.001)
and remained low even in the post-IA control at day
60 ± 10. In
parallel,
soluble
CD5L
(sCD5L)
levels
decreased to 50.5 ± 15.0% (P < 0.001) after the first IA
and remained subsequently low. Time-courses of sCD5L
and MBP are shown in Figure 3B. Table 2 Moderate adverse events occurring during
immunoadsorption that were classified as being related
to vascular access, the immunoadsorption procedure or
immunosuppression
Patients
(n = 11)
Immunoadsorption
therapies (n = 55)
Vascular access-related
Central venous catheter exit site
infection
1 (9.1%)
1 (1.8%)
Jugular vein thrombosis
1 (9.1%)
1 (1.8%)
Side effects during immunoadsorption therapy
Chest pain
1 (9.1%)
1 (1.8%)
Dyspnoe
1 (9.1%)
1 (1.8%)
Transient hypotension
4 (36.5%)
5 (9.1%)
Urticaria
1 (9.1%)
1 (1.8%)
Putatively related to immunosuppression both by steroids and
immunoadsorption
Febrile infection
2 (18.2%)
2 (3.6%) Table 2 Moderate adverse events occurring during
immunoadsorption that were classified as being related
to vascular access, the immunoadsorption procedure or
immunosuppression
Patients
(n = 11)
Immunoadsorption
therapies (n = 55)
Vascular access-related
Central venous catheter exit site
infection
1 (9.1%)
1 (1.8%)
Jugular vein thrombosis
1 (9.1%)
1 (1.8%)
Side effects during immunoadsorption therapy
Chest pain
1 (9.1%)
1 (1.8%)
Dyspnoe
1 (9.1%)
1 (1.8%)
Transient hypotension
4 (36.5%)
5 (9.1%)
Urticaria
1 (9.1%)
1 (1.8%)
Putatively related to immunosuppression both by steroids and
immunoadsorption
Febrile infection
2 (18.2%)
2 (3.6%) Table 2 Moderate adverse events occurring during
immunoadsorption that were classified as being related
to vascular access, the immunoadsorption procedure or
immunosuppression Discussion PE is an efficient treatment in acute central nervous sys-
tem inflammatory demyelinating diseases [4], including
severe optic neuritis, motor impairment or ataxia [5,6]
after steroid-refractory relapses, successful in about 40%
to 50% of cases. Here, we report on the first prospective
investigation of tryptophan-IA in 11 relapsing MS
patients with optic neuritis refractory to corticosteroid
pulses in an open prospective study. Overall, eight of Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Page 6 of 10 A
B
C
sign. sign. sign. sign. sign. sign. sign. 2R (U/ml) A
sign. sign. sign. sign. (See figure on previous page.)
Figure 2 Clinical chemistry. Time-courses of (A) IgG; (B) C3c and
(C) sIL-2R. sIL-2R is shown in responders (blue) and non-responders
(red). Box plots: sign: significant (pairwise comparison to baseline). A eleven patients (72.7%) achieved a remission. One patient
gradually improved but deteriorated again along with the
development of jugular venous thrombosis, and two
patients did not respond at all. The response to IA seems
to be comparable to the best results achieved in PE series
[5,6] and to two very recent retrospective analyses of the
effect of IA in steroid-refractory MS cases [18,19]. As in
our own PE study [10], significant clinical improvement
was seen after the third extracorporeal treatment session
with a trend in favor of early IA initiation compared to
delayed IA initiation. Our treatment protocol was limited
to a total of five IA sessions. Additional experience
showed that increasing the number of apheresis sessions
did not correlate with further improvement of outcome
(data not shown). This observation is in accord with
results of IA in acute autoimmune neuropathies like
Guillain-Barré Syndrome [20]. B
sign. sign. sign. B Most side effects were typical of any apheresis proced-
ure using central venous lines as vascular access, but not
characteristic of IA. Compared with the safety data from
previously published PE studies [15,21], the incidence of
mild adverse events was higher in our study, and moder-
ate side effects were slightly more frequent. However,
moderate side effects were almost level with our own
study of neurological patients treated with PE [15]. Potential side effects of PE known to be related to the
substitution of human plasma products were completely
avoided [12]. Apart from clinical data, we analyzed possible therapeutic
effects of IA with the help of proteomic analyses. Several
relevant proteins, particularly fibrinogen and the immuno-
globulins, were monitored. Discussion The decrease of fibrinogen is
one limiting factor in the use of tryptophan-IA that makes
regular controls necessary. Our protocol with five IA
sessions on alternate days did not decrease fibrinogen to
critical levels. Moreover, immunoglobulin depletion along
with prior corticosteroid pulse therapy reflects a strong
immunosuppression, which makes close controls of clinical
and laboratory infection signs necessary. Previous investiga-
tions reported that the restoration of serum IgG levels until
day 5 after IA does not result from increased antibody
synthesis, but is probably related to changes of catabolism
and immunoglobulin backflow [7]. Interestingly enough, we
found a significant immunoglobulin increase beyond base-
line values until day 180±10 after the start of IA, suggest-
ing additional mechanisms other than backflow alone. C
sign. sIL2R (U/ml)
Figure 2 (See legend on next page.) C Several mechanisms of PE action in neuroimmunological
disorders have been described, such as a removal of Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Page 7 of 10 Page 7 of 10 pH 3-10
Molecular mass
A
FGB
IGLC1
SERPINA 1
KNG1
PLG
FGB
APCS
GSN
HAP1
IGHM
ITIP
FGA
ALB
ZNF706
CD5L
TTR
C1R
IGCK
IGHC1
CLU
C4A
APOA1
CFHR2
KNG1
IGHA1
~21.5 kDa MBP
~40 kDa sCD5L
B
sign. sign. sign. sign. sign. sign. sign. sign. Figure 3 Protein pattern of the eluate from the tryptophan column after the first immunoadsorption treatment in a single patient
analyzed by two-dimensional gel electrophoresis. (A) The protein spots were visualized by Flamingo fluorescence staining. (B) Time-course of
plasma myelin basic protein fragment (molecular mass approximately 21 kDa) and soluble CD5L levels determined by western blot from a single
patient with MS (upper panels) and densitometric analyses with baseline values set to 100% in the plasma of all 11 treated patients with MS
before, during and after IA (lower panels). sign: significant (pairwise comparison to baseline). H 3 10
Molecular mass
A
FGB
IGLC1
SERPINA 1
KNG1
PLG
FGB
APCS
GSN
HAP1
IGHM
ITIP
FGA
ALB
ZNF706
CD5L
TTR
C1R
IGCK
IGHC1
CLU
C4A
APOA1
CFHR2
KNG1
IGHA1 A pH 3-10 pH 3-10
~21.5 kDa MBP
~40 kDa sCD5L
B
sign. sign. sign. sign. sign. sign. sign. sign. Figure 3 Protein pattern of the eluate from the tryptophan column after the first immunoadsorption treatment in a single patient B ~21.5 kDa MBP
~40 kDa sCD5L sign. sign. sign. sign. Discussion sign. sign. sign. sign. Figure 3 Protein pattern of the eluate from the tryptophan column after the first immunoadsorption treatment in a single patient
analyzed by two-dimensional gel electrophoresis. (A) The protein spots were visualized by Flamingo fluorescence staining. (B) Time-course of
plasma myelin basic protein fragment (molecular mass approximately 21 kDa) and soluble CD5L levels determined by western blot from a single
patient with MS (upper panels) and densitometric analyses with baseline values set to 100% in the plasma of all 11 treated patients with MS
before, during and after IA (lower panels). sign: significant (pairwise comparison to baseline). Figure 3 Protein pattern of the eluate from the tryptophan column after the first immunoadsorption treatment in a single patient
analyzed by two-dimensional gel electrophoresis. (A) The protein spots were visualized by Flamingo fluorescence staining. (B) Time-course of
plasma myelin basic protein fragment (molecular mass approximately 21 kDa) and soluble CD5L levels determined by western blot from a single
patient with MS (upper panels) and densitometric analyses with baseline values set to 100% in the plasma of all 11 treated patients with MS
before, during and after IA (lower panels). sign: significant (pairwise comparison to baseline). Koziolek et al. Discussion Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Page 8 of 10 Page 8 of 10 Table 3 List of identified proteins from immunoadsorption column elutes pooled from five patients with multiple
sclerosis after the first immunoadsorption therapy
Protein name
Gene name
UniProt
SwissProt
Nominal mass
PMF-sco
Alpha-1-antitrypsin
SERPINA1
A1AT HUMAN
57.5
46878
94
Apolipoprotein A-I
APOA1
APOA1 HUMAN
56.6
30759
152
Apolipoprotein A-IV
APOA4
APOA4 HUMAN
56.6
45371
67
CD5 antigen-like
CD5L
CD5L HUMAN
56.8
39603
120
Cleavage stimulation factor, subunit, tau variant
CSTF2T
CSTFT HUMAN
57.2
64624
57
Clusterin
CLU
CLUS HUMAN
56.8
53031
160
Complement C1r
C1R
C1R HUMAN
57.5
81606
72
Complement factor I
CF1
CFA1 HUMAN
56.8
68072
86
Complement factor H
CFH
CFAH
57.1
143680
61
Complement factor H-related protein 2
CFHR2
FHR2 HUMAN
57.4
30631
60
Complement C4-A
C4A
CO4A
57.4
192650
213
Fibrin alpha C term fragment
gi 223057
NCBI nr
14443
85
Fibrinogen alpha chain
FGA
FIBA HUMAN
57.1
95656
103
Fibrinogen beta chain
FGB
FIBB HUMAN
57.1
56577
170
Fibrinogen gamma chain
FGG
FIBG HUMAN
57.2
52106
146
Gelsolin
GSN
GELS HUMAN
57.2
86043
95
Histidine-rich glycoprotein
HRG
HRG HUMAN
57.0
60510
152
Huntingtin-associated protein 1
HAP1
HAP1 HUMAN
57.5
76208
65
Kininogen 1
KNG1
KNG1 HUMAN
56.8
72996
96
Transthyretin
TTR
TTHY HUMAN
56.6
15991
89
Immunoglobulin alpha-1 chain C region
IGHA1
IGHA1 HUMAN
57.4
37631
130
Immunoglobulin lamda chain C region
IGLC1
LAC HUMAN
57.4
11230
218
Immunoglobulin mu chain C region
IGHM
IGHM HUMAN
57.4
49960
90
Immunoglobulin kappa constant protein
gi 49258112
25915
78
Immunoglobulin kappa chain, C region
IGCK
IGCK HUMAN
57.2
11773
287
Immunoglobulin kappa light chain variable region
gi 48475436
NCBI nr
21251
80
Immunglobulin kappa light chain VLJ region
gi 21669479
NCBI nr
29086
76
Inter-alpha-trypsin inhibitor
ITIP
ITIH4 HUMAN
57.5
103521
67
Monoclonal immunoglobulin M antibody light chain
gi 41388186
NCBI nr
26008
65
Microtubule-actin crosslinking factor 1
MACF
MACF1
57.0
623626
57
Mps one binder kinase activator-like2A
MOBKL2A
MOL2A
57.0
25676
55
Myelin basic protein
MBP
MBP HUMAN
56.6
33097
55
Malate dehydrogenase cytoplasmic
MDH1
MDHC HUMAN
56.6
36631
57
Plasminogen
PLG
PLMN HUMAN
56.6
93247
89
Serum albumin
ALB
ALBU HUMAN
57.5
71317
101
Serum amyloid P-component
APCS
SAMP HUMAN
57.5
25485
68
Small ubiquitin-related modifier
SUMO1
SUMO1 HUMAN
57.2
11607
59
Transmembrane and tetratricopeptide repeat-containing protein 1
TMTC1
TMTC1 HUMAN
57.2
88209
62
Zinc finger protein basonuclin-2
BNC2
BNC2 HUMAN
57.2
1236677
62
Zinc fincer protein 706
ZNF706
ZN706 HUMAN
57.4
8606
64
39 S ribosomal protein L13 mitochondrial
MRPL13
RM13 HUMAN
57.0
20736
58
Gene names, accession number and identification score are given. Gene names, accession number and identification score are given. PMF: peptide mass finger printing. Acknowledgment The authors thank the nursing staff from the medical intensive care unit
1022 as well as E. Brunst-Knoblich and A. Krüger (both Department of
Nephrology & Rheumatology, Georg-August-University Göttingen, Germany)
for excellent technical support. Received: 24 October 2011 Accepted: 28 February 2012
Published: 26 April 2012 sIL-2R, a marker of TH1 cell activation, is increased in
the serum of patients with relapsing MS [14]. We induced
a significant decrease of sIL-2R after IA in responders, but
not in non-responders. Decreased sIL-2R levels might
reflect the silencing of cellular autoimmune responses
effective only in responders. Competing interests This work was supported by a research grant of Diamed (Cologne, Germany)
to MK and BK. The authors declare lecture fees and/or travel funding from
Diamed (Cologne, Germany) to MK, BK and GAM. MK received research
funds from Novartis, Germany, and RK from Asahi Kasei Kuraray Medical,
Japan. Discussion According to our proteomics data, several proteins that are
possibly involved in MS pathogenesis are removed from
the plasma by IA, for example, transthyretin [24], serum
amyloid P [24], complement factors [24], clusterin [24],
gelsolin [24], kininogen-1 [24], MBP [25,26], CD5L [27]
and immunoglobulins [1,3,8]. We confirmed a decrease
of serum levels by IA in two of them, MBP and sCD5L. MBP-like material has been detected in several body fluids
including cerebral spinal fluid and the urine of patients
with MS [28]. Since MBP and other myelin proteins have
been shown to be encephalitogenic in animal models of
MS, they could drive the systemic autoimmune response
in patients with MS. Other investigations have demon-
strated the prevalence of MBP-specific memory B-cells in
the peripheral blood of relapsing patients with remitting
MS that might prime T-cells in lymphoid organs to migrate
into the central nervous system and to elicit IFN-γ secre-
tion [25]. These data were further corroborated with the
evidence of MBP-reactive T-cells among IL-2 expanded
lymphocytes in patients with MS [26]. Thus, removal
of MBP from the plasma by IA might interrupt these
autoimmune mechanisms, although more research in this
hypothesis is definitely needed. References
L
h 1. Lucchinetti CF, Bruck W, Parisi J, Scheithauer B, Rodriguez M,
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1 1Department of Nephrology and Rheumatology, Georg-August-University
Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. 2Department of Neurology, Georg-August-University Göttingen, Robert-Koch-
Strasse 40, D-37075 Göttingen, Germany. 3Department of Medical Statistics,
Georg-August-University Göttingen, Robert-Koch-Strasse 40, D-37075
Göttingen, Germany. 4Apheresis Research Institute, Stadtwaldguertel 77,
50935 Cologne, Germany. Abbreviations AE: Affected eye; CD5L: CD5 ligand; EDSS: Expanded Disability Status Scale;
IA: Immunoadsorption; IFN: Interferon; Ig: Immunoglobulin; IL-2R: Interleukin-2
receptor; ISS: Incapacity Status Scale; kDa: kiloDalton; MBP: Myelin basic protein;
MS: Multiple sclerosis; PBS: Phosphate-buffered saline; PE: Plasma exchange;
PMF: Peptide mass fingerprinting; sIL-2R: Soluble interleukin-2 receptor. 5.
Gold R, Hartung HP: Towards individualised multiple-sclerosis therapy.
Lancet Neurol 2005, 4:693–694. Discussion PMF: peptide mass finger printing. Table 3 List of identified proteins from immunoadsorption column elutes pooled from five patients with multiple
sclerosis after the first immunoadsorption therapy proteins from immunoadsorption column elutes pooled from five patients with multiple
munoadsorption therapy Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Koziolek et al. Journal of Neuroinflammation 2012, 9:80
http://www.jneuroinflammation.com/content/9/1/80 Page 9 of 10 Page 9 of 10 pathogenic autoantibodies, a redistribution of pathogens
from the extravascular to the intravascular compartment,
increased proliferation of immune cells, an enhanced
production of immunoglobulins, a promotion of suppres-
sor T-cell function, and a deviation of cytokine patterns
redressing a disturbed T-helper type 1 and T-helper type 2
balance [3]. Although IA has been termed specific, several
studies have demonstrated additional binding properties
of ligands other than immunoglobulins alone [22,23]. According to our proteomics data, several proteins that are
possibly involved in MS pathogenesis are removed from
the plasma by IA, for example, transthyretin [24], serum
amyloid P [24], complement factors [24], clusterin [24],
gelsolin [24], kininogen-1 [24], MBP [25,26], CD5L [27]
and immunoglobulins [1,3,8]. We confirmed a decrease
of serum levels by IA in two of them, MBP and sCD5L. MBP-like material has been detected in several body fluids
including cerebral spinal fluid and the urine of patients
with MS [28]. Since MBP and other myelin proteins have
been shown to be encephalitogenic in animal models of
MS, they could drive the systemic autoimmune response
in patients with MS. Other investigations have demon-
strated the prevalence of MBP-specific memory B-cells in
the peripheral blood of relapsing patients with remitting
MS that might prime T-cells in lymphoid organs to migrate
into the central nervous system and to elicit IFN-γ secre-
tion [25]. These data were further corroborated with the
evidence of MBP-reactive T-cells among IL-2 expanded
lymphocytes in patients with MS [26]. Thus, removal
of MBP from the plasma by IA might interrupt these
autoimmune mechanisms, although more research in this
hypothesis is definitely needed. pathogenic autoantibodies, a redistribution of pathogens
from the extravascular to the intravascular compartment,
increased proliferation of immune cells, an enhanced
production of immunoglobulins, a promotion of suppres-
sor T-cell function, and a deviation of cytokine patterns
redressing a disturbed T-helper type 1 and T-helper type 2
balance [3]. Although IA has been termed specific, several
studies have demonstrated additional binding properties
of ligands other than immunoglobulins alone [22,23]. Authors’ contributions MJK: design of the study, acquisition, analyses and interpretation of the data,
drafting the manuscript, final approval. DT: acquisition and analyses of the
data. MB: interpretation of the data, revising the manuscript. HD: analyses of
the data. KJ: analyses of the data, revising the manuscript. DF: acquisition of
the data, revising the manuscript. RK: design of the study, revising the
manuscript. GAM: interpretation of the data, revising the manuscript. BK:
design of the study, acquisition, analyses and interpretation of the data,
drafting the manuscript, final approval. All authors read and approved the
final manuscript. 6.
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Neuroinflammation 2012 9:80. doi:10.1186/1742-2094-9-80
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and complement from plasma, but also reduced levels
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http://www.jneuroinflammation.com/content/9/1/80 Submit your next manuscript to BioMed Central
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Submit your manuscript at
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and take full advantage of: Submit your next manuscript to BioMed Central
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Development and validation of an occurrence-based healthy dietary diversity (ORCHID) score easy to operationalise in dietary prevention interventions in older adults: a French study
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British Journal of Nutrition (2024), 131, 1053–1063
doi:10.1017/S0007114523002520
© The Author(s), 2023. Published by Cambridge University Press on behalf of The Nutrition Society. This is an Open Access article, distributed
under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use,
distribution and reproduction, provided the original article is properly cited. British Journal of Nutrition (2024), 131, 1053–1063 J
f
(
), 3 ,
3
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3
© The Author(s), 2023. Published by Cambridge University Press on behalf of The Nutrition Society. This is an Open Access article, distributed
under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use,
distribution and reproduction, provided the original article is properly cited. Development and validation of an occurrence-based healthy dietary diversity
(ORCHID) score easy to operationalise in dietary prevention interventions
in older adults: a French study Development and validation of an occurrence-based healthy dietary diversity
(ORCHID) score easy to operationalise in dietary prevention interventions
in older adults: a French study Anne-Fleur Jacquemot1,2*, Rosalie Prat3, Rozenn Gazan3, Christophe Dubois4, Nicole Darmon5,
Catherine Feart1 and Eric O. Verger5 1Université de Bordeaux, INSERM, BPH, UMR1219, F-33000 Bordeaux, France
2ORS PACA, Faculté des Sciences Médicales et Paramédicales, Observatoire Régional de la Santé Provence–Alpes–Côte d’Azur,
Marseille, 13385, France 1Université de Bordeaux, INSERM, BPH, UMR1219, F-33000 Bordeaux, France
2ORS PACA, Faculté des Sciences Médicales et Paramédicales, Observatoire Régional de la Santé Provence–Alpes–Côte d’Azur,
Marseille, 13385, France 4Trophis, 13170 Les Pennes Mirabeau, France MoISA, Université de Montpellier, CIRAD, CIHEAM-IAMM, INRAE, Institut Agro, IRD, Montpellier, France (Submitted 17 March 2023 – Final revision received 30 October 2023 – Accepted 30 October 2023 – First published online 8 November 2023) ubmitted 17 March 2023 – Final revision received 30 October 2023 – Accepted 30 October 2023 – First published online 8 Novembe Abstract Healthy diet and dietary diversity have been associated with healthy ageing. Several scores have been developed to assess dietary diversity or
healthy diets in epidemiological studies, but they are not adapted to be used in the context of preventive nutrition interventions. This study aimed
to develop an occurrence-based healthy dietary diversity (ORCHID) score easy to implement in the field and to validate it using dietary data from
older participants in the latest French food consumption survey (INCA3). The ORCHID score was made of several components representing the
consumption occurrences of twenty food groups, in line with French dietary guidelines. The score was then validated using dietary data (namely
three 24-h recalls and a food propensity questionnaire) from 696 participants aged 60 years and over in the INCA3 survey. Score validity was
evaluated by describing the association of the score with its components, as well as with energy intakes, solid energy density (SED) and the
probability of adequate nutrient intakes (assessed by the PANDiet). Higher scores were associated with more points in healthy components such
as ‘fruits’ and ‘vegetables’ (r = 0·51, and r = 0·54, respectively). The score was positively associated with the PANDiet (r = 0·43) and inversely
associated with SED (r = −0·37), while no significant association was found with energy intakes. The ORCHID score was validated as a good
proxy of the nutritional quality of French older adults’ diets. It could therefore be a useful tool for both public health research and nutrition
interventions. * Corresponding author: A.-F. Jacquemot, email anne-fleur.jacquemot@u-bordeaux.fr
Abbreviations: FPQ, food propensity questionnaire; MAR, mean adequacy ratio; SED, solid energy density. Principle of the ORCHID score To obtain a tool for measuring dietary diversity that could be
easily used in nutritional prevention interventions, we chose to
develop a score based on the occurrence of consumption of
certain food groups, which would not require information on the
weight of the food consumed. In addition, we aimed to develop a
score that reflected both the concept of healthy dietary diversity
(i.e. diet adequacy and moderation) in which the whole diet is
considered(18) and the 2021 French dietary guidelines for older
adults(19), with an emphasis on protein-rich foods due to the
importance of adequate protein intake for healthy ageing(19). The remaining seven ORCHID components are related to
food groups known to be infrequently consumed by the French
population (e.g. wholemeal products) and/or with a weekly
consumption recommendation (e.g. the recommendation to eat
fatty fish once a week). Those seven ORCHID food groups are
the following: ‘poultry’; ‘eggs’; ‘fatty fish’; ‘lean fish and shellfish’;
‘legumes’; ‘nuts’ and ‘wholemeal or semi-wholemeal products’. Keywords: Healthy dietary diversity: Older adults: Interventional research: Consumption occurrence ORCHID component = (Number of occurrence) * weight
If the French dietary guidelines advice limiting the consumption
of a food group, then a threshold rating is used: • If
number
of
occurrences
=<
threshold,
ORCHID
component = (Number of occurrences) • If
number
of
occurrences
>
threshold,
ORCHID
component = (Number of occurrences - threshold) * weight The thresholds and the weights were decided based on the
expert judgement. Then, the twenty ORCHID components are summed up to
obtain the final ORCHID score. Among the twenty ORCHID components, thirteen are
related to food groups known to be consumed on a daily
basis in the French population (e.g. starches and potatoes)
and/or that have a daily consumption recommendation in the
2021 French dietary guidelines(19) (e.g. the recommendation to
eat five portions of vegetables every day). Those thirteen
ORCHID food groups are the following: ‘meat excluding
poultry’; ‘cooked ham’; ‘deli meats excluding cooked ham’;
‘milk and fresh dairy products’; ‘cheese’; ‘refined starches and
potatoes’; ‘vegetables’; ‘fruits’; ‘oils’; ‘butter, margarine and
fresh cream’; ‘salted aperitif products’, ‘sweetened products’
and ‘sweetened beverages’. Keywords: Healthy dietary diversity: Older adults: Interventional research: Consumption occurrence Nevertheless, a
major dimension of diet quality, moderation – defined as the
avoidance or limited consumption of nutrients, foods or food
groups considered to be unhealthy when consumed in excess –
is rarely considered by existing indicators of dietary diversity(15). To overcome this pitfall, certain authors have proposed the
concept of healthy dietary diversity, that is, balanced diversity,
with a high consumption of a variety of healthful, nutrient-dense
foods and a low consumption of energy-dense, nutrient-poor
and less healthful foods based on dietary guideline of the
country(17,18). Some scores have been developed and validated
by evaluating their content and construct validity for the general
population(17,18). To calculate these healthy diversity scores as
for other type of healthy diet scores(5,6), precise data, including
weighted food portions, are needed. No measure of healthy
dietary diversity or healthy diet that can be easily operationalised
in intervention studies has been proposed, especially for older
adults. The aims of this study are to develop an occurrence-
based healthy dietary diversity (ORCHID) score easy to
implement in the field and to validate it using dietary data
from older participants in the latest French food consumption
survey (INCA3). intakes with nutrient-based recommendations. Nevertheless, a
major dimension of diet quality, moderation – defined as the
avoidance or limited consumption of nutrients, foods or food
groups considered to be unhealthy when consumed in excess –
is rarely considered by existing indicators of dietary diversity(15). To overcome this pitfall, certain authors have proposed the
concept of healthy dietary diversity, that is, balanced diversity,
with a high consumption of a variety of healthful, nutrient-dense
foods and a low consumption of energy-dense, nutrient-poor
and less healthful foods based on dietary guideline of the
country(17,18). Some scores have been developed and validated
by evaluating their content and construct validity for the general
population(17,18). To calculate these healthy diversity scores as
for other type of healthy diet scores(5,6), precise data, including
weighted food portions, are needed. No measure of healthy
dietary diversity or healthy diet that can be easily operationalised
in intervention studies has been proposed, especially for older
adults. The aims of this study are to develop an occurrence-
based healthy dietary diversity (ORCHID) score easy to
implement in the field and to validate it using dietary data
from older participants in the latest French food consumption
survey (INCA3). Keywords: Healthy dietary diversity: Older adults: Interventional research: Consumption occurrence undernutrition(7,8). Diversity is one sub-constructs of healthy
diet according to Seligman’s paradigm(9). Dietary diversity
(typically defined as the number of different foods or food
groups consumed over a given period) may help postpone the
onset of age-related chronic diseases. For instance, longitudinal
studies exploring links between dietary diversity and health for
older adults report that greater dietary diversity is associated
with a reduced risk of all-cause mortality(10,11), frailty(12) and
cognitive decline(13). Furthermore, dietary diversity is promoted
in most Western countries’ dietary guidelines for the general
population(14,15). Increased longevity, lower fertility rates and progress in
medicine and pharmacology are contributing to the ageing of
the world’s population. In France, the current share of the
population aged 65 years and over is approximately 20·5 %(1)
and is expected to steadily increase up to 30 % in 2050(2). With
this rise, age-related chronic illnesses increase proportion-
ately(3). Healthy dietary patterns throughout life may delay the
onset of late-life chronic diseases(4) and many healthy diet scores
exist(5,6) but not especially for older people. The ability to
maintain such patterns appears even more critical during
later life stages, when socio-demographic changes associated
with ageing (e.g. widowhood, clinical disorders, poor oral
health, etc.) may lead to a loss of appetite and the risk of Increased longevity, lower fertility rates and progress in
medicine and pharmacology are contributing to the ageing of
the world’s population. In France, the current share of the
population aged 65 years and over is approximately 20·5 %(1)
and is expected to steadily increase up to 30 % in 2050(2). With
this rise, age-related chronic illnesses increase proportion-
ately(3). Healthy dietary patterns throughout life may delay the
onset of late-life chronic diseases(4) and many healthy diet scores
exist(5,6) but not especially for older people. The ability to
maintain such patterns appears even more critical during
later life stages, when socio-demographic changes associated
with ageing (e.g. widowhood, clinical disorders, poor oral
health, etc.) may lead to a loss of appetite and the risk of Dietary diversity in the general population is associated with
nutritional quality(16), assessed by the adequacy of nutrient A.-F. Jacquemot et al. 1054 guidelines advice favouring the consumption of a food group,
then a positive rating is used: intakes with nutrient-based recommendations. Population sample and dietary data Essentially, the ORCHID score involves twenty ORCHID
components corresponding to twenty ORCHID food groups. To test the validity of the ORCHID, we made secondary analyses
on data from the Third French Individual and National Food
Consumption Survey 2014–2015 (INCA3) led by ANSES. INCA3 is a cross-sectional survey aimed at estimating the food
consumption and eating habits of individuals living in France. The study was carried out between February 2014 and
September 2015 among a representative sample of individuals
living in mainland France (excluding Corsica and overseas
French territories). A total of 3157 eighteen- to seventy-nine–
year-old adults participated in the study. Because the ORCHID score was created for older adults, an
emphasis on rich proteins food was made. To create the twenty
ORCHID food groups, some food groups rich in proteins from
2021 French dietary guidelines had been reshaped: the food
group ‘poultry-fish-eggs-meat’ has been modified into ‘fatty
fish’, ‘lean fish and shellfish’, ‘poultry’, ‘eggs’ and ‘meat excluding
poultry’; ‘deli meat’ has been modified into ‘cooked ham’ and
‘deli meat excluding cooked ham’ and ‘dairy products’ have
been modified into ‘cheese’ and ‘milk and fresh dairy product’. Moreover, because of the importance of pleasure and the risk of
energy malnutrition in older adults(20), energy-dense food
groups that the 2021 French dietary guidelines advise to avoid
excessive consumption (i.e. energy-dense food groups such as
‘sweetened products’ and ‘salted aperitif product’) have been
considered positively until a threshold decided with a group of
experts. Individuals were selected according to a three-stage cluster
sampling design (geographical units, households and individ-
uals), based on the 2011 annual national census, with
geographical stratification (region, size of urban area) to ensure
national representativeness. The study sample included participants 60 years of age and
over (n 1008). Older adults who responded to less than three
24-h dietary recalls or who failed to respond to the self-
administered
food
propensity
questionnaire
(FPQ)
were
excluded from the analyses, leading to a sample of 696 older
adults (293 men and 403 women). Each ORCHID component corresponds to the total number of
points calculated for each consumption occurrence of the
corresponding ORCHID food group. If the 2021 French dietary Occurrence-based dietary diversity score 1055 Participants received a home visit to take anthropometric
measures and explain how and when to complete the self-
questionnaire. Calculation of ORCHID score in INCA3 Evaluation of ORCHID score validity. To evaluate the content
and construct validity of the score, we applied a strategy
commonly used for diet quality scores(5,17,21) summarised by
Bland and Altam(22). Content validity is met if the score seems
complete (i.e. it does not lack any important component), and
construct validity is met if the score is associated with the same
variables than the object it measures, that is, diet quality. First,
content validity was evaluated by (i) examining the distribution
of the ORCHID score and its components throughout the sample
and (ii) examining the correlation between the ORCHID score
and its components. Examining the correlations of each of the
components with the total score is important to understand how
the score works and what it could imply for its interpretation in
various research applications(6). Second, the construct validity of
the ORCHID score was evaluated by (i) examining the
correlation between the score and daily consumption (in g/d)
of the twenty ORCHID food groups, based on the hypothesis that
participants with a higher ORCHID score would more likely
consume more fruits and vegetables and eat fewer products with
a high fat, salt and sugar content than participants with an
ORCHID lower score, (ii) examining the correlation between the
energy, total food and nutrient intakes, as well as diet quality
measures, based on the hypothesis that participants with a
higher score would likely have a more nutritionally adequate
diet and (iii) examining the relation with socio-demographic
and anthropometric variables known to correlate with diet
quality:
educational
level(23),
sex(5),
BMI(5,23,24),
monthly Calculation of the thirteen ORCHID components based on
data from the 24-h dietary recalls. Each food associated with
the 24-h dietary recalls was categorised into one of the thirteen
ORCHID food groups. Each food declaration was considered
only if its ORCHID food group’s daily consumption was
more than half of a standard portion (in g) as defined in the
French Nutrition and Health Survey (ENNS) and the French
‘Market Research Group for Collective Catering and Nutrition’
(GEMRCN)(21) (Table 1). For example, if less than 25 g of ‘cooked
ham’ per day is declared whereas the standard portion size is
50 g, then the ‘cooked ham’ consumption is not taken into
account. Food taken into account consisted of both simple foods and
complex dishes. The categorisation of simple foods into
ORCHID food groups was based on the existing INCA3 food
categorisation. Population sample and dietary data Dietary intakes were then assessed using a
series of three non-consecutive 24-h dietary recalls (two on
weekdays and one during the weekend) over the 3 weeks after
the home visit. During phone interviews conducted by
professional investigators, participants were asked to declare
all foods and beverages they had consumed the day prior to the
call. Every declaration was computerised using GloboDiet
(previously known as EPIC-Soft(17)) by professional inter-
viewers specifically trained in the methods and the software
used. Foods included both simple foods (e.g. apples, meat, etc.)
and complex dishes with multiple ingredients (e.g. apple pie,
lasagne, meat and vegetable stew, etc.). Portion sizes were
estimated by participants during the call, using validated
photographs from a picture book of food portion sizes and
household measures(16). The nutrient contents of the foods and
beverages cited by participants were extracted from the 2016
food composition database of the French Information Centre
on Food Quality(18). Participants were also asked to complete
the FPQ describing their usual consumption frequency of
approximately sixty foods or food groups over 12 months. FPQ
are qualitative FFQ that may be used alongside 24-h dietary
recalls to estimate long-term food intake. The FPQ was
completed either on paper or directly online. Daily nutritional
intakes were estimated (without taking into consideration
alcohol consumption and without excluding the consumption
of food supplements) for each individual over the three 24-h
dietary recalls. the quantity consumed of the ORCHID food group represent
more than half a standard portion. For each individual and each of the thirteen ORCHID food
groups, the ORCHID components are calculated from the total
number of consumption occurrences in three 24 recalls. The
details of the scorings are fully described in Table 1 and had been
decided based on expert judgements. Calculation of consumption occurrences based on data from
the food propensity questionnaire. For the remaining seven
ORCHID food groups, the associated ORCHID components are
based on data from the FPQ. To overcome missing occurrences
due to a lack of response in the FPQ, an imputation was
computed using the random forest method(22), taking into
consideration individual characteristics, INCA3, food group
quantities and FPQ frequencies with no missing values. To maintain consistency with weekly dietary recommendations,
the monthly consumption frequencies collected by the FPQ
were transformed into weekly frequencies. Population sample and dietary data For each individual and each of the seven ORCHID food
groups, the total number of consumption occurrences (based on
weekly frequencies) is used to calculate the ORCHID compo-
nents, using a positive, proportional rating system, as French
dietary guidelines advice favouring the consumption of foods in
these food groups (Table 1). An example of an ORCHID score’s individual calculation is
presented in online Supplementary Material 1. A toy data set created from the INCA3 data and the R code are
available as online Supplementary Materials 2, 3 and 4. Calculation of ORCHID score in INCA3 About 70 % of the foods were successfully
classified among the thirteen ORCHID food groups. The
remaining foods were not included in the ORCHID calculation
(mostly water, spices, herbs and sauces). Approximately 5 % of
the data collected over the three non-consecutive 24-h dietary
recalls concerned complex dishes. Complex dishes were disaggregated into ingredients using
recipes, and the main ingredients (i.e. those whose sum
represented half of the recipe) were identified. Only the main
ingredients were sorted into one of the ORCHID food group and
each main ingredient was considered as one occurrence only if calculation of
es
Scoring
2021 French dietary guidelines(19)
quency
OC‡ = number of occurrences*2
Eat at least once a day, choosing from poultry, fish, eggs or meat
ber of occurrences
24 h recalls
• If number of occurrences =< 1·5,
OC = number of occurrences
• If number of occurrences > 1·5,
OC = (number of occurrences – 1·5)*(-1)
Eat at least once a day, choosing from poultry, fish, eggs or meat
For high consumers of meat other than poultry, there is no nutritional
benefit in eating it systematically at every meal
ber of occurrences
24 h recalls
• If number of occurrences =< 1·5,
OC= number of occurrences
• If number of occurrences > 1·5,
OC= (number of occurrences – 1·5)*(-1)
Avoid consuming it every day and avoid excessive consumption
ber of occurrences
24 h recalls
• If number of occurrences =< 1·5,
OC = number of occurrences
• If number of occurrences > 1·5,
OC = (number of occurrence – 1·5)*(-2)
Avoid consuming it every day and avoid excessive consumption
quency
OC = number of occurrences
Eat at least once a day, choosing from poultry, fish, eggs or meat
quency
OC = number of occurrences*2
For large consumers of fish, vary the species and places of supply to
limit exposure to environmental contaminants
quency
OC = number of occurrences*2
For large consumers of fish, vary the species and places of supply to
limit exposure to environmental contaminants
ber of occurrences
24 h recalls
OC = number of occurrences
2–3 dairy products a day
ber of occurrences
24 h recalls
OC = number of occurrences
2–3 dairy products a day
quency
OC = number of occurrences*2
At least 2 times/week
quency
OC = number of occurrences*2
A handful per day
Method of calculation
of occurrences
Scoring
2021 French dietary guidelines(19)
NS. Calculation of ORCHID score in INCA3 Potatoes 150 g ENNS
Total number of
occurrences on
three 24 h recalls
OC = number of occurrences
To be eaten every day, with a preference for
wholemeal or slightly refined products over
refined products
S
Weekly frequency
OC = number of occurrences*2
To be eaten every day, with a preference for
wholemeal or slightly refined products over
refined products
BP-FAIBP
Total number of
occurrences on
three 24 h recalls
OC = number of occurrences*2
At least five portions of vegetables or fruits/d
anada
Total number of
occurrences on
three 24 h recalls
OC = number of occurrences*2
At least five portions of vegetables or fruits/d
Total number of oils on
three 24 h recalls
OC = ∑different type of oil þ
∑(different mixed oil)*2
Favour colza, nut and olive oil. Other added fat
can be consumed every day in small
quantities
CN
Total number of
occurrences on
three 24 h recalls
• If number of occurrences =<3,
OC= number of occurrences
• If number of occurrences > 3,
OC = (number of occurrences
- 3)*(-2)
Favour colza, nut and olive oil. Other added fat
can be consumed every day in small
quantities
056
A.-F. Jacquemot et al. rg/10.1017/S0007114523002520 Published online by Cambridge University Press Occurrence-based dietary diversity score 1057 Table 1. (Continued )
ORCHID food group
Standard portion size
Method of calculation
of occurrences
Scoring
2021 French dietary guidelines(19)
Salted aperitif
products
(30 g) GEMRCN
Total number of
occurrences on
three 24 h recalls
• If number of occurrences =<3,
OC= number of occurrences
• If number of occurrences > 3,
OC = (number of occurrences
– 3)*(-2)
Avoid excessive salt consumption
Sweetened products
(including sugar)
Sugar (5 g), dairy desserts (100 g) GEMRCN, biscuits (30 g)
ENNS, cereals for breakfast (30 g) ENNS, candies, chocolate,
etc. Calculation of ORCHID score in INCA3 (30 g) GEMRCN
Total number of
occurrences on
three 24 h recalls
• If number of occurrences =<9,
OC= number of occurrences
• If number of occurrences > 9,
OC = (number of occurrences
– 9)*(-2)
Avoid excessive consumption of sweet products
Sweetened drinks
(including juice)
(200 g) GEMRCN
Total number of
occurrences on
three 24 h recalls
• If number of occurrences =<3,
OC = number of occurrences
• If number of occurrences > 3,
OC = (number of occurrences
– 3)*(-2)
Avoid excessive consumption of sweet products household income(25), number of persons living in the house-
hold(25), smoking status(26) and physical activity level(5). Retained diet quality measures consisted of the solid energy
density (SED), a proxy of low nutritional quality, the mean
adequacy ratio (MAR), a measure of nutritional adequacy and
the PANDiet, a measure of overall nutritional quality, on
individual diets. These diet quality measures (SED, MAR,
PANDiet) were estimated from the daily nutritional intakes
calculated in INCA3. The SED (kcal/100 g) was defined as the
ratio of total energy over the total weight consumed, including
solid foods only. A low SED has been associated with good
overall nutritional quality(27). The MAR was calculated for each
individual as the mean percentage of sex- and age-specific
French RDA for twenty-two key nutrients. MAR values ranged
from 0 % to 100 %, and the higher the MAR, the better the
nutritional quality of the diet(28). The PANDiet was calculated for
each individual based on probabilities of adequacy for twenty-
seven nutrients grouped into two sub-scores: the adequacy sub-
score and the moderation sub-score. The adequacy sub-score
assesses the probability of adequacy for food items whose usual
intake should be above a reference value, whereas the
moderation sub-score evaluates the probability of adequacy
for food items whose usual intake should not exceed a reference
value. PANDiet scores ranged from 0 to 100, where 100
represents 100 % of the usual intake adequacy for the twenty-
seven nutrients(21). Anthropometric and socio-demographic characteristics
according to the ORCHID score quartile The ORCHID score was significantly associated with educational
level, smoking status, BMI and physical activity level. In the
highest ORCHID score quartile, a larger proportion of individ-
uals had a higher educational level and physical activity level
compared with the other quartiles. The prevalence of current
smokers was significantly higher in Q1 compared with Q4 of the
ORCHID score. Sex, total monthly household income and the
number of persons living in the household were not significantly
associated with the ORCHID score (Table 4). No significant ORCHID component differences were found
between men and women with the exception of ‘refined
starches and potatoes’ (men had þ2·25 points), ‘cheese’
(þ0·61 point), ‘poultry’, ‘fatty fish’ and ‘oils’. The differences
between men and women for the three last ORCHID
components are less than 0·5 points (online Supplementary
Table 2). ORCHID score description and correlation with ORCHID
components The score was normally distributed. The average score was
53·5 (SD 17·8) and ranged from −36 to 115. The average score
was not significantly different between women and men
(53·7 (SD 16·5) and 52·6 (SD 19·1), respectively). The range of
values was smaller for men (-13·5 to 99) as compared with
women (-40 to 112). Diet quality measures and nutrients are different depending
on sex with the exception of the PANDiet and proteins when
adjusted by the ORCHID score quartile. Men have lower
moderation PANDiet sub-score than women (-10 %), higher
scores in other diet quality measures than women and higher
nutrient intakes than women (online Supplementary Table 4). Among the whole sample, three of the twenty components
were positively correlated with the score with a high strength of
correlation (online Supplementary Table 1), that is, the
components ‘fruits’, ‘vegetables’ and ‘wholemeal or semi-
wholemeal products’. Six components were positively corre-
lated with the score with a moderate strength of correlation. The
other components were either not correlated with the score or
the correlation was of low strength. Discussion Among the whole sample, the intakes of ‘fruits’, ‘vegetables’ and
‘wholemeal or semi-wholemeal products’ were positively
correlated with the score with a moderate strength of correlation:
for ‘fruits’, Q4 = 324·1 (296·6, 351·7) and Q1 = 169·3 (132·0,
206·6); for ‘vegetables’, Q4 = 214·4 (187·3, 241·5) and Q1 = 110·8
(96·8, 124·7) and for ‘wholemeal or semi-wholemeal Products’,
Q4 = 30·7 (23·2, 38·1) and Q1 = 19·4 (11·2, 27·6) (Table 2). The
intakes of ‘nuts’, ‘lean fish and shellfish’, ‘milk and fresh dairy
products’ and ‘oils’ were positively correlated with the score with
a low strength of correlation. This study describes an innovative and pragmatic healthy dietary
diversity score that is easy to implement in intervention studies
and adapted to the challenges of healthy ageing. Based on a
sample of French individuals aged 60 years of age and over, we
constructed an ORCHID score whose higher score was
associated with higher consumption of the most healthful food
groups and lower consumption of less healthful food groups. Furthermore, a higher ORCHID score was associated with a
more nutritionally adequate diet, independent from energy
intake. Construct validity was confirmed by expected relation-
ships between the ORCHID score and socio-demographic
characteristics: higher education, higher physical activity level,
lower BMI and non-smoking status are significantly associated
with higher ORCHID scores. The intakes of ‘refined starches and potatoes’, ‘meat
excluding poultry’, ‘sweetened products’ and ‘butter, margarine
and fresh cream’ were negatively correlated with the score with a
low strength of correlation. The score was not significantly
associated with the consumption of other ORCHID food groups
such as ‘legumes’. Correlations between nutrient intakes, diet quality
measures and the ORCHID score Correlations between nutrient intakes, diet quality
measures and the ORCHID score Correlations between nutrient intakes, diet quality
measures and the ORCHID score Total food intake was significantly and positively associated with
the ORCHID score, but energy intake was not. The ORCHID
score was observed to be moderately and positively associated
with fibres, proteins and good diet quality measures (PANDiet,
adequacy sub-score and MAR). A negative association was
found between the ORCHID score and SED, but no significant
associations were found in terms of the moderation sub-score of
the PANDiet or nutrients that should be limited, namely sugars,
saturated fats and Na (Table 3). Statistical analysis ORCHID components, average daily ORCHID food group intake
(in g/d), energy intake (kcal/d), total food intake (g/d), SED,
MAR, PANDiet and nutrient intake were first presented across
the ORCHID score quartiles, calculated for the whole sample
and then stratified based on sex, given known dietary sex
differences. The means of the score quartiles and their CI were
calculated for each variable. Because ORCHID food group intake and ORCHID compo-
nents did not follow a normal distribution, non-parametric tests
were used for these variables in analyses. Associations between
the score and ORCHID components and the score and the
ORCHID food group intake were assessed using Spearman
correlations. Associations between the score and total food
intake, energy intake, nutrient intake, SED, MAR and PANDiet
were assessed using Pearson correlations. Wilcoxon tests were
used to assess the effect of sex on ORCHID components and
ORCHID food group intake, and the relative difference between
the men and women means was calculated. Energy intake,
nutrient intake, SED, MAR and PANDiet differences between
sexes were assessed using analyses of variance adjusted on the
score quartiles, and the absolute difference between men and
women means was calculated. *The French Nutrition and Health Survey (ENNS). †French market research group for collective catering and nutrition (GEMRCN). ‡ORCHID
t In terms of correlations, we describe them as being of the
following strengths: high strength (|r|>0·5), moderate strength
(0·5>=|r|>0·2) or low strength (|r|<0·2). Socio-demographic
characteristics
were
described
and
statistically compared between the score quartiles using the
χ2test. 1058 A.-F. Jacquemot et al. (the
difference
is
negligible)
(online
Supplementary
Table 3). All means and SD are presented as follows: mean ± SD, and
95 % confidence intervals are presented as follows: (,). All
analyses accounted for the complex INCA3 sampling frame
design (R package (survey)(29) except for the Spearman
correlations and all analyses were performed using R, version
R-4.1.2(30). ORCHID score validity (Mean values and standard deviations; 95 % confidence intervals)
All
Q1
Q2
Q3
Q4
Indicators
Mean
SD
Cor-Pearson
p-value Pearson
Mean
95 % CI
Mean
95 % CI
Mean
95 % CI
Mean
95 % CI
PANDiet
64·5
6·0
0·43
P < 0·001
61·2
60·1, 62·3
63·4
62·3, 64·4
66·0
64·8, 67·1
67·3
66·5, 68·1
SED (kcal/100 g)
141·4
33·8
-0·37
P < 0·001
158·0
149·3, 166·6
145·4
138·9, 151·8
134·0
127·6, 140·3
128·4
123,0 133·8
Fibres (g/d)
20·2
7·3
0·33
P < 0·001
17·4
15·9, 18·9
18·9
17·7, 20·0
21·1
18·8, 23·4
23·6
22·4, 24·8
Adequacy sub-score
66·3
16·1
0·31
P < 0·001
59·8
55·8, 63·9
63·9
60·6, 67·1
68·2
64·3, 72·1
73·2
71·0, 75·5
MAR (% adequacy)
84·6
10·7
0·30
P < 0·001
79·9
76·9, 82·9
83·4
81·3, 85·6
86·2
84·0, 88·3
89·1
87·8, 90·4
Total food intakes (g/d)
2698·4
809·5
0·18
P < 0·001
2534·5
2353·9, 2715·2
2606·8
2441·6, 2772·0
2754·4
2561·0, 2947·8
2900·1
2774·9, 3025·3
Proteins (g/kg/d)
1·1
0·4
0·12
P < 0·001
1·0
0·9, 1·1
1·1
1·0, 1·2
1·1
1·1, 1·2
1·2
1·1, 1·2
Na (mg/d)
3231·3
1418·0
0·07
0·06
3019·4
2711·3, 3327·5
3114·1
2820·7, 3407·6
3428·0
2997·3, 3858·6
3369·5
3142·3, 3596·6
Saturated fat (% energy)
29·9
13·7
-0·06
0·13
30·6
27·0, 34·1
29·4
26·5, 32·3
29·7
26·1, 33·3
30·0
27·8, 32·1
Sugars (g/d)
83·4
37·3
0·02
0·72
81·5
71·2, 91·9
81·4
74·8, 88·1
77·8
70·9, 84·7
92·6
87·1, 98·1
Energy intake (kcal/d)
1878·7
660·7
0·02
0·62
1846·8
1679·6, 2014·0
1837·2
1718·1, 1956·2
1893·3
1685·5, 2101·0
1938·8
1841·3, 2036·4
Moderation sub-score
62·7
11·9
0·02
0·67
62·7
59·7, 65·6
62·9
60·2, 65·5
63·8
61·0, 66·5
61·3
59·5, 63·2 measures, with higher ORCHID, PANDiet(21) and PNNS-GS2(5)
(index based solely on French recommendations) scores all
being correlated with higher fruit and vegetable intakes and
lower processed meat intake. Our results were also consistent
with a study validating the US Healthy Food Diversity Index,
which is based on the concept of healthy diversity(17). As for Chaltiel et al.(5), certain ORCHID food groups
(‘legumes’ and ‘nuts’) in our data were scarcely consumed,
regardless of the diversity score. This explains why no
associations between these ORCHID food groups and the
ORCHID score were detected. In any case, incentives to increase
‘legume’ and ‘nut’ consumption would no doubt be beneficial to
all older adults. ORCHID score validity The effect of sex was statistically significant for the intake of
‘refined starches and potatoes’ (men consume þ50 g/d than
women),
‘wholemeal
or
semi-wholemeal
products’(men
consume −1·8 g/d than women), ‘legumes’ (men consume
−1·83 g/d than women), ‘cheese’ (þ6·69 g/d), ‘butter, margarine
and fresh cream’ (-1·53 g/d) and ‘lean fish and shellfish’ As no gold standard exists in nutrition, we chose to compare the
ORCHID score to French scores, although neither is specific to
older adults. Associations between the ORCHID score and
ORCHID food group intakes went in the same direction as those
reported in previous French studies validating nutritional 3
95 % CI
4·3, 303
7·8, 186
1·6, 25·
2·6, 17·
1·0, 2·7
7·7, 207
4·4, 8·3
0·3, 35·
4·4, 91·
1·3, 74·
8·2, 305
9·0, 37·4
6·4, 14·
9·7, 18·
9·9, 23·
8·3, 14·
5 1 104 Q1
Q2
Q3
e Spearman*
Mean
95 % CI
Mean
95 % CI
Mean
9
< 0·001
169·3
132·0, 206·6
221·5
176·2, 266·7
268·9
234
< 0·001
110·8
96·8, 124·7
156·9
128·9, 184·9
167·0
147
< 0·001
19·4
11·2, 27·6
14·0
8·4, 19·7
18·5
11
< 0·001
22·6
18·4, 25·9
16·8
13·0, 20·6
15·2
12
< 0·001
3·2
0·5, 5·8
1·7
0·7, 2·7
1·9
1
< 0·001
127·4
101·8, 153·0
174·7
139·3, 210·1
167·8
127
< 0·001
5·3
4·2, 6·4
8·3
6·3, 10·2
6·3
4
0·007
18·4
12·2, 24·6
19·7
13·3, 26·1
28·1
20
0·006
104·6
85·1, 124·2
89·8
76·7, 102·9
78·1
64
0·02
55·1
44·8, 65·5
60·7
49·5, 71·9
52·6
31
0·04
225·8
196·6, 255·1
226·8
199·8, 253·8
236·9
168
0·06
26·8
19·6, 34·0
25·4
20·9, 30·0
28·2
19
0·23
9·7
6·0, 13·4
9·9
5·6, 14·3
10·5
6
0·22
18·7
13·3, 24·0
16·7
11·3, 22·2
14·2
9
0·16
26·1
18·2, 34·0
20·5
10·7, 30·3
16·6
9
0·26
5·8
3·4, 8·3
11·4
7·4, 15·3
11·2
8
0·25
120·8
77·2, 164·4
81·7
49·2, 114·2
69·5
35
0·42
1·8
1·0, 2·7
2·9
1·2, 4·7
2·4
0
0·85
7·7
2·9, 12·5
7·3
3·6, 11·1
4·1
1
0·97
35·8
29·3, 42·3
39·4
33·2, 45·5
41·2
35
Cambridge University Press A.-F. Jacquemot et al. ORCHID score validity Associations between the ORCHID score and nutrient
intakes were also generally the same as those reported in
previous studies validating diet quality or diet diversity
measures using data from French(5), USA(17), German(18) or
Danish(31) surveys. Higher dietary diversity scores were
associated with more favourable nutrient intakes. Regarding
nutrients to limit, the German Healthy Food Diversity index was
negatively associated with Na and sugars, but the associations
were near zero(18). The ORCHID score was not significantly
associated with Na and sugars. The ORCHID score was not
correlated with energy intake: a desirable attribute for a healthy
diversity index given that higher diversity may solely reflect a
higher energy intake(32). We also found that higher ORCHID
scores were correlated with diet quality measures including the
SED (a proxy of low nutritional quality), the MAR (a measure of
nutritional adequacy) and the PANDiet (a measure of overall
nutritional quality). Taken together, these results underline the
ORCHID score’s ability to reflect the nutritional quality of
the diet. Associations between the ORCHID score and socio-
demographic and anthropometric characteristics, including
educational level, physical activity level, BMI and smoking
status, were similar to those reported for other scores in studies
based on French dietary surveys(5). On the other hand, certain
socio-demographic characteristics that are typically linked to
better dietary habits for older adults (i.e. being female as
opposed to male, a higher total monthly household income and
living with someone) were not associated with the ORCHID
score(25,33). Concerning sex, the PANDiet in the French
population is not associated with sex(21) and the ORCHID is
well associated with the PANDiet (see Table 4). Moreover, men
consume more energy, Na, sugars and fat but more fibres than
women. Men consume more but in an unhealthy way
compared with women, so they have a higher adequacy score
but a lower moderation score (online Supplementary Table 4). This explains why they have the same PANDiet and ORCHID
scores. Table 3. Diet quality indicators by whole and by quartile of ORCHID score (n 696)
(Mean values and standard deviations; 95 % confidence intervals) A healthy dietary diversity score Unlike most diversity scores, which are based on the number of
food categories or foods consumed(15), the ORCHID score is
favourable to health given that (1) it relies on French nutrition
recommendations for older adults(19) and (2) it allows moder-
ation dimension of the diet to be taken into account as shown Occurrence-based dietary diversity score 1061 Table 4. Description of the 696 individuals of the sample by quartile of ORCHID score
All
Q1
Q2
Q3
Q4
P value of χ2 test
%
Number of individuals (60 years old and over)
696
168
162
160
206
Sex
0·89
Man
42·10
49·07
36·42
38·13
44·13
Woman
57·90
50·93
63·58
61·88
55·87
Level of study
0·02
Primary þ middle school
51·72
57·14
64·20
51·88
38·03
High school
17·39
17·39
9·88
17·50
23·00
Bachelor
17·82
14·91
14·81
16·25
23·47
Master and over
13·07
10·56
11·11
14·38
15·49
BMI (kg/m2)
27·25
27·69
27·16
27·66
26·53
Body condition
0·02
Thinness
2·44
3·11
0·62
3·13
2·82
Normal
36·93
31·68
37·04
33·75
43·19
Overweight
40·23
39·13
40·12
45·00
37·56
Obesity
20·11
26·09
22·22
16·88
16·43
NA
0·29
0·00
0·00
1·25
0·00
Total monthly household income per consumption unit (CU) in four classes
0·31
<900 €/month/CU
9·77
13·04
13·58
6·25
7·04
900–1340 €/month/CU
18·10
21·12
22·22
14·38
15·49
1340–1850 €/month/CU
21·70
21·74
17·90
25·63
21·60
≥1850 €/month/CU
41·67
35·40
36·42
44·38
48·36
NA
8·76
8·70
9·88
9·38
7·51
Number of persons living in the household
0·18
1
33·91
37·89
32·72
38·13
28·64
2
60·92
57·76
60·49
56·25
67·14
3
4·60
3·73
4·94
5·63
4·23
Smoking
0·02
Yes, daily
9·63
13·66
8·64
10·00
7·04
Yes, sometimes
1·58
1·86
0·62
2·50
1·41
No, but I have ever smoked
36·93
37·89
38·27
32·50
38·50
No, never smoked
50·86
44·72
51·85
53·75
52·58
NA
1·01
1·86
0·62
1·25
0·47
Physical activity
0·05
Sedentary
31·18
40·37
30·86
31·25
24·41
Active
58·62
52·17
58·64
60·63
61·97
Very active
6·18
4·97
7·41
4·38
7·51
NA
4·02
2·48
3·09
3·75
6·10 Table 4. Description of the 696 individuals of the sample by quartile of ORCHID score by its negative correlation with the SED and its independence
from energy. These results are in line with the healthy diversity
scores developed by Drescher and Vadiveloo(17,18). limited consumption of energy-dense products often considered
pleasant to consume(20). Strengths and limitations As opposed to other healthy dietary diversity scores requiring
heavy data like portion weights(14,15), the ORCHID score is based
on consumption occurrences that can be easily reported,
facilitating its implementation in public health intervention
studies. For example, the Diversity ALAPAGE Score (an
adaptation of the ORCHID score) was used in an intervention
that aimed to increase the dietary diversity of older adults(34). First, this study uses INCA3 data. Collected between 2014 and
2015, it is the most recent and comprehensive dietary data
available in France. Food consumption patterns do not change
rapidly over time(35). Furthermore, the INCA3 data were of
particular relevance for our study, as it provided us with a sample
of adults aged 60 years and over at the national level. However, it
should be noted that the recipes used to disaggregate complex
dishes into ingredients were either standard or those most
popular on the internet, meaning they may or may not
correspond to the real consumption of study participants. The
recipes
were
reduced
only
to
their
main
ingredients. Nevertheless, the consumption of complex dishes was quite
low among participants 60 years of age and over. Also, the FPQ is
self-reported. Given that the population in question is of A healthy dietary diversity score The ORCHID score fills a gap by
offering a healthy diversity score adapted to older adults and
easy to collect in nutrition prevention interventions. Acknowledgements 12. Wang XM, Zhong WF, Li ZH, et al. (2022) Dietary diversity and
frailty among older Chinese people: evidence from the Chinese
longitudinal healthy longevity study. Am J Clin Nutr 117,
383–391. AFJ would like to thank the ORS PACA and LEHA statistician
teams and members of MS-nutrition for their advice. This research received no specific grant from any funding
agency, commercial or not-for-profit sectors. 13. Otsuka R, Nishita Y, Tange C, et al. (2017) Dietary diversity
decreases the risk of cognitive decline among Japanese older
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designed the ORCHID study. R. P. and A. F. J. performed the
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English
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Effects of Stress on Phase Transformations in Grinding by FE Modeling and Experimental Approaches
|
Materials
| 2,019
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cc-by
| 11,177
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Received: 13 June 2019; Accepted: 16 July 2019; Published: 22 July 2019 Received: 13 June 2019; Accepted: 16 July 2019; Published: 22 July 2019 Abstract: In the grinding process, the materials within the surface layer may undergo phase
transformation and finally form a strengthened layer. It is of great significance to model the phase
transformation and predict the characteristics of the strengthened layer accurately. The phase
transformations occur under the varying temperature and high stress–strain in grinding, so the
effects of stress on the transformations are inescapable. This paper focuses on revealing the effects of
stress on phase transformations in grinding. For this purpose, a thermal–mechanical–metallurgical
direct coupling finite element (FE) model of grinding was established in Abaqus. The coupling
interactions such as the latent heat, the volume change strain caused by phase transformation, and
the stress-induced phase transformation were considered in the modeling procedure. Grinding
experiments were carried out and proved the model could accurately predict the microstructure
distribution and thickness of the strengthened layer. Further, the evolution of the phase transformation
was discussed, and the effects of stress on the transformations were revealed. Keywords: grinding; phase transformation; strengthened layer; stress-induced phase transformation;
thermal–mechanical–metallurgical coupling; material processing; phase evolution Materials 2019, 12, 2327; doi:10.3390/ma12142327 materials materials materials Article
Effects of Stress on Phase Transformations in
Grinding by FE Modeling and
Experimental Approaches
Shichao Xiu, Yansheng Deng * and Xiangna Kong
Mechanical Engineering & Automation, Northeastern University, Shenyang 110819, China
* Correspondence: dysstudy@163.com Shichao Xiu, Yansheng Deng * and Xiangna Kong
Mechanical Engineering & Automation, Northeastern University, Shenyang 110819, China
* Correspondence: dysstudy@163.com Received: 13 June 2019; Accepted: 16 July 2019; Published: 22 July 2019 1. Introduction Grinding is a widely used machining process in engineering because it can create a precise
surface and a surface layer with good surface integrity [1]. In the process, there is a large amount
of heat transfer into the workpiece, so the temperature of the surface layer rises sharply and a great
temperature gradient forms within the layer. The grinding heat is the main reason for the phase
transformation and the formation of the strengthened layer. The grinding heat is usually considered as
a negative factor, and various cooling techniques are used to cool the workpiece. In 1994, German
scholars proposed a new technology called grinding hardening, in which the grinding heat is directly
used to quench the surface material [2]. In fact, whether in traditional grinding or grinding hardening,
surface materials may undergo phase transformation because of the excessive temperature, so studying
the phase transformation in grinding has universal significance. Some theoretical and experimental studies about phase transformation and strengthened layer in
grinding have been carried out. Brinksmeier and Brokhoff[2] first found the microstructure of the
surface layer after grinding could be divided into two layers. The nearest surface is a white etching
layer a few microns thick, and the next layer is the strengthened layer comprised of martensite and
carbides. Furthermore, they pointed out the materials undergo short austenitization and self-quenching. Zarudi and Zhang [3,4] took the quenched steel as the experimental material and carried out grinding
hardening experiments. They observed the strengthened layer by a transmission electron microscope
(TEM) and found there was no ferrite in the depth range of 0–0.9 mm, but when the depth was greater Materials 2019, 12, 2327; doi:10.3390/ma12142327 www.mdpi.com/journal/materials www.mdpi.com/journal/materials 2 of 18 Materials 2019, 12, 2327 than 0.3 mm, bainite appeared. Then, they thought the microstructure composition of the strengthened
layer was complex, the nearest surface layer was the refined martensite, and more and more bainite
appeared with increasing depth. Nguyen and Zhang found the martensite was finer after cooling with
liquid nitrogen in grinding hardening by observing the microstructure of the surface layer. This is
because the temperature is lower than the room temperature after the usage of the liquid nitrogen,
and the martensite transformation occurs below the room temperature [5]. Mao et al. [6] studied
the formation of the white layer in grinding AISI 52100 steel. 1. Introduction Furthermore, they found the phase
transformation, retained austenite, and white layer could occur below the nominal temperature of
the phase transformation. They thought the plastic deformation caused by the mechanical effect
could affect the formation of the white layer. Liu et al. [7] studied the effects of grinding depth and
initial microstructure of the workpiece on the depth, microstructure, and micro-hardness distribution
of the strengthened layer in grinding AISI 1060 steel. The results show the strengthened layer can
divide into the complete hardening region and the transition region. The complete hardening region
consists of martensite, retained austenite, and little cementite. Furthermore, the grinding depth and
initial microstructure have no obvious effect on the martensite morphology and micro-hardness in the
complete hardening region. However, the depth of the strengthened layer increases with the grinding
depth or uniformity of the initial microstructure increase. Simulation is economical, efficient, and beneficial to understanding the process mechanism. The simulation of phase transformation is widely used in heat treatment, welding, and other
research [8,9]. Since the phase transformation mainly depends on temperature, it is necessary
to simulate the temperature. Based on finite element (FE) thermal analysis, Zhang and Mahdi [10]
studied the effects of feeding speed, heat flux, material thermal properties, and convective ability
of cooling medium on phase transformation in grinding. Brosse et al. [11] used the FE software
Sysweld in which the Leblond’s transformation model is adopted to calculate the phase transformation
in grinding. Ding et al. [12] theoretically analyzed the phase transformation of martensitic steel in
grinding. Moreover, they considered that temperature is not the only factor affecting the phase
transformation in grinding, and the strain rate also plays an important role. Therefore, they established
a phase transformation model incorporating the effect of strain rate based on the experimental data and
regression analysis. Deng et al. proposed a microstructure evolution model based on cellular automata
(CA), and then in detail discussed the austenite transformation in grinding [13]. Salonitis [14] first
proposed a hybrid CA–FE model for simulating the phase transformation in grinding hardening, then
discussed the grain size and the phase composition. So far, most of the studies of the phase transformation in grinding are experimental and theoretical. 2.1. Thermal–Mechanical Model of Grinding In grinding, the grinding force and heat are the causes of deformation, material removal, and
phase transformation. The heat conduction satisfies the following differential equation: ρCp
∂T
∂t −∂
∂x
k∂T
∂x
! −∂
∂y
k∂T
∂y
! −∂
∂z
k∂T
∂z
! −
. Q = 0
(1) (1) where ρ, Cp, and k are the density, the specific heat, and the heat conductivity of the workpiece,
respectively. Table 1 shows the temperature-dependent ρ, Cp, and k used in the present work. . Q is
the heat flux of the inner heat source. Considering the release or absorption of heat due to phase
transformation,
. Q can be estimated by [15]: . Q =
X
Hx
dfx
dt
(2) (2) where Hx is the changing latent heat per unit transformed volume, and dfx is the increment of
transformed volume during the time increment dt. Subscript x in Hx and dfx refers to the transformation
type. Table 2 shows Hx used in the present work [16]. The subroutine *HETVAL provided in Abaqus is
used and written to calculate the latent heat of phase transformation. Table 1. Thermal properties of AISI 1045 steel. Table 1. Thermal properties of AISI 1045 steel. Table 1. Thermal properties of AISI 1045 steel. Temp (◦C)
ρ (kg/m3)
Cp (J/kg ◦C)
k (W/m ◦C)
20
7850
460
49.77
100
7830
480
46.76
200
7800
498
43.24
300
7770
524
40.29
400
7740
524
37.87
500
7700
615
35.96
600
7680
690
33.18
700
7672
720
30.52
800
7660
682
27.96
900
7651
637
25.92
1000
7649
602
24.02
Table 2. Latent heat changes in phase transformation. Transformation Type
Hx (J/m3)
Ferrite to austenite
595 × 106
Pearlite to austenite
526 × 106
Austenite to martensite
640 × 106 Table 1. Thermal properties of AISI 1045 steel. Temp (◦C)
ρ (kg/m3)
Cp (J/kg ◦C)
k (W/m ◦C)
20
7850
460
49.77
100
7830
480
46.76
200
7800
498
43.24
300
7770
524
40.29
400
7740
524
37.87
500
7700
615
35.96
600
7680
690
33.18
700
7672
720
30.52
800
7660
682
27.96
900
7651
637
25.92
1000
7649
602
24.02 Table 2. Latent heat changes in phase transformation. Table 2. Latent heat changes in phase transformation. Table 2. Latent heat changes in phase transformation. 1. Introduction The existing models and simulations also need to improve due to: (1) the temperature, stress–strain,
and phase transformation are directly coupled in grinding, but most studies ignore or simplify
the coupling relations; and (2) the surface materials undergo austenite transformation during the
heating period, and may undergo various phase transformations during the cooling period, but these
transformations are not always fully accounted for. This paper focuses on revealing the effects of stress
on the phase transformations in grinding. The following works were carried out: first, the thermal
and mechanical loads, initial and boundary, in the grinding were analyzed or modeled. The phase
transformation incorporating the effects of stress in grinding was analyzed theoretically and modeled
mathematically; then, the thermal–mechanical–metallurgical model was implemented by developing
several user subroutines in Abaqus. Next, the reliability of the model was verified by conducting
grinding experiments of AISI 1045 steel. The microstructure distribution and the thickness of the
strengthened layer were observed or measured. Finally, the evolutions of the temperature and the
phase transformation in grinding are discussed, and the effects of stress on phase transformations
are revealed. Materials 2019, 12, 2327 3 of 18 2. Thermal–Mechanical–Metallurgical Coupling Model of Grinding 2. Thermal–Mechanical–Metallurgical Coupling Model of Grinding 2. Thermal–Mechanical–Metallurgical Coupling Model of Grinding 2. Thermal–Mechanical–Metallurgical Coupling Model of Grinding 2.1. Thermal–Mechanical Model of Grinding 2.1. Thermal–Mechanical Model of Grinding 2.1. Thermal–Mechanical Model of Grinding Transformation Type
Hx (J/m3)
Ferrite to austenite
595 × 106
Pearlite to austenite
526 × 106
Austenite to martensite
640 × 106 To solve Equation (1), it is necessary to define the initial and boundary conditions in grinding. The initial temperature of the workpiece is equal to the room temperature: T(x, y, z)|t=0 = T∞= 20◦C
(3) (3) Figure 1a shows the diagram of the thermal boundary conditions in grinding. The distributed heat
source moves and acts on the workpiece surface, and it is assumed that the heat source is distributed
in a right triangle:
(
)
l qw(x) = 2qm
(x −vwt) + lg
lg
, vwt −lg ≤x ≤vwt
(4) (4) 4 of 18 Materials 2019, 12, 2327 where qw is the heat flux of the heat source, vw is the feeding speed and lg is the contact chord length,
which can be estimated by
p
apds, where ds is the wheel diameter and ap is the grinding depth. qm is
the mean heat flux within the contact zone and can be expressed as: where qw is the heat flux of the heat source, vw is the feeding speed and lg is the contact chord length,
which can be estimated by
p
apds, where ds is the wheel diameter and ap is the grinding depth. qm is
the mean heat flux within the contact zone and can be expressed as: qm = εFtvs
blg
(5) (5) where Ft is the tangential grinding force, vs is the wheel speed, and b is the grinding width. ε is the
heat partition ratio transferred into the workpiece and can be estimated by [17]: where Ft is the tangential grinding force, vs is the wheel speed, and b is the grinding width. ε is the
heat partition ratio transferred into the workpiece and can be estimated by [17]: ε =
1
1 + (kρc)svs
(kρc)wvw
AR
As
1/2
(6) (6) where (kρc)s and (kρc)w are the thermal properties of the wheel and the workpiece, respectively. AR
and As are real and geometrical contact areas between the workpiece and the wheel, respectively. Furthermore, the heat convection and heat radiation occur on the workpiece surfaces, and the equivalent
heat flux of the convection and radiation is: where (kρc)s and (kρc)w are the thermal properties of the wheel and the workpiece, respectively. 2.1. Thermal–Mechanical Model of Grinding AR
and As are real and geometrical contact areas between the workpiece and the wheel, respectively. Furthermore, the heat convection and heat radiation occur on the workpiece surfaces, and the equivalent
heat flux of the convection and radiation is: qe = hc(Ts −T∞) + σbεb
Ts4 −T∞4
(7) (7) where hc is the convection coefficient, Ts is the surface temperature, εb is the emissivity (0.21), and εb is
the Stefan–Boltzmann constant (5.67 × 10−8 W/(m2 ◦C4)). In grinding, the surface materials have elastic–plastic deformation due to the actions of the grinding
force and heat. According to Prandtl–Reuss plastic increment theory, the constitutive relationship
within the elastic zone is: d{σ} = [D]ed{ε} −[C]edT
(8) (8) and in the plastic zone is: d{σ} = [D]epd{ε} −[C]epdT
(9) (9) where [D]e and [D]ep are the elastic matrix and plastic matrix, respectively, and [C]e and [C]ep are,
respectively, elastic vector and plastic vector related to the temperature, and dT is the temperature
increment. d{σ} and d{ε} are the total stress increment and strain increment, respectively. The major
strain sources, such as mechanical strain, thermal strain, and the volume change strain caused by phase
transformation, were considered, and some less important strain sources, such as stress relaxation and
strain caused by transformation plasticity, were ignored in the present paper [18,19]. Then, d{ε} can be
expressed as: d{ε} = dεe + dεp + d
n
εtho
+ d
n
ε∆Vo
(10) (10) where d{εe} is the elastic strain, which can be calculated by Hooke’s law with the temperature-dependent
elastic modulus E and constant Poisson’s ratio v (as shown in Table 3). d{εp} is the plastic strain
increment following Prandtl–Reuss flow rule, the von Mises yield criterion, and the Johnson-Cook
model. Table 4 shows the Johnson-Cook coefficients used in the present work [20]. d
n
εtho
is the thermal
strain increment and can be estimated by: d
n
εtho
= αdT
(11) (11) where α is the thermal expansion coefficient; the temperature-dependent α used in the present work is
listed in Table 3. d
n
ε∆Vo
is the volume strain increment caused by phase transformation and can be
estimated by [21]:
1 ∆V d
n
ε∆Vo
= 1
3
∆V
V dfx
(12) (12) Materials 2019, 12, 2327 5 of 18 where ∆V/V is the volume change ratio caused by phase transformation and is listed in Table 5 [21]. d
n
ε∆Vo
was incorporated into the model by using and writing the subroutine *UEXPAN. where ∆V/V is the volume change ratio caused by phase transformation and is listed in Table 5 [21]. d
n
ε∆Vo
was incorporated into the model by using and writing the subroutine *UEXPAN. n
o
Figure 1b shows the mechanical boundary conditions in grinding. The upper surface is under the
actions of moving normal grinding force and tangential grinding force. and in the plastic zone is: The grinding forces distribute
in the contact zone in a right triangle and can be expressed as follows: ft(x) = 2 Ft
blg
(x −vwt) + lg
lg
, vwt −lg ≤x ≤vwt
(13)
fn(x) = 2 Fn
blg
(x −vwt) + lg
lg
, vwt −lg ≤x ≤vwt
(14) (13) (14) where Fn is the normal grinding force. The bottom surface is fixed on the worktable, and its
displacements are set as 0. Table 3. Mechanical properties of AISI 1045 steel. Temp (◦C)
E (GPa)
α (×10−6/◦C)
v
20
206
11.56
0.3
100
200
11.90
200
192
12.32
300
184
13.09
500
163
14.18
800
125
14.50
1000
99
14.40
Table 4. Johnson–Cook coefficients of AISI 1045 steel. A
B
C
m
n
553.1
600.8
0.0134
1
0.234
Table 5. Volume change ratio in phase transformation. Transformation Type
∆V/V
Ferrite to austenite
−3.8 × 10−3
Pearlite to austenite
−3.3 × 10−3
Austenite to martensite
1.026 × 10−2
Materials 2019, 12, x FOR PEER REVIEW
6 of 1
(a)
(b)
Figure 1. Boundary conditions in the grinding: (a) thermal boundary conditions; (b) mechanical
boundary conditions. H is the height of the workpiece, L is the length of the workpiece,
eq is
the equivalent heat flux of the convection and radiation. Figure 1. Boundary conditions in the grinding: (a) thermal boundary conditions; (b) mechanical
boundary conditions. H is the height of the workpiece, L is the length of the workpiece, qe is the
equivalent heat flux of the convection and radiation. Table 3. Mechanical properties of AISI 1045 steel. (b) (b) (a) Figure 1. Boundary conditions in the grinding: (a) thermal boundary conditions; (b) mechanical
boundary conditions. H is the height of the workpiece, L is the length of the workpiece,
eq is
the equivalent heat flux of the convection and radiation. Figure 1. Boundary conditions in the grinding: (a) thermal boundary conditions; (b) mechanical
boundary conditions. H is the height of the workpiece, L is the length of the workpiece, qe is the
equivalent heat flux of the convection and radiation. Materials 2019, 12, 2327 6 of 18 2.2. Metallurgical Model for Grinding 2.2.1. Diffusive Transformations 2.2.1. Diffusive Transformations For the diffusive transformations, such as austenite transformation, ferrite transformation, pearlite
transformation, and bainite transformation under isothermal condition, a kinetic equation proposed
by Avrami is widely used [15,22]: by Avrami is widely used [15,22]: fx = 1 −exp
−b(t −τ)n
(15) (15) where fx is the transformation fraction, t is isothermal time, τ is the incubation time, b is the growth
factor, and n is the growth index. b and n at a certain isothermal temperature can be estimated by: where fx is the transformation fraction, t is isothermal time, τ is the incubation time, b is the growth
factor, and n is the growth index. b and n at a certain isothermal temperature can be estimated by: n =
ln
ln(1−fx1)
ln(1−fx2)
ln
t1
t2
(16)
b = −ln(1 −fx1)
tn
1
(17) (16) b = −ln(1 −fx1)
tn
1
(17) (17) where fx1 and fx2 are two different transformation fractions at an isothermal temperature, and their
corresponding isothermal time are t1 and t2. fx1 and fx2 are usually set, respectively, as 0.1% and
99.9% in calculations. fx1, fx2, t1, and t2 can be obtained from a time–temperature-austenitization (TTA)
diagram (isothermal kinetic curves of austenite formation) or time–temperature-transformation (TTT)
diagram (isothermal transformation curves of undercooled austenite). Figure 2 shows the TTA and
TTT diagrams of AISI 1045 steel used in the present paper. The TTT diagram was directly obtained
from JmatPro and the TTA diagram was transformed from the CHA diagram (continuous heating
kinetic curves of austenite formation), which was also obtained from JmatPro (Public Release Version
7.0.0, Sente Software Ltd., Guildford, United Kingdom). Materials 2019, 12, x FOR PEER REVIEW
7 of 19 (a)
(b)
Figure 2. Transformation kinetic curves of AISI 1045 steel: (a) time–temperature-austenitization
(TTA) diagram; (b) time–temperature-transformation (TTT) diagram. Figure 2. Transformation kinetic curves of AISI 1045 steel: (a) time–temperature-austenitization (TTA)
diagram; (b) time–temperature-transformation (TTT) diagram. (a) (b) (b) (a) Figure 2. Transformation kinetic curves of AISI 1045 steel: (a) time–temperature-austenitization
(TTA) diagram; (b) time–temperature-transformation (TTT) diagram. Figure 2. Transformation kinetic curves of AISI 1045 steel: (a) time–temperature-austenitization (TTA)
diagram; (b) time–temperature-transformation (TTT) diagram. 2.2.2. Martensite Transformation Martensite transformation occurs when the temperature is less than the starting temperature of
martensite transformation Ms in the cooing period. The undercooled austenite constantly transforms
into martensite with the temperature decreasing. The transformed fraction can be calculated by the
Koistinen–Marburger equation [26]: fM = f ∗
γ(1 −exp(−α(Ms −T)))
(23) (23) where fM is the formed martensite fraction, f ∗
γ is the retained austenite fraction before martensite
transformation, and α is a constant depending on the material and is equal to 0.011 for most Fe–C
alloy steels. where fM is the formed martensite fraction, f ∗
γ is the retained austenite fraction before martensite
transformation, and α is a constant depending on the material and is equal to 0.011 for most Fe–C
alloy steels. 2.2.1. Diffusive Transformations According to Cheng’s work [23], the temperature-dependent b and n can be obtained by
tti
i
th f ll
i
t
f
According to Cheng’s work [23], the temperature-dependent b and n can be obtained by fitting in
he following two forms: According to Cheng’s work [23], the temperature-dependent b and n can be obtained by
fitting in the following two forms:
According to Cheng’s work [23], the temperature-dependent b and n can be obtained by fitting in
the following two forms: rms:
( )
3
2
3
2
1
0
ln
x
x
x
x
b T
C T
C T
C T
C
=
+
+
+
(18)
ln b(T) = C3xT3 + C2xT2 + C1xT + C0x
(18)
n(T) = D3xT3 + D2xT2 + D1xT + D0x
(19) (18)
(18)
(19) (18) (18)
(19) Materials 2019, 12, 2327 7 of 18 where C0x,1x,2x,3x and D0x,1x,2x,3x are constants. The incubation time τ related to temperature can also
be obtained by fitting in the form: ln τ(T) = E3xT3 + E2xT2 + E1xT + E0x
(20) (20) where E0x,1x,2x,3x are constants. where E0x,1x,2x,3x are constants. To describe the phase transformation under non-isothermal condition, Scheil’s additivity rule
based on fictitious time is used [24,25]. A non-isothermal process could be divided into many isothermal
steps with a time interval ∆t; the transformed fraction after the ith. isothermal step is: fi = 1 −exp
−b
t∗
i + ∆t
n
(21) (21) fi = 1 −exp
−b
t∗
i + ∆t
n where t∗
i is the fictitious time used in the ith isothermal step: where t∗
i is the fictitious time used in the ith isothermal step: where t∗
i is the fictitious time used in the ith isothermal step: t∗
i =
−ln(1 −fi−1)
b
!1/n
(22) (22) where fi−1 is the transformed fraction after the i −1th. isothermal step. where fi−1 is the transformed fraction after the i −1th. isothermal step. 2.2.3. Effect of Stress on Phase Transformation Austenite transformation and martensite transformation are dominant in grinding, so the effects of
stress on the two transformations were considered in the present work. For AISI 1045 steel, if ignoring
the effect of stress, the austenite transformation begins when the temperature exceeds the nominal
equilibrium temperature A1(722.0 ◦C). However, the transformation in grinding happens under high
stress, which could change A1. The Clausius–Clapeyron equation can be used to incorporate the effect
of stress [27,28]. Material is under a complex stress state, so the pressure in the Clausius–Clapeyron
equation is replaced by the hydrostatic stress: d(−σm)
dT
=
∆Hαγ
T∆Vαγ
(24) (24) where σm is the hydrostatic stress, and σm = (σ1 + σ2 + σ3)/3 and σm < 0 indicate that the stress is
compressive. ∆Hαγ (920.5J/mol) is the enthalpy of ferrite–austenite transformation per mole, and
∆Vαγ (−0.06cm3/mol) [28] is the volume change of the transformation per mole: ∆Vαγ = M
ργ
−M
ρα
(25) (25) Materials 2019, 12, 2327 8 of 18 where ρα and ργ are the densities of ferrite and austenite, respectively. Through deduction and
integration of Equation (25), the equilibrium temperature A1 incorporating the effect of stress
expresses as: where ρα and ργ are the densities of ferrite and austenite, respectively. Through deduction and
integration of Equation (25), the equilibrium temperature A1 incorporating the effect of stress
expresses as: T = T0 exp
∆Vαγ(−σm)
∆Hαγ
! . (26) (26) Besides A1, the stress also changes the equilibrium temperatures A3. For example, Figure 2a shows
A1 and A3 under the action of σm = −500Mpa (minus represents compressive stress). From Figure 2b,
the kinetic curves have movement in the temperature decreasing direction, which indicates the
hydrostatic compressive stress could promote the austenite transformation by reducing A1 and A3. The original TTT and TTA diagrams obtained from JMatPro are used to calculate transformation
coefficients, and then the transformation without the effect of stress can be calculated by Equations
(21) and (22) with the coefficients. In order to incorporate the effect of stress on the transformation,
the TTT and TTA diagrams are adjusted according to Equations (24)–(26), then the adjusted TTT and
TTA diagrams are used to calculate transformation coefficients, and finally, the transformation with the
effect of stress is calculated by Equations (21) and (22) with the coefficients. 2.2.3. Effect of Stress on Phase Transformation When the mechanical driving force produced by the internal stress and chemical driving force
exceeds a critical value, martensite transformation will occur beyond the nominal starting temperature
Ms (321.9 ◦C). This is called stress-induced martensite transformation [29]. In general, the effects of
stress on martensite transformation include the effect of hydrostatic stress and the effect of uniaxial
stress. The hydrostatic compressive stress decreases the equilibrium temperature Ms, and both uniaxial
tension stress and compression stress increase the equilibrium temperature Ms. To calculate the effects
of shear stress and normal stress on Ms under complex stress conditions, Inoue proposed a model for
calculating the variation of Ms [30,31]: ∆MS = Aσm + B
p
J2
(27) (27) where J2 is second deviatoric invariant of the stress tensor and can be expressed as: J2 = (1/6)
(σ1 −σ2)2 + (σ2 −σ3)2 + (σ3 −σ1)2
(28) J2 = (1/6)
(σ1 −σ2)2 + (σ2 −σ3)2 + (σ3 −σ1)2
(28) (28) A (0.05) and B (0.033) are coefficients related to the material [28]. A (0.05) and B (0.033) are coefficients related to the material [28]. A (0.05) and B (0.033) are coefficients related to the material [28]. 3. Experimental Details The grinding experiments were conducted to validate the model. Figure 3a shows the experiment
setup. The grinder used was the BLOHM ORBIT 36 plane and forming grinder (ORBIT 36, United
Grinding (Shanghai) Ltd., Shanghai, China). The material used in the experiments was AISI 1045 steel (Anshan Steel Group, Anshan, China),
the chemical composition of which is shown in Table 6. Figure 3b shows the initial microstructure of the
material, and the material consisted of 56.3% pearlite and 43.7% ferrite. The dimensions of the grinding
region of the specimen were 50 mm (length) × 10 mm (width) × 20 mm (height). An aluminum oxide
wheel with a grain size number of 60, diameter of 340 mm, and width of 40 mm, was used. g
Table 7 shows the experimental grinding parameters used in present work. To obtain a metallographic, a part of each ground workpiece was first cut as the metallographic
sample. Then, the samples had treatments with lapping, polishing, cleaning, and corrosion. Finally,
the microstructure distribution and thickness of the strengthened layer were observed or measured by
the LEICA-DMIRM multifunctional metallographic microscope (LEICA-DMIRM, Leica Microsystems
Inc., Buffalo Grove, United States). Table 6. Chemical composition of AISI 1045 steel (wt%). Elements
C
Si
Mn
Cr
Ni
Cu
wt%
0.42–0.45%
0.17–0.37%
0.5–0.8%
0.25%
0.3%
0.25%
Table 7. Experimental grinding parameters. No. ap (µm)
vw (m/min)
vs (m/s)
1
450
9
30
2
400
9
3
350
9
2019, 12, x FOR PEER REVIEW
1
450
9
30
2
400
9
3
350
9
Figure 3. (a) Experimental setup; (b) initial microstructure of AISI 1045 steel. Figure 3. (a) Experimental setup; (b) initial microstructure of AISI 1045 steel. Table 6. Chemical composition of AISI 1045 steel (wt%). Elements
C
Si
Mn
Cr
Ni
Cu
wt%
0.42–0.45%
0.17–0.37%
0.5–0.8%
0.25%
0.3%
0.25% Table 6. Chemical composition of AISI 1045 steel (wt%). Table 7. Experimental grinding parameters. EW Figure 3. (a) Experimental setup; (b) initial microstructure of AISI 1045 steel. Figure 3. (a) Experimental setup; (b) initial microstructure of AISI 1045 steel. 2.3. Model Implementation in FE The workpiece was modeled with the size of 50 mm (length) × 16 mm (height). Since the
phase transformation only occurs within the shallow surface layer in the grinding, the depth range
of 0–1 mm of the workpiece was finely meshed and the rest was coarsely meshed. The bottom
of the workpiece was fixed on workbench. The heat convection and heat radiation were applied
on the surfaces by writing subroutine *FILM and setting emissivity. User subroutines *DFLUX,
*DLOAD, and *UTRACLOAD were defined to achieve the applications of moving grinding heat flux,
normal grinding force, and tangential grinding force, respectively. The purpose of this paper was to
establish a thermal–mechanical–metallurgical direct coupling FE model for grinding and reveal the
effects of stress on the phase transformation. However, the Abaqus does not provide the model of
thermal–mechanical–metallurgical coupling analysis. In order to accomplish the coupling analysis,
the temp-displacement coupling analysis was chosen and several user subroutines were used. The
phase transformations were calculated by defining user subroutine *USDFLD, the latent heat of the
phase transformation was defined by writing user subroutine *HETVAL, and the volume change strain
caused by phase transformation was defined in user subroutine *UEXPAN. Materials 2019, 12, 2327 9 of 18 4.1. Comparison of Experimental Results and Simulated Results
4.1. Comparison of Experimental Results and Simulated Results 4.1. Comparison of Experimental Results and Simulated Results
4.1. Comparison of Experimental Results and Simulated Results p
f
p
Figure 4 shows the microstructure distribution of the strengthened layer under the no. 1
grinding parameter. Figure 4a is a metallographic obtained by experimental observation and Figure
4b is a map obtained from the simulation. Considering the phase transition occurs only in the
shallow surface layer, only four elements nearest to the surface are selected to show the simulated
results. As seen in Figure 4a, the microstructure of the strengthened layer was obviously different
from the original microstructure. The ferrite was bright white, martensite was bright black, and the
Figure 4 shows the microstructure distribution of the strengthened layer under the no. 1 grinding
parameter. Figure 4a is a metallographic obtained by experimental observation and Figure 4b is a map
obtained from the simulation. Considering the phase transition occurs only in the shallow surface
layer, only four elements nearest to the surface are selected to show the simulated results. As seen
in Figure 4a, the microstructure of the strengthened layer was obviously different from the original
microstructure. The ferrite was bright white, martensite was bright black, and the pearlite was black 10 of 18 Materials 2019, 12, 2327 10 of 18 under current corrosion conditions. The strengthened layer visually presented dark–bright–dark
with the depth increasing because of the different degrees of phase transformation along the depth
direction. The strengthened layer could be divided into complete strengthened layer and transition
layer according to the degree of phase transformation. The upper dark layer almost comprised of 100%
martensite was the complete strengthened layer (Figure 4c), and the next bright layer comprised of
martensite and pearlite + ferrite was the transition layer (Figure 4d). In the deeper depth, the materials
did not undergo phase transformation, and the boundary between the strengthened layer and the
bulk region is shown in Figure 4e. As seen in Figure 4a, the martensite decreased and the ferrite +
pearlite increased along the depth within the strengthened layer. As seen in Figure 4b, the retained
austenite and bainite of the strengthened layer were small and changeless along the depth, so we
only discuss the variations of the martensite and ferrite + pearlite. Furthermore, the martensite
fraction was about 90–100% within the depth range of 0–120 µm. 4.1. Comparison of Experimental Results and Simulated Results
4.1. Comparison of Experimental Results and Simulated Results This shows the materials within the
range underwent complete austenite transformation during the heating period and mainly underwent
martensite transformation during the cooling period. Comparing to the experimental result (Figure 4a),
the range corresponded to the complete strengthened layer. There were martensite and ferrite +
pearlite within the depth range of 120–250 µm, and the martensite increased and ferrite + pearlite
decreased with the increases in depth. The range corresponded to the transition layer in Figure 4a. In summary, the simulated microstructure distribution was consistent with the experimental result. Materials 2019, 12, x FOR PEER REVIEW
11 of 19 Figure 4. Microstructure distribution of the strengthened layer under no. 1 grinding parameter: (a)
experimental result; (b) simulated result; (c) the complete strengthened layer; (d) the transition layer;
(e) the boundary between the strengthened layer and the bulk region. Figure 4. Microstructure distribution of the strengthened layer under no. 1 grinding parameter:
(a) experimental result; (b) simulated result; (c) the complete strengthened layer; (d) the transition
layer; (e) the boundary between the strengthened layer and the bulk region. Figure 4. Microstructure distribution of the strengthened layer under no. 1 grinding parameter: (a)
experimental result; (b) simulated result; (c) the complete strengthened layer; (d) the transition layer;
(e) the boundary between the strengthened layer and the bulk region. Figure 4. Microstructure distribution of the strengthened layer under no. 1 grinding parameter:
(a) experimental result; (b) simulated result; (c) the complete strengthened layer; (d) the transition
layer; (e) the boundary between the strengthened layer and the bulk region. Figure 5 and Figure 6 show the microstructure distribution of the strengthened layer under the
no. 2 and no. 3 grinding parameters. These shows the microstructure distributions under the two
grinding parameters were similar to that under the no. 1 grinding parameter. However, the
thicknesses of the strengthened layers were different under different grinding parameters. The
predicting accuracy of the established model could be further verified by comparing the thicknesses
of the strengthened layers. In the experiments, to overcome the measuring error and the variability
of the thickness, we measured the thickness at three different positions by a microscope, and used
the average to compare with the simulated results. Figure 7 shows the comparison results of the
thicknesses. This means that the simulated thicknesses were in good agreement with the
Figures 5 and 6 show the microstructure distribution of the strengthened layer under the no. 4.1. Comparison of Experimental Results and Simulated Results
4.1. Comparison of Experimental Results and Simulated Results 2
and no. 3 grinding parameters. These shows the microstructure distributions under the two grinding
parameters were similar to that under the no. 1 grinding parameter. However, the thicknesses of the
strengthened layers were different under different grinding parameters. The predicting accuracy of the
established model could be further verified by comparing the thicknesses of the strengthened layers. In the experiments, to overcome the measuring error and the variability of the thickness, we measured
the thickness at three different positions by a microscope, and used the average to compare with the
simulated results. Figure 7 shows the comparison results of the thicknesses. This means that the 11 of 18
esses
bility 11 of 18
esses
bility Materials 2019, 12, 2327
predicting accurac
of the strengthene simulated thicknesses were in good agreement with the experimental results and the maximum error
was 10.6%. the average to compare with the simulated results. Figure 7 shows the comparison results of the
thicknesses. This means that the simulated thicknesses were in good agreement with the
experimental results and the maximum error was 10 6% Figure 5. Microstructure distribution of the strengthened layer under no. 2 grinding parameter: (a)
experimental result; (b) simulated result. Figure 5. Microstructure distribution of the strengthened layer under no. 2 grinding parameter: (a)
experimental result; (b) simulated result. Materials 2019, 12, x FOR PEER REVIEW
12 of 19
Materials 2019 12 x FOR PEER REVIEW
12 of 19 Figure 5. Microstructure distribution of the strengthened layer under no. 2 grinding parameter: (a)
experimental result; (b) simulated result. Figure 5. Microstructure distribution of the strengthened layer under no. 2 grinding parameter: (a)
experimental result; (b) simulated result. Materials 2019, 12, x FOR PEER REVIEW
12 of 1
M t i l 2019 12
FOR PEER REVIEW
12
f 19 Figure 6. Microstructure of the strengthened layer under no. 3 grinding parameter: (a) experimental
result; (b) simulated result. Figure 6. Microstructure of the strengthened layer under no. 3 grinding parameter: (a) experimental
result; (b) simulated result. Figure 6. Microstructure of the strengthened layer under no. 3 grinding parameter: (a) experimental
result; (b) simulated result. Figure 6. Microstructure of the strengthened layer under no. 3 grinding parameter: (a) experimental
result; (b) simulated result. Figure 6. Microstructure of the strengthened layer under no. 3 grinding parameter: (a) experimental
result; (b) simulated result. Figure 6. Microstructure of the strengthened layer under no. 4.1. Comparison of Experimental Results and Simulated Results
4.1. Comparison of Experimental Results and Simulated Results 3 grinding parameter: (a) experimental
result; (b) simulated result. Figure 7. The thickness of the strengthened layer under different grinding depths. 4 2 Temperature and Phase Distributions
Figure 7. The thickness of the strengthened layer under different grinding depths. 2 Temperature and Phase Distributions Figure 7. The thickness of the strengthened layer under different grinding depths. Figure 7. The thickness of the strengthened layer under different grinding depths. 4.3. Evolution of the Phase Transformation Figure 9a shows the time–temperature histories of four material points at different depths. The maximum temperatures were 1188.9, 970.3, 864.3, and 810.6 ◦C, respectively, which exceeded
the actual starting temperature A1. The temperature increased rapidly during the temperature rising
period and the temperature dropping rate was also high during the cooling period. The type of
phase transformation occurring during the cooling period was relevant to the temperature dropping
rate, so the average rates within 400–800 ◦C were calculated and used to judge the possible phase
transformation. The average rates at different depths were within 260–300 ◦C/s, which were higher
than the critical temperature dropping rates of bainite transformation of AISI 1045 steel, which is
100 ◦C/s. This illustrates the undercooled austenite would not experience ferrite transformation,
pearlite transformation, and bainite transformation before martensite transformation. Figure 9b–d show the evolution of each phase at different depths. We ignored the evolution of the
bainite because the simulation results showed that bainite transformation rarely occurs. The austenite
transformation started since the temperature reached the actual A1. For the material consisting
of pearlite and ferrite grains, the pearlite first underwent austenite transformation, and the ferrite
underwent austenite transformation after pearlite dissolution. Figure 9c shows the austenite fractions
increased over time within a short time range after the austenite transformation began. Closer to
the surface, the austenite transformation began earlier, which was due to the temperature of the
material closer to the surface rising to A1 earlier. Austenite transformation is a time-consuming process. Complete austenite transformation occurs only when the temperature lasts long enough above A1. At the depths of 0 µm and 400 µm, complete austenite transformation happened and forms 100%
austenite at the moments of 2.636 s and 2.836 s, respectively. At the depth of 650 µm, the austenite
transformation was incomplete, and formed 86.4% austenite. Furthermore, as seen in Figure 9b, all
pearlite transformed into austenite while the remaining 13.6% ferrite did not transform into austenite
at the depth. The austenite transformation was also incomplete, and only formed 6.4% austenite at the
depth of 800 µm. As seen in Figure 9b, the remaining 93.6% pearlite + ferrite did not transform into
austenite at the depth. This is because the duration times beyond A1 at the depths of 650 and 800 µm
were not enough. Fi
7 Th thi k
f th
4.2. Temperature and Phase Distributions
4.2. Temperature and Phase Distributions Fi
7 Th
hi k
f h
4.2. Temperature and Phase Distributions
4.2. Temperature and Phase Distributions Figure 7. The thickness of the strengthened layer under different grinding depths. 4.2. Temperature and Phase Distributions
Figure 8a shows the temperature distribution at the moment of 3.576 s under the grinding
Figure 8a shows the temperature distribution at the moment of 3.576 s under the grinding
parameters,
200 μm
p
a =
,
0.6 m/ min
w
v =
, and
30 m/s
sv =
. It shows a temperature field with a large
gradient formed in the surface layer of the workpiece. The maximum temperature appeared on the
workpiece surface in the contact zone, reaching 1191
oC . In the rear region of the contact zone, the
Figure 8a shows the temperature distribution at the moment of 3.576 s under the grinding
parameters, ap = 200µm, vw = 0.6m/min, and vs = 30m/s. It shows a temperature field with a large
gradient formed in the surface layer of the workpiece. The maximum temperature appeared on 12 of 18 12 of 18 Materials 2019, 12, 2327 the workpiece surface in the contact zone, reaching 1191 ◦C. In the rear region of the contact zone,
the temperature was generally higher, which was due to the heat source having just passed. In the
region farther away from the contact zone, the grinding heat source had passed for some time, so the
temperature was lower. In the front region of the contact zone, the temperature was only high around
the contact zone, but close to room temperature at a farther position because the heat source had not
passed and the heat could not transfer to a deeper and farther position. Materials 2019, 12, x FOR PEER REVIEW
13 of 19 Figure 8. Temperature and microstructure distributions at the moment of 3.576 s: (a) temperature; (b
ferrite + pearlite; (c) austenite; (d) martensite. Figure 8. Temperature and microstructure distributions at the moment of 3.576 s: (a) temperature
errite + pearlite; (c) austenite; (d) martensite. Figure 8. Temperature and microstructure distributions at the moment of 3.576 s: (a) temperature; (b)
ferrite + pearlite; (c) austenite; (d) martensite. Figure 8. Temperature and microstructure distributions at the moment of 3.576 s: (a) temperature; (b)
ferrite + pearlite; (c) austenite; (d) martensite. Fi
7 Th thi k
f th
4.2. Temperature and Phase Distributions
4.2. Temperature and Phase Distributions Materials 2019, 12, 2327 13 of 18 Figure 8b–d show the distributions of ferrite + pearlite, austenite, and martensite at the moment
of 3.576 s. As seen in Figure 8b,c, the fractions of ferrite + pearlite and austenite of the region in and
behind the contact zone were 0–10% and 90–100%, respectively. This means that the materials in the
region underwent austenite transformation. In particular, as shown in Figure 8d, martensite appeared
in zone 1 with a maximum fraction of 56.1%. This shows that the martensite transformation began in
zone 1. This was because the temperature of the materials in the zone dropped below 300 ◦C at this
moment, as shown in Figure 8a. 4.3. Evolution of the Phase Transformation As seen in Figure 9c, the fractions of the austenite at all the depths were stable for a period during
which the factions of other phases were changeless. This indicates no phase transformation took place
during the period. When the temperature dropped below Ms, the martensite transformation began. As seen in Figure 9d, the martensite transformations took place almost at the same time at different
depths. At the depths of 0 and 400 µm, the evolution curves of martensite almost coincided, because
their temperatures below Ms almost coincided during the cooling process, as shown in Figure 9a. The fraction of austenite decreased and the fraction of martensite increased over time because of the
continual decline of the temperature. At the moment of 14 s, the fractions of martensite at the depths
were about 81.2%, 81.2%, 70.8%, and 5.2%, respectively. Materials 2019, 12, 2327 14 of 18 14 of 18 Materials 2019, 12, x FOR PEER REVIEW
15 of 19
(a)
(b)
(c)
(d)
Figure 9. Evolution of temperature and each phase at different depths: (a) temperature; (b) ferrite +
pearlite; (c) austenite; (d) martensite. Figure 9. Evolution of temperature and each phase at different depths: (a) temperature; (b) ferrite +
pearlite; (c) austenite; (d) martensite. (a)
(b) (b) (a) ( )
(d) (c) (d) (c) Figure 9. Evolution of temperature and each phase at different depths: (a) temperature; (b) ferrite +
pearlite; (c) austenite; (d) martensite. Figure 9. Evolution of temperature and each phase at different depths: (a) temperature; (b) ferrite +
pearlite; (c) austenite; (d) martensite. 4.4. Effect of Stress on Phase Transformation in Grinding
4.4. Effect of Stress on Phase Transformation in Grinding 4.4. Effect of Stress on Phase Transformation in Grinding
4.4. Effect of Stress on Phase Transformation in Grinding Figure 11 shows the hydrostatic stress history and
2
J history of the material point at the
d
h
f
d
d
d h
f
h Figure 10. Hydrostatic stress histories and
1
A
with stress. Figure 10. Hydrostatic stress histories and A1 with stress. the hydrostatic stress history and
2
J history of th Figure 11 shows the hydrostatic stress history and
2
J history of the material point at the
depth of 0 μm during grinding and the martensite transformation starting temperature Ms with
stress. As seen in the figure, the hydrostatic stress was tensile in the time range of 3.50–5.21 s (a
period before martensite transformation), and increased over time. 2
J also increased over time in
the time range. Ms with stress increased relative to the nominal Ms in the time range due to the
effects of hydrostatic tensile stress and
2
J
, and increased over time. This is because both
hydrostatic tension stress and
2
J could increase Ms . As seen in Figure 9a, the temperature of the
material point decreased during the period. The temperature began to be lower than the equilibrium
temperature Ms at the moment of 5.21 s, and the martensite transformation began. At the moment,
the hydrostatic stress was 421.44 MPa,
2
J was 368.36 MPa, and Ms with stress was 355.13 oC
Figure 11 shows the hydrostatic stress history and √J2 history of the material point at the depth
of 0 µm during grinding and the martensite transformation starting temperature Ms with stress. As seen in the figure, the hydrostatic stress was tensile in the time range of 3.50–5.21 s (a period before
martensite transformation), and increased over time. √J2 also increased over time in the time range. Ms with stress increased relative to the nominal Ms in the time range due to the effects of hydrostatic
tensile stress and √J2, and increased over time. This is because both hydrostatic tension stress and √J2
could increase Ms. As seen in Figure 9a, the temperature of the material point decreased during the
period. The temperature began to be lower than the equilibrium temperature Ms at the moment of
5.21 s, and the martensite transformation began. At the moment, the hydrostatic stress was 421.44 MPa,
√J2 was 368.36 MPa, and Ms with stress was 355.13 ◦C (actual starting temperature of martensite
transformation of the material point). 4.4. Effect of Stress on Phase Transformation in Grinding
4.4. Effect of Stress on Phase Transformation in Grinding Figure 10 shows the hydrostatic stress history at the depth of 0 μm during grinding and
1
A
considering the stress. In the figure, negative stress represents compressive stress and positive stress
represents tensile stress. It shows the hydrostatic stress was compressive within the time range of
0–2.57 s (a period before austenite transformation), and firstly increased and then decreased in value
over time. As such, 1
A with stress decreased relative to the nominal
1
A , and the changing trend
was consistent with the hydrostatic stress. This is because hydrostatic compressive stress reduces
1
A . Figure 9a shows the temperature of the material point kept rising within the time range of 0–2.57
s and exceeded the equilibrium temperature
1
A at the moment of 2.57 s, and then the austenite
transformation began. The hydrostatic stress was –278.96 MPa at the moment, and
1
A with stress
was 709.0 oC (the actual starting temperature of austenite transformation for the material point). Compared to the nominal
1
A
the actual
1
A
decreased by 13 0
oC due to the effect of the
Figure 10 shows the hydrostatic stress history at the depth of 0 µm during grinding and A1
considering the stress. In the figure, negative stress represents compressive stress and positive stress
represents tensile stress. It shows the hydrostatic stress was compressive within the time range of
0–2.57 s (a period before austenite transformation), and firstly increased and then decreased in value
over time. As such, A1 with stress decreased relative to the nominal A1, and the changing trend
was consistent with the hydrostatic stress. This is because hydrostatic compressive stress reduces A1. Figure 9a shows the temperature of the material point kept rising within the time range of 0–2.57 s and
exceeded the equilibrium temperature A1 at the moment of 2.57 s, and then the austenite transformation
began. The hydrostatic stress was −278.96 MPa at the moment, and A1 with stress was 709.0 ◦C
(the actual starting temperature of austenite transformation for the material point). Compared to the
nominal A1, the actual A1 decreased by 13.0 ◦C due to the effect of the hydrostatic compressive stress. 15 of 18 Materials 2019, 12, 2327 Figure 10. Hydrostatic stress histories and
1
A with stress. Figure 10. Hydrostatic stress histories and A1 with stress. Figure 10. Hydrostatic stress histories and
1
A with stress. 4.4. Effect of Stress on Phase Transformation in Grinding
4.4. Effect of Stress on Phase Transformation in Grinding Figure 12 shows the evolution of each phase in grinding with and without Materials 2019, 12, 2327
Materials 2019, 12, x FOR
considering the effe
without considering 16 of 18
17 of 19
ith and
not, the 16 of 18
17 of 19
ith and
not, the considering stress. As seen in the figure, no matter whether considering the stress or not, the material
point underwent complete austenite transformation and resulted in 100% austenite. The difference was
the austenite transformation began earlier when considering stress. This is because hydrostatic stress
is compressive and reduces A1. Then, the austenite transformation occurred at lower temperature. The martensite transformation took place earlier with considering the stress than without considering
the stress. This was because both the hydrostatic tensile stress and √J2 could increase Ms; then the
martensite transformation occurred at a higher temperature. without considering stress. As seen in the figure, no matter whether considering the stress or not, the
material point underwent complete austenite transformation and resulted in 100% austenite. The
difference was the austenite transformation began earlier when considering stress. This is because
hydrostatic stress is compressive and reduces
1
A . Then, the austenite transformation occurred at
lower temperature. The martensite transformation took place earlier with considering the stress than
without considering the stress. This was because both the hydrostatic tensile stress and
2
J could
increase Ms ; then the martensite transformation occurred at a higher temperature. difference was the austenite transformation began earlier when considering stress. This is because
hydrostatic stress is compressive and reduces
1
A . Then, the austenite transformation occurred at
lower temperature. The martensite transformation took place earlier with considering the stress than
without considering the stress. This was because both the hydrostatic tensile stress and
2
J could
increase Ms ; then the martensite transformation occurred at a higher temperature. Figure 12. Effect of stress on the evolution of each phase. Figure 12. Effect of stress on the evolution of each phase. Figure 12. Effect of stress on the evolution of each phase. The microstructure distribution of the strengthened layer in grinding is shown in Figure 1 Figure 12. Effect of stress on the evolution of each phase. Figure 12. Effect of stress on the evolution of each phase. e distribution of the strengthened layer in grinding is The microstructure distribution of the strengthened layer in grinding is shown in Figure 13. 4.4. Effect of Stress on Phase Transformation in Grinding
4.4. Effect of Stress on Phase Transformation in Grinding As
seen in the figure, the thickness of the strengthened layer was 933 μm with considering the stress,
which was larger than 856 μm without considering stress. This indicated the phase transformation
occurred at a deeper position with a lower temperature when considering the effect of stress. Within
the strengthened layer, the fraction of martensite decreased and the fraction of ferrite + pearlite
increased with the depth increasing no matter whether considering the stress or not. The figure also
shows more martensite formed when considering the stress, but more austenite remained when
ignoring the stress. The result illustrates that considering the effect of stress on phase transformation
is necessary for predicting the microstructure distribution and thickness of the strengthened layer
The microstructure distribution of the strengthened layer in grinding is shown in Figure 13. As seen in the figure, the thickness of the strengthened layer was 933 µm with considering the stress,
which was larger than 856 µm without considering stress. This indicated the phase transformation
occurred at a deeper position with a lower temperature when considering the effect of stress. Within the
strengthened layer, the fraction of martensite decreased and the fraction of ferrite + pearlite increased
with the depth increasing no matter whether considering the stress or not. The figure also shows
more martensite formed when considering the stress, but more austenite remained when ignoring the
stress. The result illustrates that considering the effect of stress on phase transformation is necessary
for predicting the microstructure distribution and thickness of the strengthened layer more accurately. seen in the figure, the thickness of the strengthened layer was 933 μm with considering the stress,
which was larger than 856 μm without considering stress. This indicated the phase transformation
occurred at a deeper position with a lower temperature when considering the effect of stress. Within
the strengthened layer, the fraction of martensite decreased and the fraction of ferrite + pearlite
increased with the depth increasing no matter whether considering the stress or not. The figure also
shows more martensite formed when considering the stress, but more austenite remained when
ignoring the stress. The result illustrates that considering the effect of stress on phase transformation
is necessary for predicting the microstructure distribution and thickness of the strengthened layer
more accurately. ore accurately. Figure 13. Effect of stress on the microstructure distribution and thickness of the strengthened layer. Figure 13. 4.4. Effect of Stress on Phase Transformation in Grinding
4.4. Effect of Stress on Phase Transformation in Grinding Compared with the nominal Ms, the actual Ms increased by
33.23 ◦C due to the effects of hydrostatic tensile stress and √J2. depth of 0 μm during grinding and the martensite transformation starting temperature Ms with
stress. As seen in the figure, the hydrostatic stress was tensile in the time range of 3.50–5.21 s (a
period before martensite transformation), and increased over time. 2
J also increased over time in
the time range. Ms with stress increased relative to the nominal Ms in the time range due to the
effects of hydrostatic tensile stress and
2
J
, and increased over time. This is because both
hydrostatic tension stress and
2
J could increase Ms . As seen in Figure 9a, the temperature of the
material point decreased during the period. The temperature began to be lower than the equilibrium
temperature Ms at the moment of 5.21 s, and the martensite transformation began. At the moment,
the hydrostatic stress was 421.44 MPa,
2
J was 368.36 MPa, and Ms with stress was 355.13 oC
(actual starting temperature of martensite transformation of the material point). Compared with the
nominal Ms , the actual Ms increased by 33.23 oC due to the effects of hydrostatic tensile stress p
p
)
p
e actual Ms increased by 33.23 oC due to the effects of hydrostatic ten
Figure 11. Hydrostatic stress and
2
J
histories, and Ms with stress. Figure 11. Hydrostatic stress and √J2 histories, and Ms with stress. igure 11. Hydrostatic stress and
2
J
histories, and Ms with stres
Figure 11. Hydrostatic stress and √J2 histories, and Ms with stress. Figure 11. Hydrostatic stress and
2
J
histories, and Ms with stress. In order to analyze the effects of stress on the phase transformations and the final characteristics
In order to analyze the effects of stress on the phase transformations and the final characteristics
of the strengthened layer in the grinding, the simulations were carried out with and without
In order to analyze the effects of stress on the phase transformations and the final characteristics of
the strengthened layer in the grinding, the simulations were carried out with and without considering
the effect of stress. 4.4. Effect of Stress on Phase Transformation in Grinding
4.4. Effect of Stress on Phase Transformation in Grinding Effect of stress on the microstructure distribution and thickness of the strengthened layer. Materials 2019, 12, 2327 17 of 18 17 of 18 5. Conclusions A thermal–mechanical–metallurgical directly coupling FE model of grinding was established. In the modeling procedure, the latent heat, volume change strain caused by phase transformation,
and stress-induced phase transformation were considered. The comparison between the experimental
and simulated results proved that the established model can accurately simulate the phase
transformation in grinding and predict the microstructure distribution and the thickness. Based on the model, the evolution of the phase transformation in grinding was studied. The austenite transformation in grinding may be incomplete because the heating rate is extremely
high and the duration time above A1 is short. Furthermore, the temperature drops quickly, so the
undercooled austenite does not undergo other transformation before martensite transformation during
the cooling period. The effects of stress on phase transformation in grinding were revealed. Stress promotes
austenite transformation because the hydrostatic stress is compressive in the period before austenite
transformation and could reduce A1. The stress also promotes the martensite transformation because
the hydrostatic stress is tensile in the period before martensite transformation, and both the hydrostatic
tensile stress and √J2 could increase Ms. To predict the microstructure distribution and thickness of the strengthened layer more accurately,
consideration of the effects of stress on phase transformations is necessary. Author Contributions: Conceptualization, S.X. and Y.D.; methodology, S.X.; software, Y.D.; validation, Y.D.;
formal analysis, X.K.; investigation, S.X.; resources, S.X.; data curation, Y.D.; writing—original draft preparation,
Y.D.; writing—review and editing, S.X. and X.K.; visualization, Y.D.; supervision, S.X.; project administration, S.X.;
funding acquisition, S.X. Funding: This research was funded by the National Natural Science Foundation of China, Grant No. 51775101
and the Fundamental Research Funds for the Central Universities of China, Grant No. N180306003. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Tang, J.; Du, J.; Chen, Y. Modeling and experimental study of grinding forces in surface grinding. J. Mater. Process. Technol. 2009, 209, 2847–2854. [CrossRef] 1. Tang, J.; Du, J.; Chen, Y. Modeling and experimental study of grinding forces in surface grinding. J. Mater. Process. Technol. 2009, 209, 2847–2854. [CrossRef] 2. Brinksmeier, E.; Brockhoff, T. Utilization of grinding heat as a new heat treatment process. CIRP Ann. Manuf. Technol. 1996, 45, 283–286. [CrossRef] 2. Brinksmeier, E.; Brockhoff, T. Utilization of grinding heat as a new heat treatment process. CIRP Ann. Manuf. Technol. 1996, 45, 283–286. [CrossRef] 3. Zarudi, I.; Zhang, L.C. Mechanical property improvement of quenchable steel by grinding. J. Mater. Sci. 2002, 37, 3935–3943. [CrossRef] 3. Zarudi, I.; Zhang, L.C. Mechanical property improvement of quenchable steel by grinding. J. Mater. Sci. 2002, 37, 3935–3943. [CrossRef] 4. Zarudi, I.; Zhang, L.C. Modelling the structure changes in quenchable steel subjected to grinding. J. Mater. Sci. 2002, 37, 4333–4341. [CrossRef] 4. Zarudi, I.; Zhang, L.C. Modelling the structure changes in quenchable steel subjected to grinding. J. Mater. Sci. 2002, 37, 4333–4341. [CrossRef] 5. Nguyen, T.; Zhang, L. Grinding–hardening using dry air and liquid nitrogen: Prediction and verification of
temperature fields and hardened layer thickness. Int. J. Mach. Tools Manuf. 2010, 50, 901–910. [CrossRef] 5. Nguyen, T.; Zhang, L. Grinding–hardening using dry air and liquid nitrogen: Prediction and verification of
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y
J
f
,
,
[
]
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31. Denis, S. Considering stress-phase transformation interactions in the calculation of heat treatment residual
stresses. In Mechanics of Solids with Phase Changes; Springer: Vienna, Austria, 1997; pp. 293–317. 31. Denis, S. Considering stress-phase transformation interactions in the calculation of heat treatment residual
stresses. In Mechanics of Solids with Phase Changes; Springer: Vienna, Austria, 1997; pp. 293–317. 31. Denis, S. Considering stress-phase transformation interactions in the calculation of heat treatment residual
stresses. In Mechanics of Solids with Phase Changes; Springer: Vienna, Austria, 1997; pp. 293–317. © 2019 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 (http://creativecommons.org/licenses/by/4.0/). © 2019 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 (http://creativecommons.org/licenses/by/4.0/). © 2019 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 (http://creativecommons.org/licenses/by/4.0/).
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CONSORT 2010 Statement: updated guidelines for reporting parallel group randomised trials
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Introduction Randomised controlled trials, when appropriately
designed, conducted, and reported, represent the gold
standard in evaluating healthcare interventions. How-
ever, randomised trials can yield biased results if they
lack methodological rigour [1]. To assess a trial accu-
rately, readers of a published report need complete,
clear, and transparent information on its methodology
and findings. Unfortunately, attempted assessments fre-
quently fail because authors of many trial reports
neglect to provide lucid and complete descriptions of
that critical information [2-4]. That lack of adequate reporting fuelled the develop-
ment of the original CONSORT (Consolidated Stan-
dards of Reporting Trials) statement in 1996 [5] and its
revision five years later [6-8]. While those statements
improved the reporting quality for some randomised
controlled trials, [9,10] many trial reports still remain
inadequate [2]. Furthermore, new methodological evi-
dence and additional experience has accumulated since
the last revision in 2001. Consequently, we organised a
CONSORT Group meeting to update the 2001 state-
ment [6-8]. We introduce here the result of that pro-
cess, CONSORT 2010. Diligent adherence by authors to the checklist items
facilitates clarity, completeness, and transparency of
reporting. Explicit descriptions, not ambiguity or omis-
sion, best serve the interests of all readers. Note that the
CONSORT 2010 Statement does not include recom-
mendations for designing, conducting, and analysing
trials. It solely addresses the reporting of what was done
and what was found. Nevertheless, CONSORT does indirectly affect design
and conduct. Transparent reporting reveals deficiencies
in research if they exist. Thus, investigators who con-
duct inadequate trials, but who must transparently
report, should not be able to pass through the publica-
tion process without revelation of their trial’s inadequa-
cies. That emerging reality should provide impetus to
improved trial design and conduct in the future, a © 2010 Schulz 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. Abstract The CONSORT statement is used worldwide to improve the reporting of randomised controlled trials. Kenneth
Schulz and colleagues describe the latest version, CONSORT 2010, which updates the reporting guideline based on
new methodological evidence and accumulating experience. The CONSORT statement is used worldwide to improve the reporting of randomised controlled trials. Kenneth
Schulz and colleagues describe the latest version, CONSORT 2010, which updates the reporting guideline based on
new methodological evidence and accumulating experience. To encourage dissemination of the CONSORT 2010 Statement, this article is freely accessible on bmj.com and will
also be published in the Lancet Obstetrics and Gynecology PLoS Medicine Annals of Internal Medicine Open The CONSORT statement is used worldwide to improve the reporting of randomised controlled trials. Kenneth
Schulz and colleagues describe the latest version, CONSORT 2010, which updates the reporting guideline based on
new methodological evidence and accumulating experience. To encourage dissemination of the CONSORT 2010 Statement, this article is freely accessible on bmj.com and will
also be published in the Lancet, Obstetrics and Gynecology, PLoS Medicine, Annals of Internal Medicine, Open
Medicine, Journal of Clinical Epidemiology, BMC Medicine, and Trials. g
g
p
To encourage dissemination of the CONSORT 2010 Statement, this article is freely accessible on bmj.com and will
also be published in the Lancet, Obstetrics and Gynecology, PLoS Medicine, Annals of Internal Medicine, Open
Medicine, Journal of Clinical Epidemiology, BMC Medicine, and Trials. diagram (Figure 1). It provides guidance for reporting all
randomised controlled trials, but focuses on the most
common design type-individually randomised, two
group, parallel trials. Other trial designs, such as cluster
randomised trials and non-inferiority trials, require vary-
ing amounts of additional information. CONSORT
extensions for these designs, [11,12] and other CON-
SORT products, can be found through the CONSORT
website http://www.consort-statement.org. Along with
the CONSORT statement, we have updated the explana-
tion and elaboration article, [13] which explains the
inclusion of each checklist item, provides methodologi-
cal background, and gives published examples of trans-
parent reporting. Kenneth F Schulz1*, Douglas G Altman2, David Moher3, the CONSORT Group Kenneth F Schulz1*, Douglas G Altman2, David Moher3, the CONSORT Group * Correspondence: kschulz@fhi.org
1Family Health International, Research Triangle Park, NC 27709, USA Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 TRIALS Open Access Intent of CONSORT 2010 The CONSORT 2010 Statement is this paper including
the 25 item checklist in the table (Table 1) and the flow * Correspondence: kschulz@fhi.org
1Family Health International, Research Triangle Park, NC 27709, USA Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Page 2 of 8 Figure 1 Flow diagram of the progress through the phases of a parallel randomised trial of two groups (that is, enrolment,
intervention allocation, follow-up, and data analysis). Figure 1 Flow diagram of the progress through the phases of a parallel randomised trial of two groups (that is, enrolment,
intervention allocation, follow-up, and data analysis). secondary indirect goal of our work. Moreover, CON-
SORT can help researchers in designing their trial. efforts provides an evidence base on which to update
the CONSORT statement. We add, drop, or modify
items based on that evidence and the recommendations
of the CONSORT Group, an international and eclectic
group of clinical trialists, statisticians, epidemiologists,
and biomedical editors. The CONSORT Executive (KFS,
DGA, DM) strives for a balance of established and
emerging researchers. The membership of the group is
dynamic. As our work expands in response to emerging
projects and needed expertise, we invite new members
to contribute. As such, CONSORT continually assimi-
lates new ideas and perspectives. That process informs
the continually evolving CONSORT statement. Implications and limitations We developed CONSORT 2010 to assist authors in
writing reports of randomised controlled trials, editors
and peer reviewers in reviewing manuscripts for publica-
tion, and readers in critically appraising published arti-
cles. The CONSORT 2010 Explanation and Elaboration
provides elucidation and context to the checklist items. We strongly recommend using the explanation and ela-
boration in conjunction with the checklist to foster
complete, clear, and transparent reporting and aid
appraisal of published trial reports. CONSORT 2010 focuses predominantly on the two
group, parallel randomised controlled trial, which
accounts for over half of trials in the literature [2]. Most
of the items from the CONSORT 2010 Statement, how-
ever, pertain to all types of randomised trials. Neverthe-
less, some types of trials or trial situations dictate the
need for additional information in the trial report. When in doubt, authors, editors, and readers should
consult the CONSORT website for any CONSORT
extensions, expansions (amplifications), implementa-
tions, or other guidance that may be relevant. After the meeting, the CONSORT Executive convened
teleconferences and meetings to revise the checklist. After seven major iterations, a revised checklist was dis-
tributed to the larger group for feedback. With that
feedback, the executive met twice in person to consider
all the comments and to produce a penultimate version. That served as the basis for writing the first draft of this
paper, which was then distributed to the group for feed-
back. After consideration of their comments, the execu-
tive finalised the statement. The evidence based approach we have used for CON-
SORT also served as a model for development of other
reporting guidelines, such as for reporting systematic
reviews and meta-analyses of studies evaluating inter-
ventions [16], diagnostic studies [17], and observational
studies [18]. The explicit goal of all these initiatives is to
improve reporting. The Enhancing the Quality and
Transparency of Health Research (EQUATOR) Network
will facilitate development of reporting guidelines and
help disseminate the guidelines: http://www.equator-
network.org provides information on all reporting guide-
lines in health research. The CONSORT Executive then drafted an updated
explanation and elaboration manuscript, with assistance
from other members of the larger group. The substance
of the 2007 CONSORT meeting provided the material
for the update. The updated explanation and elaboration
manuscript was distributed to the entire group for addi-
tions, deletions, and changes. That final iterative process
converged to the CONSORT 2010 Explanation and Ela-
boration [13]. Background to CONSORT We
added additional items either as a sub-item under an
existing item, an entirely new item number at the end
of the checklist, or (with item 3) an interjected item into
a renumbered segment. We have summarised the note-
worthy general changes in Appendix 1 and specific
changes in Appendix 2. The CONSORT website con-
tains a side by side comparison of the 2001 and 2010
versions. Development of CONSORT 2010 p
Thirty one members of the CONSORT 2010 Group met
in Montebello, Canada, in January 2007 to update the
2001 CONSORT statement. In addition to the accumu-
lating evidence relating to existing checklist items, sev-
eral new issues had come to prominence since 2001. Some participants were given primary responsibility for
aggregating and synthesising the relevant evidence on a
particular checklist item of interest. Based on that evi-
dence, the group deliberated the value of each item. As
in prior CONSORT versions, we kept only those items
deemed absolutely fundamental to reporting a rando-
mised controlled trial. Moreover, an item may be funda-
mental to a trial but not included, such as approval by
an institutional ethical review board, because funding
bodies strictly enforce ethical review and medical jour-
nals usually address reporting ethical review in their
instructions for authors. Other items may seem desir-
able, such as reporting on whether on-site monitoring
was done, but a lack of empirical evidence or any con-
sensus on their value cautions against inclusion at this
point. The CONSORT 2010 Statement thus addresses
the minimum criteria, although that should not deter
authors from including other information if they con-
sider it important. Implications and limitations With CONSORT 2010, we again intentionally declined
to produce a rigid structure for the reporting of rando-
mised trials. Indeed, SORT [19] tried a rigid format, and
it failed in a pilot run with an editor and authors [20]. Consequently, the format of articles should abide by
journal style, editorial directions, the traditions of the
research field addressed, and, where possible, author
preferences. We do not wish to standardise the structure
of reporting. Authors should simply address checklist Background to CONSORT Efforts to improve the reporting of randomised con-
trolled trials accelerated in the mid-1990s, spurred
partly by methodological research. Researchers had
shown for many years that authors reported such trials
poorly, and empirical evidence began to accumulate
that some poorly conducted or poorly reported aspects
of trials were associated with bias [14]. Two initiatives
aimed at developing reporting guidelines culminated in
one of us (DM) and Drummond Rennie organising the
first CONSORT statement in 1996 [5]. Further metho-
dological research on similar topics reinforced earlier
findings [15] and fed into the revision of 2001 [6-8]. Subsequently, the expanding body of methodological
research informed the refinement of CONSORT 2010. More than 700 studies comprise the CONSORT data-
base (located on the CONSORT website), which pro-
vides
the
empirical
evidence
to
underpin
the
CONSORT initiative. Over time, CONSORT has garnered much support. More than 400 journals, published around the world
and in many languages, have explicitly supported the
CONSORT statement. Many other healthcare journals
support it without our knowledge. Moreover, thousands
more have implicitly supported it with the endorsement
of the CONSORT statement by the International Com-
mittee of Medical Journal Editors http://www.icmje.org. Other prominent editorial groups, the Council of
Science Editors and the World Association of Medical
Editors, officially support CONSORT. That support Indeed, CONSORT Group members continually
monitor the literature. Information gleaned from these Page 3 of 8 Page 3 of 8 Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 seems warranted: when used by authors and journals,
CONSORT seems to improve reporting [9]. seems warranted: when used by authors and journals,
CONSORT seems to improve reporting [9]. kept the existing items under their previous item num-
bers except for some renumbering of items 2 to 5. We
added additional items either as a sub-item under an
existing item, an entirely new item number at the end
of the checklist, or (with item 3) an interjected item into
a renumbered segment. We have summarised the note-
worthy general changes in Appendix 1 and specific
changes in Appendix 2. The CONSORT website con-
tains a side by side comparison of the 2001 and 2010
versions. kept the existing items under their previous item num-
bers except for some renumbering of items 2 to 5. Changes in CONSORT 2010 The revision process resulted in evolutionary, not revo-
lutionary, changes to the checklist (Table 1), and the
flow diagram was not modified except for one word
(Figure 1). Moreover, because other reporting guidelines
augmenting the checklist refer to item numbers, we Page 4 of 8 Schulz et al. Changes in CONSORT 2010 Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Page 5 of 8 Table 1: CONSORT 2010 checklist of information to include when reporting a randomised trial* (Continued)
Interpretation
22
Interpretation consistent with results, balancing benefits and harms, and considering other
relevant evidence
Other information
Registration
23
Registration number and name of trial registry
Protocol
24
Where the full trial protocol can be accessed, if available
Funding
25
Sources of funding and other support (such as supply of drugs), role of funders
*We strongly recommend reading this statement in conjunction with the CONSORT 2010 Explanation and Elaboration [13] for important clarifications on all the
items. If relevant, we also recommend reading CONSORT extensions for cluster randomised trials [11], non-inferiority and equivalence trials [12], non-
pharmacological treatments [32], herbal interventions [33], and pragmatic trials [34]. Additional extensions are forthcoming: for those and for up to date
references relevant to this checklist, see http://www.consort-statement.org/. Table 1: CONSORT 2010 checklist of information to include when reporting a randomised trial* (Continued)
Interpretation
22
Interpretation consistent with results, balancing benefits and harms, and considering other
relevant evidence
Other information
Registration
23
Registration number and name of trial registry
Protocol
24
Where the full trial protocol can be accessed, if available
Funding
25
Sources of funding and other support (such as supply of drugs), role of funders CONSORT 2010 checklist of information to include when reporting a randomised trial* (Continued) Table 1: CONSORT 2010 checklist of information to include when reporting a randomised trial* (Continued)
Interpretation
22
Interpretation consistent with results, balancing benefits and harms, and considering other *We strongly recommend reading this statement in conjunction with the CONSORT 2010 Explanation and Elaboration [13] for impor
items. If relevant, we also recommend reading CONSORT extensions for cluster randomised trials [11], non-inferiority and equivalenc
pharmacological treatments [32], herbal interventions [33], and pragmatic trials [34]. Additional extensions are forthcoming: for thos
references relevant to this checklist, see http://www.consort-statement.org/. versions of CONSORT 2010, the CONSORT 2010
Explanation and Elaboration, and the CONSORT web-
site in their “Instructions to authors.” We suggest that
authors who wish to cite CONSORT should cite this or
another of the original journal versions of CONSORT
2010 Statement, and, if appropriate, the CONSORT
2010 Explanation and Elaboration [13]. All CONSORT
material can be accessed through the original publishing
journals or the CONSORT website. Appendix 1: Noteworthy general changes in
CONSORT 2010 Statement q
y
Nevertheless, we suggest that researchers begin trials
with their end publication in mind. Poor reporting
allows authors, intentionally or inadvertently, to escape
scrutiny of any weak aspects of their trials. However,
with wide adoption of CONSORT by journals and edi-
torial groups, most authors should have to report trans-
parently all important aspects of their trial. The ensuing
scrutiny rewards well conducted trials and penalises
poorly conducted trials. Thus, investigators should
understand the CONSORT 2010 reporting guidelines
before starting a trial as a further incentive to design
and conduct their trials according to rigorous standards. CONSORT 2010 supplants the prior version published
in 2001. Any support for the earlier version accumulated
from journals or editorial groups will automatically
extend to this newer version, unless specifically
requested otherwise. Journals that do not currently sup-
port CONSORT may do so by registering on the CON-
SORT website. If a journal supports or endorses
CONSORT 2010, it should cite one of the original • We simplified and clarified the wording, such as in
items 1, 8, 10, 13, 15, 16, 18, 19, and 21 Nevertheless, we suggest that researchers begin trials
with their end publication in mind. Poor reporting
allows authors, intentionally or inadvertently, to escape
scrutiny of any weak aspects of their trials. However,
with wide adoption of CONSORT by journals and edi-
torial groups, most authors should have to report trans-
parently all important aspects of their trial. The ensuing
scrutiny rewards well conducted trials and penalises
poorly conducted trials. Thus, investigators should
understand the CONSORT 2010 reporting guidelines
before starting a trial as a further incentive to design
and conduct their trials according to rigorous standards. • We improved consistency of style across the items
by removing the imperative verbs that were in the 2001
version • We enhanced specificity of appraisal by breaking
some items into sub-items. Many journals expect
authors to complete a CONSORT checklist indicating
where in the manuscript the items have been
addressed. Experience with the checklist noted prag-
matic difficulties when an item comprised multiple
elements. For example, item 4 addresses eligibility of
participants and the settings and locations of data
collection. With the 2001 version, an author could
provide a page number for that item on the checklist,
but might have reported only eligibility in the paper,
for example, and not reported the settings and
locations. Changes in CONSORT 2010 Groups or indivi-
duals who desire to translate the CONSORT 2010 State-
ment into other languages should first consult the
CONSORT policy statement on the website. items somewhere in the article, with ample detail and
lucidity. That stated, we think that manuscripts benefit
from frequent subheadings within the major sections,
especially the methods and results sections. CONSORT urges completeness, clarity, and transpar-
ency of reporting, which simply reflects the actual trial
design and conduct. However, as a potential drawback,
a reporting guideline might encourage some authors to
report fictitiously the information suggested by the gui-
dance rather than what was actually done. Authors, peer
reviewers, and editors should vigilantly guard against
that potential drawback and refer, for example, to trial
protocols, to information on trial registers, and to regu-
latory agency websites. Moreover, the CONSORT 2010
Statement does not include recommendations for
designing and conducting randomised trials. The items
should elicit clear pronouncements of how and what the
authors did, but do not contain any judgments on how
and what the authors should have done. Thus, CON-
SORT 2010 is not intended as an instrument to evaluate
the quality of a trial. Nor is it appropriate to use the
checklist to construct a “quality score.” We emphasise that CONSORT 2010 represents an
evolving guideline. It requires perpetual reappraisal and,
if necessary, modifications. In the future we will further
revise the CONSORT material considering comments,
criticisms, experiences, and accumulating new evidence. We invite readers to submit recommendations via the
CONSORT website. Changes in CONSORT 2010 Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Page 4 of 8 Table 1 CONSORT 2010 checklist of information to include when reporting a randomised trial*
Section/Topic
Item
No
Checklist item
Re
pa
Title and abstract
1a
Identification as a randomised trial in the title
1b
Structured summary of trial design, methods, results, and conclusions (for specific
guidance see CONSORT for abstracts [21,31])
Introduction
Background and objectives
2a
Scientific background and explanation of rationale
2b
Specific objectives or hypotheses
Methods
Trial design
3a
Description of trial design (such as parallel, factorial) including allocation ratio
3b
Important changes to methods after trial commencement (such as eligibility criteria), with
reasons
Participants
4a
Eligibility criteria for participants
4b
Settings and locations where the data were collected
Interventions
5
The interventions for each group with sufficient details to allow replication, including how
and when they were actually administered
Outcomes
6a
Completely defined pre-specified primary and secondary outcome measures, including
how and when they were assessed
6b
Any changes to trial outcomes after the trial commenced, with reasons
Sample size
7a
How sample size was determined
7b
When applicable, explanation of any interim analyses and stopping guidelines
Randomisation:
Sequence generation
8a
Method used to generate the random allocation sequence
8b
Type of randomisation; details of any restriction (such as blocking and block size)
Allocation concealment
mechanism
9
Mechanism used to implement the random allocation sequence (such as sequentially
numbered containers), describing any steps taken to conceal the sequence until
interventions were assigned
Implementation
10
Who generated the random allocation sequence, who enrolled participants, and who
assigned participants to interventions
Blinding
11a
If done, who was blinded after assignment to interventions (for example, participants, care
providers, those assessing outcomes) and how
11b
If relevant, description of the similarity of interventions
Statistical methods
12a
Statistical methods used to compare groups for primary and secondary outcomes
12b
Methods for additional analyses, such as subgroup analyses and adjusted analyses
Results
Participant flow (a diagram is
strongly recommended)
13a
For each group, the numbers of participants who were randomly assigned, received
intended treatment, and were analysed for the primary outcome
13b
For each group, losses and exclusions after randomisation, together with reasons
Recruitment
14a
Dates defining the periods of recruitment and follow-up
14b
Why the trial ended or was stopped
Baseline data
15
A table showing baseline demographic and clinical characteristics for each group
Numbers analysed
16
For each group, number of participants (denominator) included in each analysis and
whether the analysis was by original assigned groups
Outcomes and estimation
17a
For each primary and secondary outcome, results for each group, and the estimated
effect size and its precision (such as 95% confidence interval)
17b
For binary outcomes, presentation of both absolute and relative effect sizes is
recommended
Ancillary analyses
18
Results of any other analyses performed, including subgroup analyses and adjusted
analyses, distinguishing pre-specified from exploratory
Harms
19
All important harms or unintended effects in each group (for specific guidance see
CONSORT for harms [28])
Discussion
Limitations
20
Trial limitations, addressing sources of potential bias, imprecision, and, if relevant,
multiplicity of analyses
Generalisability
21
Generalisability (external validity, applicability) of the trial findings Reported on
page No 20
Trial limitations, addressing sources of potential bias, imprecision, and, if relevant,
multiplicity of analyses 21
Generalisability (external validity, applicability) of the trial findings Schulz et al. Appendix 2: Noteworthy specific changes in
CONSORT 2010 Statement Item 1b (title and abstract)-We added a sub-item on
providing a structured summary of trial design, meth-
ods, results, and conclusions and referenced the CON-
SORT for abstracts article [21]. Sub-item 17b (outcomes and estimation)-For appropri-
ate clinical interpretability, prevailing experience sug-
gested
the
addition
of
“For
binary
outcomes,
presentation of both relative and absolute effect sizes is
recommended” [27] Item 2b (introduction)-We added a new sub-item (for-
merly item 5 in CONSORT 2001) on “Specific objectives
or hypotheses” Item 19 (harms)-We included a reference to the CON-
SORT paper on harms [28] Item 3a (trial design)-We added a new item including
this sub-item to clarify the basic trial design (such as par-
allel group, crossover, cluster) and the allocation ratio Item 20 (limitations)-We changed the topic from
“Interpretation” and supplanted the prior text with a
sentence focusing on the reporting of sources of poten-
tial bias and imprecision Item 3b (trial design)-We added a new sub-item that
addresses any important changes to methods after trial
commencement, with a discussion of reasons Item 4 (participants)-Formerly item 3 in CONSORT
2001 Item 22 (interpretation)-We changed the topic from
“Overall evidence.” Indeed, we understand that authors
should be allowed leeway for interpretation under this
nebulous heading. However, the CONSORT Group
expressed concerns that conclusions in papers fre-
quently misrepresented the actual analytical results and
that harms were ignored or marginalised. Therefore, we
changed the checklist item to include the concepts of
results matching interpretations and of benefits being
balanced with harms Item 5 (interventions)-Formerly item 4 in CONSORT
2001. We encouraged greater specificity by stating that
descriptions of interventions should include “sufficient
details to allow replication”[3] Item 6 (outcomes)-We added a sub-item on identifying
any changes to the primary and secondary outcome
(endpoint) measures after the trial started. This followed
from empirical evidence that authors frequently provide
analyses of outcomes in their published papers that
were not the prespecified primary and secondary out-
comes in their protocols, while ignoring their prespeci-
fied outcomes (that is, selective outcome reporting)
[4,22]. We eliminated text on any methods used to
enhance the quality of measurements Item 23 (registration)-We added a new item on trial
registration. Empirical evidence supports the need for
trial registration, and recent requirements by journal
editors have fostered compliance [29] Item 24 (protocol)-We added a new item on availabil-
ity of the trial protocol. Appendix 1: Noteworthy general changes in
CONSORT 2010 Statement CONSORT 2010 relieves obfuscations
and forces authors to provide page numbers in the
checklist for both eligibility and settings. CONSORT 2010 supplants the prior version published
in 2001. Any support for the earlier version accumulated
from journals or editorial groups will automatically
extend to this newer version, unless specifically
requested otherwise. Journals that do not currently sup-
port CONSORT may do so by registering on the CON-
SORT website. If a journal supports or endorses
CONSORT 2010, it should cite one of the original Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Page 6 of 8 Item 16 (numbers analysed)-We replaced mention of
“intention to treat” analysis, a widely misused term, by a
more explicit request for information about retaining
participants in their original assigned groups [26] Appendix 2: Noteworthy specific changes in
CONSORT 2010 Statement Empirical evidence suggests that
authors often ignore, in the conduct and reporting of
their trial, what they stated in the protocol [4,22]. Hence, availability of the protocol can instigate adher-
ence to the protocol before publication and facilitate
assessment of adherence after publication Item 9 (allocation concealment mechanism)-We
reworded this to include mechanism in both the report
topic and the descriptor to reinforce that authors should
report the actual steps taken to ensure allocation con-
cealment rather than simply report imprecise, perhaps
banal, assurances of concealment Item 25 (funding)-We added a new item on funding. Empirical evidence points toward funding source
sometimes being associated with estimatedeatment
effects [30] Item 11 (blinding)-We added the specification of how
blinding was done and, if relevant, a description of the
similarity of interventions and procedures. We also elimi-
nated text on “how the success of blinding (masking) was
assessed” because of a lack of empirical evidence support-
ing the practice as well as theoretical concerns about the
validity of any such assessment [23,24] g
The CONSORT Group contributors to CONSORT 2010: DG Altman, Centre for
Statistics in Medicine, University of Oxford; Virginia Barbour, PLoS Medicine;
Jesse A Berlin, Johnson & Johnson Pharmaceutical Research and
Development, USA; Isabelle Boutron, University Paris 7 Denis Diderot,
Assistance Publique des Hôpitaux de Paris, INSERM, France; PJ Devereaux,
McMaster University, Canada; Kay Dickersin, Johns Hopkins Bloomberg
School of Public Health, USA; Diana Elbourne, London School of Hygiene &
Tropical Medicine; Susan Ellenberg, University of Pennsylvania School of
Medicine, USA; Val Gebski, University of Sydney, Australia; Steven Goodman,
Journal of the Society for Clinical Trials, USA; Peter C Gøtzsche, Nordic
Cochrane Centre, Denmark; Trish Groves, BMJ; Steven Grunberg, American
Society of Clinical Oncology, USA; Brian Haynes, McMaster University,
Canada; Sally Hopewell, Centre for Statistics in Medicine, University of
Oxford; Astrid James, Lancet; Peter Juhn, Johnson & Johnson, USA; Philippa Competing interests Uniform disclosure of potential conflicts of interest: all authors have
completed the ICMJE unified competing interest form at http://www.icmje. org/coi_disclosure.pdf (available from the corresponding author) and declare
(1) DM received grants for this work from Johnson & Johnson, BMJ, and
American Society for Clinical Oncology; KFS and DGA received support for
travel to meetings for this work from Johnson & Johnson, BMJ, and
American Society for Clinical Oncology; (2) KFS and DA had travel expenses
reimbursed by the EQUATOR Network; KFS has received honoraria for
delivering educational presentations for the American Board of Obstetrics
and Gynecology Foundation for Excellence in Women’s Health Care, Ortho-
McNeil Janssen Scientific Affairs, and the American College of Obstetrics and
Gynecology; and has done consultancy for Wyeth. All authors also declare
(3) no spouses, partners, or children with relationships with commercial
entities that might have an interest in the submitted work; (4) no non-
financial interests that may be relevant to the submitted work. 18. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC,
Vandenbroucke JP, for the STROBE Initiative: Strengthening the reporting
of observational studies in epidemiology (STROBE) statement: guidelines
for reporting observational studies. BMJ 2007, 335:806-8. 19. Standards of Reporting Trials Group: A proposal for structured reporting
of randomized controlled trials. JAMA 1994, 272:1926-31. 19. Standards of Reporting Trials Group: A proposal for structured reporting
of randomized controlled trials. JAMA 1994, 272:1926-31. 20. Rennie D: Reporting randomized controlled trials. An experiment and a
call for responses from readers. JAMA 1995, 273:1054-5. 20. Rennie D: Reporting randomized controlled trials. An experiment and a
call for responses from readers. JAMA 1995, 273:1054-5. 21. Hopewell S, Clarke M, Moher D, Wager E, Middleton P, Altman DG,
Schulz KF, CONSORT Group: CONSORT for reporting randomised trials in
journal and conference abstracts. Lancet 2008, 371:281-3. 22. Chan AW, Hróbjartsson A, Haahr MT, Gøtzsche PC, Altman DG: Empirical
evidence for selective reporting of outcomes in randomized trials:
comparison of protocols to published articles. JAMA 2004, 291:2457-65. 23. Sackett DL: Commentary: Measuring the success of blinding in RCTs:
don’t, must, can’t or needn’t?. Int J Epidemiol 2007, 36:664-5. Received: 9 March 2010 Accepted: 24 March 2010
Published: 24 March 2010 Received: 9 March 2010 Accepted: 24 March 2010
Published: 24 March 2010 24. Schulz KF, Grimes DA: Blinding in randomised trials: hiding who got
what. Lancet 2002, 359:696-700. 25. Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 3. Glasziou P, Meats E, Heneghan C, Shepperd S: What is missing from
descriptions of treatment in trials and reviews? BMJ 2008, 336:1472-4. Middleton, University of Adelaide, Australia; Don Minckler, University of
California Irvine, USA; D Moher, Ottawa Methods Centre, Clinical
Epidemiology Program, Ottawa Hospital Research Institute, Canada; Victor M
Montori, Knowledge and Encounter Research Unit, Mayo Clinic College of
Medicine, USA; Cynthia Mulrow, Annals of Internal Medicine, USA; Stuart
Pocock, London School of Hygiene & Tropical Medicine; Drummond Rennie,
JAMA, USA; David L Schriger, Annals of Emergency Medicine, USA; KF Schulz,
Family Health International, USA; Iveta Simera, EQUATOR Network; Elizabeth
Wager, Sideview. 4. Dwan K, Altman DG, Arnaiz JA, Bloom J, Chan AW, Cronin E, Decullier E,
Easterbrook PJ, Von Elm E, Gamble C, Ghersi D, Ioannidis JP, Simes J,
Williamson PR: Systematic review of the empirical evidence of study
publication bias and outcome reporting bias. PLoS ONE 2008, 3:e3081. 5. Begg C, Cho M, Eastwood S, Horton R, Moher D, Olkin I, Pitkin R, Rennie D
Schulz KF, Simel D, Stroup DF: Improving the quality of reporting of
randomized controlled trials. The CONSORT statement. JAMA 1996,
276:637-9. Contributors to CONSORT 2010 who did not attend the Montebello
meeting: Mike Clarke, UK Cochrane Centre; Gordon Guyatt, McMaster
University, Canada. 6. Moher D, Schulz KF, Altman DG: The CONSORT statement: revised
recommendations for improving the quality of reports of parallel-group
randomised trials. Lancet 2001, 357:1191-4. Funding: We received financial support from United Kingdom National
Institute for Health Research and the Medical Research Council; Canadian
Institutes of Health Research; Presidents Fund, Canadian Institutes of Health
Research; Johnson & Johnson; BMJ; and the American Society for Clinical
Oncology. DGA is supported by Cancer Research UK, DM by a University of
Ottawa Research Chair, and KFS by Family Health International. None of the
sponsors had any involvement in the planning, execution, or writing of the
CONSORT documents. Additionally, no funder played a role in drafting the
manuscript. 7. Moher D, Schulz KF, Altman DG: The CONSORT statement: revised
recommendations for improving the quality of reports of parallel-group
randomized trials. Ann Intern Med 2001, 134:657-62. 8. Moher D, Schulz KF, Altman D: The CONSORT statement: revised
recommendations for improving the quality of reports of parallel-group
randomized trials. JAMA 2001, 285:1987-91. 9. Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Plint AC, Moher D, Morrison A, Schulz K, Altman DG, Hill C, Gaboury : Does
the CONSORT checklist improve the quality of reports of randomised
controlled trials? A systematic review. Med J Aust 2006, 185:263-7. In order to encourage dissemination of the CONSORT 2010 Statement, this
article is freely accessible on bmj.com and will also be published in the
Lancet, Obstetrics and Gynecology, PLoS Medicine, Annals of Internal
Medicine, Open Medicine, Journal of Clinical Epidemiology, BMC Medicine,
and Trials. The authors jointly hold the copyright of this article. For details
on further use, see the CONSORT website http://www.consort-statement.org. 10. Hopewell S, Dutton S, Yu L-M, Chan A-W, Altman DG: The quality of
reports of randomised trials in 2000 and 2006: a comparative study of
articles indexed by PubMed. BMJ 2010, 340:c723. 11. Campbell MK, Elbourne DR, Altman DG: CONSORT statement: extension to
cluster randomised trials. BMJ 2004, 328:702-8. 12. Piaggio G, Elbourne DR, Altman DG, Pocock SJ, Evans SJ: Reporting of
noninferiority and equivalence randomized trials: an extension of the
CONSORT statement. JAMA 2006, 295:1152-60. Author details
1 1Family Health International, Research Triangle Park, NC 27709, USA. 2Centre
for Statistics in Medicine, University of Oxford, Wolfson College, Oxford, UK. 3Ottawa Methods Centre, Clinical Epidemiology Program, Ottawa Hospital
Research Institute, Department of Epidemiology and Community Medicine,
University of Ottawa, Ottawa, Canada. 13. Moher D, Hopewell S, Schulz KF, Montori V, Gøtzsche PC, Devereaux PJ,
et al: CONSORT 2010 Explanation and Elaboration: updated guidelines
for reporting parallel group randomised trials. BMJ 2010, 340:c869. 14. Schulz KF, Chalmers I, Hayes RJ, Altman DG: Empirical evidence of bias. Dimensions of methodological quality associated with estimates of
treatment effects in controlled trials. JAMA 1995, 273:408-12. Competing interests Montori VM, Devereaux PJ, Adhikari NK, Burns KE, Eggert CH, Briel M,
Lacchetti C, Leung TW, Darling E, Bryant DM, Bucher HC, Schünemann HJ,
Meade MO, Cook DJ, Erwin PJ, Sood A, Sood R, Lo B, Thompson CA,
Zhou Q, Mills E, Guyatt GH: Randomized trials stopped early for benefit: a
systematic review. JAMA 2005, 294:2203-9. Authors’ contributions 15. Moher D, Pham B, Jones A, Cook DJ, Jadad AR, Moher M, Tugwell P,
Klassen TP: Does quality of reports of randomised trials affect estimates
of intervention efficacy reported in meta-analyses? Lancet 1998,
352:609-13. KFS, DM, and DGA participated in meetings and regular conference calls,
planned the CONSORT 2007 meeting at Montebello, developed the agenda,
prepared background research, identified and invited participants,
contributed to the CONSORT meeting, drafted the manuscript, and, after
critical review by the CONSORT Group, finalised the text of the manuscript. Members of the CONSORT Group attended the meeting, except for those
noted below, and provided input on and review of the revised checklist and
text of this article. Some members also prepared background material. 16. Moher D, Liberati A, Tetzlaff J, Altman DG, for the PRISMA Group: Preferred
reporting items for systematic reviews and meta-analyses: the PRISMA
statement. BMJ 2009, 339:b2535. 17. Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziou PP, Irwig LM,
Lijmer JG, Moher D, Rennie D, de Vet HC, Standards for Reporting of
Diagnostic Accuracy: Towards complete and accurate reporting of studies
of diagnostic accuracy: the STARD initiative. BMJ 2003, 326:41-4. 2.
Chan AW, Altman DG: Epidemiology and reporting of randomised trials
published in PubMed journals. Lancet 2005, 365:1159-62. Acknowledgements
The CONSORT Group g
The CONSORT Group contributors to CONSORT 2010: DG Altman, Centre for
Statistics in Medicine, University of Oxford; Virginia Barbour, PLoS Medicine;
Jesse A Berlin, Johnson & Johnson Pharmaceutical Research and
Development, USA; Isabelle Boutron, University Paris 7 Denis Diderot,
Assistance Publique des Hôpitaux de Paris, INSERM, France; PJ Devereaux,
McMaster University, Canada; Kay Dickersin, Johns Hopkins Bloomberg
School of Public Health, USA; Diana Elbourne, London School of Hygiene &
Tropical Medicine; Susan Ellenberg, University of Pennsylvania School of
Medicine, USA; Val Gebski, University of Sydney, Australia; Steven Goodman,
Journal of the Society for Clinical Trials, USA; Peter C Gøtzsche, Nordic
Cochrane Centre, Denmark; Trish Groves, BMJ; Steven Grunberg, American
Society of Clinical Oncology, USA; Brian Haynes, McMaster University,
Canada; Sally Hopewell, Centre for Statistics in Medicine, University of
Oxford; Astrid James, Lancet; Peter Juhn, Johnson & Johnson, USA; Philippa Item 12a (statistical methods)-We added that statisti-
cal methods should also be provided for analysis of sec-
ondary outcomes Sub-item 14b (recruitment)-Based on empirical
research, we added a sub-item on “Why the trial ended
or was stopped” [25] Sub-item 14b (recruitment)-Based on empirical
research, we added a sub-item on “Why the trial ended
or was stopped” [25] Item 15 (baseline data)-We specified “A table” to clar-
ify that baseline and clinical characteristics of each
group are most clearly expressed in a table Page 7 of 8 Page 7 of 8 1.
Jüni P, Altman DG, Egger M: Systematic reviews in health care: assessing
the quality of controlled clinical trials. BMJ 2001, 323:42-6. Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 26. Hollis S, Campbell F: What is meant by intention to treat analysis? Survey
of published randomised controlled trials. BMJ 1999, 319:670-4. 27. Nuovo J, Melnikow J, Chang D: Reporting number needed to treat and
absolute risk reduction in randomized controlled trials. JAMA 2002,
287:2813-4. 28. Ioannidis JP, Evans SJ, Gøtzsche PC, O’Neill RT, Altman DG, Schulz K,
Moher D, CONSORT Group: Better reporting of harms in randomized
trials: an extension of the CONSORT statement. Ann Intern Med 2004,
141:781-8. 29. De Angelis C, Drazen JM, Frizelle FA, Haug C, Hoey J, Horton R, Kotzin S,
Laine C, Marusic A, Overbeke AJ, Schroeder TV, Sox HC, Weyden Van Der
MB: International Committee of Medical Journal Editors. Clinical trial
registration: a statement from the International Committee of Medical
Journal Editors. Lancet 2004, 364:911-2. 30. Lexchin J, Bero LA, Djulbegovic B, Clark O: Pharmaceutical industry
sponsorship and research outcome and quality: systematic review. BMJ
2003, 326:1167-70. 31. Hopewell S, Clarke M, Moher D, Wager E, Middleton P, Altman DG,
Schulz KF, CONSORT Group: CONSORT for reporting randomized
controlled trials in journal and conference abstracts: explanation and
elaboration. PLoS Med 2008, 5:e20. 31. Hopewell S, Clarke M, Moher D, Wager E, Middleton P, Altman DG,
Schulz KF, CONSORT Group: CONSORT for reporting randomized
controlled trials in journal and conference abstracts: explanation and
elaboration. PLoS Med 2008, 5:e20. 32. Boutron I, Moher D, Altman DG, Schulz KF, Ravaud P: Extending the
CONSORT statement to randomized trials of nonpharmacologic
treatment: explanation and elaboration. Ann Intern Med 2008,
148:295-309. 33. Gagnier JJ, Boon H, Rochon P, Moher D, Barnes J, Bombardier C: Reporting
randomized, controlled trials of herbal interventions: an elaborated
CONSORT statement. Ann Intern Med 2006, 144:364-7. 34. Zwarenstein M, Treweek S, Gagnier JJ, Altman DG, Tunis S, Haynes B,
Oxman AD, Moher D, CONSORT group: Pragmatic Trials in Healthcare
(Practihc) group. Improving the reporting of pragmatic trials: an
extension of the CONSORT statement. BMJ 2008, 11(337):a2390. doi:10.1186/1745-6215-11-32
Cite this article as: Schulz et al.: CONSORT 2010 Statement: updated
guidelines for reporting parallel group randomised trials. Trials 2010
11:32. References Page 8 of 8 Schulz et al. Trials 2010, 11:32
http://www.trialsjournal.com/content/11/1/32 Submit your next manuscript to BioMed Central
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Mary-Anne Plaatjies-van Huffel
|
Stellenbosch theological journal
| 2,024
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cc-by
| 6,814
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2
Miranda Pillay is Extraordinary Professor at the University of the Western Cape,
affiliated with the Desmond Tutu Centre for Religion and Social Justice which is
jointly supported by the Lund Mission Society and the National Research Foundation
of South Africa (Grant number 118854). The author acknowledges that the opinions,
findings, and conclusions are those of the author alone, and the NRF accepts no liability
whatsoever in this regard. Stellenbosch Theological Journal 2023, Vol 9, No 1, 1–18
DOI: http://dx.doi.org/10.17570/stj.2023.v9n1.a33
Online ISSN 2413-9467 | Print ISSN 2413-9459
2023 © The Author(s) Stellenbosch Theological Journal 2023, Vol 9, No 1, 1–18
DOI: http://dx.doi.org/10.17570/stj.2023.v9n1.a33
Online ISSN 2413-9467 | Print ISSN 2413-9459
2023 © The Author(s) 1
Paper presented at “The Fourth Mary-Anne Plaatjies-Van Huffel Memorial Lecture”,
Faculty of Theology, Stellenbosch University, 11 August 2023. Abstracth There’s no doubt that Mary-Ann Plaatjies-van Huffel is amongst the women who “have
moved into the academy, assumed religious leadership, and claimed their religious
agency and heritage”. However, as a woman of colour Plaatjies-van Huffel’s life and
work reveal that she had to navigate her leadership and exercise her agency along a
well-beaten patriarchal beaten track. In this article I foreground some “first woman
to…” milestones on Plaatjies-van Huffel’s trailblazing journey through the ecclesial
ranks of the Uniting Reformed Church in Southern Africa (URCSA), highlighting that
her academic research and community engagement reflect the social, economic, and
political realities of racism and sexism, and its complex ramifications in post-apartheid
South Africa. The main argument I make in this article is that, while women may no
longer be excluded from leadership positions, it is second-generation gender bias that
maintains the patriarchal beaten track in “the church”. Thus, I call for the debunking
of second-generation gender bias which, I argue, will require a virtue of unctuousness. Mary-Anne Plaatjies-van Huff el: Trailblazing
journey off the patriarchal beaten track1 Miranda Pillay2
University of the Western Cape
mpillay@uwc.ac.za 1
Paper presented at “The Fourth Mary-Anne Plaatjies-Van Huffel Memorial Lecture”,
Faculty of Theology, Stellenbosch University, 11 August 2023.
2
Miranda Pillay is Extraordinary Professor at the University of the Western Cape,
affiliated with the Desmond Tutu Centre for Religion and Social Justice which is
jointly supported by the Lund Mission Society and the National Research Foundation
of South Africa (Grant number 118854). The author acknowledges that the opinions,
findings, and conclusions are those of the author alone, and the NRF accepts no liability
whatsoever in this regard. Keywords Mary-Anne Plaatjies-van Huffel; debunk; second-generation gender bias; patriarchy;
church; virtue of unctuousness Pillay • STJ 2023, Vol 9, No 1, 1–18 2 5
For example, in a statement following Plaatjies van Huffel’s death, the World Council of
Churches notes that Plaatjies-van Huffel was known as a transformative church leader
in sub-Saharan Africa. Her significance was not only rooted in her leadership positions,
her many theological publications, and her lecturing status, but could also be found in
her active participation in processes to transform society. [Online]. Available: https://
www.oikoumene.org/news/wcc-mourns-passing-of-rev-prof-dr-mary-anne-plaatjies-
van-huffel [Accessed: 31 July 2023]. 3
I use “the church” here, with an understanding of the “variety of manifestation of
‘church’”. See for example Dirkie Smit (1996:119–129); cf. Pillay (2008:172).
4
See for example Landman (2021); Kgatla (2021); Zeze (2021); Nel (2021); Baloi (2022);
Flaendorp (2014).
5
For example, in a statement following Plaatjies van Huffel’s death, the World Council of
Churches notes that Plaatjies-van Huffel was known as a transformative church leader
in sub-Saharan Africa. Her significance was not only rooted in her leadership positions,
her many theological publications, and her lecturing status, but could also be found in
her active participation in processes to transform society. [Online]. Available: https://
www.oikoumene.org/news/wcc-mourns-passing-of-rev-prof-dr-mary-anne-plaatjies-
van-huffel [Accessed: 31 July 2023]. 3
I use “the church” here, with an understanding of the “variety of manifestation of
‘church’”. See for example Dirkie Smit (1996:119–129); cf. Pillay (2008:172). e for example Landman (2021); Kgatla (2021); Zeze (2021); Nel (2021); Baloi (2022);
endorp (2014). f Introductory remarks: celebrate, commemorate, commiserate This paper was presented at the Fourth Mary-Anne Plaatjies van Huffel
Memorial Lecture held at the Faculty of Theology, Stellenbosch University
on 11 August 2023. I was asked to focus on Plaatjies-van Huffel’s leadership
legacy in “the church”.3 Much has already been written about Plaatjies-
van Huffel’s leadership in the church and the academy4 – particularly
in response to her death on 19 May 2020. Many colleagues with whom
she served in the academy and the church reflect on her leadership a as
“visionary, participative and transformative”.5 In preparing to also add my voice to celebrating and commemorating
Plaatjies-van Huffel’s leadership legacy, I was reminded that her work,
like that of many other South African theologians exhibit a social justice
framing that grapple with the socio-cultural-political-economic realities
in racist and sexist contexts. It is particularly the complex socio-economic
ramifications of patriarchal normativity in post-apartheid South Africa
that were cause for great concern, frustration, and disappointment for
Plaatjies-van Huffel. This observation is made by Nel (2021:2) who says that
Plaatjies-van Huffel “wrestled with the moments of deep disappointment
and the ongoing trauma experienced through structures and ecclesial
cultures that remained deeply patriarchal – deeply insensitive and violent”. Thus, in calling to remembrance Plaatjies-van Huffel’s leadership amongst
communities who work the vineyards and those who own the vineyards
(literally and figurative speaking) – from Robertson and Scottsdene to
Stellenbosch; from Prieska to Priesthood; from classrooms to boardrooms;
from Wellington to the World Council of Churches, one can sense the Pillay • STJ 2023, Vol 9, No 1, 1–18 3 tension between celebration and commiseration. This is particularly the
case for me as the memorial lecture took place on 11 August 2023. In South
Africa, August is celebrated as Women’s Month in commemoration of the
agency of women who marched against South Africa’s apartheid laws on
9 August 1956.6 But sadly, almost seven decades later, and almost thirty
years into democracy, South African women continue to march with
placards conveying despair #AmINext; frustration #EnoughIsEnough;
hope #IAmNotNext; and solidarity (if not courage) #MeToo.7 Thus, while we pause to celebrate and commemorate the leadership legacy
of Plaatjies-van Huffel we are also called commiserate – to lament and ask:
How many more trailblazing women leaders will it take for gender justice
to be taken seriously in ecclesial and academic spaces? 6
See
https://www.sahistory.org.za/article/1956-womens-march-pretoria-9-august.
[Accessed:1 August 2023].
7
See Wilma Jakobson and Miranda Pillay (2022:6) 7
See Wilma Jakobson and Miranda Pillay (2022:6) 8
A term coined by Paul Ricoeur which, according to Felski, he notes is a commitment
to “unmasking the lies and illusions of consciousness” (2011:1), Schüssler Fiorenza
identifies a feminist hermeneutic of suspicion as an approach that questions
androcentrism and male privilege exhibited explicitly and implicitly in texts. However,
like many feminist theologians, it is my view that uncovering the oppression justified
and maintained by patriarchal realities and readings of texts is an important move
towards discovering possible liberative potential of texts in efforts to recover the full
humanity of women, men and also those who do not identify with the stereo typical
gender binaries. Introductory remarks: celebrate, commemorate, commiserate Second, there’s
need to commiserate the fact that the general, if not natural expectation,
is that women who are “allowed” into leadership positions, are expected
to lead on a patriarchal beaten track. In other words, women’s trailblazing
accomplishments are lauded as long as they do not break the barriers which
keep women in their stereotypical gendered lane. Third, there is a need
to commiserate the fact that trailblazing women are burdened over and
over again, generation after generation with the ongoing task of clearing
weeds that cushion the patriarchal beaten track of male privilege. To
commiserate, to lament in the context of celebration and commemoration,
is to give us the liberty to ask with a hermeneutic of suspicion: What is
it about the leadership of women like Plaatjies-van Huffel that we ought
to celebrate? What are the “real” celebratory milestones? Or are they
millstone adornments to keep women on the patriarchal normative
track? What might it take for trailblazing women leaders to set previously
male dominated ecclesial and academic spaces ablaze to erase the weed-
cushioned patriarchal beaten track? With these questions in mind, I will explore the virtue of unctuousness
as intervention against the subtle, pervasive, less obvious, sometimes
patronizing and often times justified patriarchal normative culture of “the Pillay • STJ 2023, Vol 9, No 1, 1–18 4 church”. First, I foreground some of Plaatjies-van Huffel’s “first woman to
…” accolades with a hermeneutic of suspicion.8 10 See for example Plaatjies-van Huffel (2011:3). 9
Her research and writing reflect her lived experiences as a woman of colour “born,
raised and educated during the hight of apartheid”. See Selaelo Thias Kgatla (2021) On navigating leadership as a woman of colour in “previously”
male-dominated spaces There’s no doubt that Plaatjies-van Huffel is amongst the women who,
according to Schüssler Fiorenza, “have moved into the academy, assumed
religious leadership, and claimed their religious agency and heritage”
(2011:4). However, as a woman of colour Plaatjies-van Huffel had to
navigate her leadership and exercise her agency along a patriarchal beaten
track where somewhat faded white privilege signposts still remain. This
observation is made on the bases of Plaatjies van Huffel’s lived experiences
as taken up in her academic writings and community engagement.9 For
example, much of her research is framed within the social, economic, and
political realities of racism and sexism in South Africa in general, and its
complex ramifications in post-apartheid South Africa, in particular.10 Second, while Plaatjies-van Huffel’s publications exhibit how the
intersections of race, class, gender, and ability continue to impact the lives
and livelihood of different people differently in a democratic South Africa,
it is with reflexivity and an awareness that, what she knows about the
experiences of “othered” persons and communities is not everything there
is to know. Employing a post-structural feminist discourse to deconstruct
dualisms operative in western epistemologies Plaatjies-van Huffel’s
work emphasises the fact that such dualisms not only serve to justify
and maintain gender binaries, but they also function as justification for Pillay • STJ 2023, Vol 9, No 1, 1–18 5 5 domination on the basis of class, race, and ethnicity (Plaatjies-van Huffel
2008:94; 2011a:110–113 Graff 1995:26;). Thus, she calls for a “move away
from a dualistic, hierarchical, anthropocentric, androcentric, patriarchal
framework of thinking” (2011b:8). Third, Plaatjies-van Huffel’s lived-experience reveals the relentless task
it is for women of colour to confront racial and gender biases. Moreover,
when pushback against such confrontations is justified by the expectation
that women leaders are required to stay on the paternalistic, androcentric
paths of the patriarchal beaten track, the task is not only taxing, but also
frustrating in the South African context where gender discrimination is
unconstitutional. In light of this observation, Plaatjies-van Huffel laments: Despite the enacted legislation, ratification of international and
regional instruments and formulation of numerous gender policies,
the engendering of society has not advanced at the desired rate. The
dominant discourses which imprisoned women and men remain
the same. Essentially, nothing has changed. No major paradigm
shifts with regard to gendered objects have come to pass. Only a few
modifications of the human image have taken place (2011:6). 11 While “gatvol” is an Afrikaans word which literally means “full to the brim” and
“fed-up”, it is also defined as a South African English adjective meaning “very upset or
extremely unhappy” (https://www.oxfordlearnersdictionaries.com/definition/english/
gatvol [Accessed: 8 August 2023]. 13 See video on “Gatvol: ACSA Women in conversation with The Most Revd Dr Thabo
Makgoba, https://www.youtube.com/watch?v=k4LIOOVkhSI [Accessed: 1 August
2023]. See the statement https://volmoedyouth.files.wordpress.com/2020/08/final-
acsa-call-to-action-1.pdf. The statement highlighted the lack of action, representation,
and transformation despite numerous ACSA resolutions, statements, and interventions
over the years. 12
See
https://volmoedyouth.org.za/gatvol-yet-hopeful-women-call-the-anglican-
church-of-southern-africa-to-action/ for the full statement. On navigating leadership as a woman of colour in “previously”
male-dominated spaces In her presentation on the First Mary-Anne Plaatjies-Van Huffel Memorial
Lecture in 2020 Landman gives some insight into the “prejudiced
background” against which Plaatjies-van Huffel had to navigate her studies,
ecclesial ministry, and academic career as a “brown Afrikaans-speaking”
woman (2021:4). My understanding of Plaatjies-van Huffel’s concerns for racial and gender
justice is shaped by my own social location as a Christian South African
woman of colour who, like her, was born during apartheid in the 1950s;
a despondent, yet feisty teenager during the 70s; a hopeful yet suspicious
young adult during the 80s. Though inspired by the promise of democracy
in the 90’s through to the 2000s, I am now at a point of being “gatvol”. 11 In July 2020, I was part of a group of lay and ordained women from
the Anglican Church of Southern Africa who published a statement titled, Pillay • STJ 2023, Vol 9, No 1, 1–18 6 Gatvol yet Hopeful! Women call the Anglican Church of Southern Africa
to Action.12 Following the release of this statement the “Gatvol Women”
(as we’ve come to be known) were invited to a public conversation with
the Anglican Archbishop of Cape Town, Thabo Makgoba. 13 In a counter-
statement, a group of women clergy expressed the view that the use of the
Afrikaans word “gatvol” is a sign of vulgarity and its use unbecoming in
Christian discourse. 14 I deliberately use the word “gatvol” here as a virtue
of unctuousness – the main argument in this article to which I shall return
later. Why am I gatvol? I am gatvol that women have to march over and over; year
after year; August after August in protest against the violence perpetrated
against them just because they are women.15 I am gatvol that women’s
agency is ignored in patriarchal contexts that portray women as powerless,
weak, and needy.16 I am gatvol that women are “allowed” to be leaders in
male dominated ecclesial spaces and then given a hamster wheel to be
“baanbrekers”.17 Yet, I am hopeful because of a new generation of academics – women and
men – who are emboldened to push forward against patriarchal pushback.18
Standing on the legendary shoulders of women leaders like Plaatjies-van
Huffel, some younger scholars have the advantage of range and perspective 12
See
https://volmoedyouth.org.za/gatvol-yet-hopeful-women-call-the-anglican-
church-of-southern-africa-to-action/ for the full statement. 15 August 2019 proved to be a month of commiseration more than commemoration and
celebration as it turned out to be the bloodiest month for women who suffered violence
and death at the hands of men. 14 See Wilma Jacobson and Miranda Pillay (2022:8). 16 Women leaders do inhabit positions of power and privilege and participate in the
production of knowledge and public opinion which ought to dispel the simplistic
view of women as powerless which, in turn, uphold patriarchal power. A feminist
consciousness would suggest reflexive use of power in a network of relative power
relations. See Dottolo and Tillery (2015). On navigating leadership as a woman of colour in “previously”
male-dominated spaces 15 August 2019 proved to be a month of commiseration more than commemoration and
celebration as it turned out to be the bloodiest month for women who suffered violence
and death at the hands of men. 16 Women leaders do inhabit positions of power and privilege and participate in the
production of knowledge and public opinion which ought to dispel the simplistic
view of women as powerless which, in turn, uphold patriarchal power. A feminist
consciousness would suggest reflexive use of power in a network of relative power
relations. See Dottolo and Tillery (2015). 16 Women leaders do inhabit positions of power and privilege and participate in the
production of knowledge and public opinion which ought to dispel the simplistic
view of women as powerless which, in turn, uphold patriarchal power. A feminist
consciousness would suggest reflexive use of power in a network of relative power
relations. See Dottolo and Tillery (2015). 17 “Baanbrekers” is an Afrikaans word meaning “trailblazers”. See Pillay (2020).h 18 See for example Ashwin Afrikanus Thyssen and Sheurl Davis (2021). Pillay • STJ 2023, Vol 9, No 1, 1–18 7 to survey the patriarchal beaten track. For example, Megan Robertson says
that it is “thanks to Black women scholars of religion in South Africa that
she has felt that there is a place for her thoughts and voice in the South
African academy”. However, Robertson “has also seen some of these same
mentors and godmothers in tears because of the micro-aggressions of
White people and men in the academy” (Nadar and Robertson 2021:11–14). 20 Though, I also agree with Thyssen and Davis (2021:2) who see the value of Flaendorp’s
contribution in drawing “together various foci present in Plaatjies-Van Huffel’s work” 19 On 14 April 1994, the Uniting Reformed Church in Southern Africa (URCSA) was
established through the union of the former Dutch Reformed Mission Church (DRMC)
and the Dutch Reformed Church in Africa (Zeze 2021:1). 19 On 14 April 1994, the Uniting Reformed Church in Southern Africa (URCSA) was
established through the union of the former Dutch Reformed Mission Church (DRMC)
and the Dutch Reformed Church in Africa (Zeze 2021:1).
20 Though, I also agree with Thyssen and Davis (2021:2) who see the value of Flaendorp’s
contribution in drawing “together various foci present in Plaatjies-Van Huffel’s work” Women’s leadership trail: on debunking second-generation
gender bias There is no doubt that Plaatjies-van Huffel’s leadership took her on
paths that opened up the way for many other women. I say this with a
mindfulness that, using “women” as a category in a way that means “all
women”, is an exclusionary and discriminatory way of constructing “an
alleged universality of women” (Kappeler 1995:21). The fact that “women”,
as a category, does not represent a homogeneous group is particularly
true of the South African context where some women benefit from white
privilege, while women of colour (black, coloured, Indian) continue to
carry a double and triple burden of having to “carve out more and more
spaces for themselves and others to come” (Nadar and Robertson 2021:11). Thyssen and Davis (2021:2) highlight the point that Plaatjies-Van Huffel
was hailed as “the first of firsts”. Plaatjies-van Huffel’s trailblazing journey through the ecclesial ranks of
the Uniting Reformed Church of Southern Africa (URCSA) 19 is registered,
amongst others, by Charles Flaendorp in 2014 and by Selaelo Kgatla and
Willie Zeze respectively, in 2021. Flaendorp, who wrote about Plaatjies-
van Huffel before her death in May 2020, concludes that her ecclesial
advancement was one of merit and not “tokenism”. While the obvious
intension is to illuminate Plaatjies-van Huffel’s leadership capabilities it
may also, in my view at least, be seen as an attempt to defend the credibility
of the institutions concerned.20In a more recent publication, Thin space: Pillay • STJ 2023, Vol 9, No 1, 1–18 8 tussen hemel en harde grond, compiled by Elna Mouton, Frederick Marais
(2023:134) also recounts some of Plaatjies-van Huffel’s “first woman to …”
achievements noting that, “she was the first female pastor to be ordained in
the Uniting Reformed Church of Southern Africa (URCSA)”. In his account of events, Kgatla (2021) also registers the frustration and
despair Plaatjies-van Huffel encountered as a woman leader in the church –
much of which Plaatjies-van Huffel herself boldly declares, almost a decade
earlier.21 For the purpose of the argument made in this article, mentioning
some on these achievements and frustrations seems appropriate. As
pointed out by Kgatla, it was only “after a long wait” that Plaatjies-van
Huffel was “allowed” into the ordained ministry and appointed to the
leadership of URCSA where she became the first woman minister of the
Word in URCSA (2021:1–8). which range from her “role as minister and church leader to being an iconic figure in
the ecumenical world” (2021:2).
21 In “Mary-Anne Elizabeth Plaatjies-Van Huffel: A First Voice on Gender Equity in
South Africa”, Landman (2021) also indicates that Plaatjies-van Huffel’s journey was
marked with frustration and disappointment.
22 See Landman (2021:6). Plaatjies van Huffel was also the first black woman to be
promoted to full professor at the Faculty of Theology at Stellenbosch University; cf.
Landman (2021:3) Women’s leadership trail: on debunking second-generation
gender bias Plaatjies-van Huffel, herself, laments the fact
her first experience as ordained minister was one of rejection as she was
“not allowed” to perform her ministerial functions as pastor (Plaatjies-
van Huffel 2011; cf. Kgatla 2021). Her presence, as a woman leader in that
particular male dominated ecclesial space was not recognised as legitimate
by many congregants who not only refused her services, but left to join
another congregation “headed by a male minister” (Plaatjies-van Huffel
2011). Plaatjies-van Huffel was also the first woman Moderator of the
Uniting Reformed Church in Southern Africa when she was elected in
2012. However, as noted by Kgatla, the fact that Plaatjies-van Huffel was not
elected for a second term was a “humiliating experience” for her (2021:1–8). Another milestone on Plaatjies-van Huffel’s trailblazing journey is that she
was the first woman to serve as Actuarius of URCSA Cape Synod.22 While the four male authors mentioned above set out to honour and
acknowledge Plaatjies-van Huffel’s “first woman to …” achievements
which implicitly praise her courage and resilience, they do so without the Pillay • STJ 2023, Vol 9, No 1, 1–18 9 necessary “reflexive practice” regarding their male-privilege, compliance
and complicity within ecclesial and academic patriarchal institutional
cultures.23 By ignoring reflexive practice, they aggravate the “inequalities of
the tridimensional phenomenon of race/class/gender oppression” (Cannon
1988:39). For example, Zeze’s article, “What does Mary-Anne Elizabeth Plaatjies-van
Huffel Have to say to Silent Partners of the Reformed World?” has many
verbatim quotations from Plaatjies-van Huffel’s publications which he uses
together with posts from her personal Facebook Page, without reflexivity
on his own positionality. He concludes with “conviction” that, “… though
she [Plaatjies-van Huffel] is deceased, she is still speaking to Reformed
women in the Reformed world” (Zeze 2021:10). 24 He writes about women
and questions their “silence” oblivious to the fact that women are explicitly
and implicitly silenced by the patriarchal normative institutional culture
of the church. To be oblivious to the lived-reality of women in patriarchal
normative paces and spaces is to be compliant with the upholding the
systemic and structural institutional culture of patriarchal normativity.. To frame “women’s experiences” outside of culturally- and politically
mediated stratification of race, class and gender is to ignore the fact that
women’s agency is continually shaped “within a system of power and
difference” (Graham 2003:27). 23 What is lacking here is a reflexive use of power in a network of relative power relations.
As Dottolo and Tillery argue, “A more careful understanding of systems of power,
institutional privilege and marginalization, and the social symbols that communicate
status and hierarchy, are necessary in order to situate oneself within political structures
in order to disclose one’s subject position in the context of research” (2015:124). 25 See for example, Pillay (2013:56; 2015:68–69; 2021:80). Moreover, it is disturbing that
‘newly liberated’ voices of women of colour continue to be silenced by ‘newly liberated’ 24 Here Zeze cites Hebrews 11:4 as a source suggesting that, “even though she is dead”
Plaatjies van Huffel has something to say to her silent partners – whom he identifies as
the “women of the Reformed world” (2021:10). 23 What is lacking here is a reflexive use of power in a network of relative power relations.
As Dottolo and Tillery argue, “A more careful understanding of systems of power,
institutional privilege and marginalization, and the social symbols that communicate
status and hierarchy, are necessary in order to situate oneself within political structures
in order to disclose one’s subject position in the context of research” (2015:124).
24 Here Zeze cites Hebrews 11:4 as a source suggesting that, “even though she is dead”
Plaatjies van Huffel has something to say to her silent partners – whom he identifies as
the “women of the Reformed world” (2021:10).
25 See for example, Pillay (2013:56; 2015:68–69; 2021:80). Moreover, it is disturbing that
‘newly liberated’ voices of women of colour continue to be silenced by ‘newly liberated’ voices of men of colour, as these men remain beholden “to the supremacist ideas when
it comes to gender relations” (Maluleke 2009:33; cf. Pillay 2015:70). 28 Moreover, African women theologians have, for many years, articulated their concerns
about the “dehumanizing effects of patriarchy on women and men” (Pillay 2017:8;
2020:2–3). This is with particular reference to the work of the Circle of African Women
Theologians. A vision and initiative of Mercy Amba Oduyoye, The Circle, as it has come
to be known, was inaugurated in 1989. Conversations had already begun in 1980 at a
meeting of African women theologians in Ibadan. For a brief overview of the history of
The Circle, see Oduyoye (1997:1–6); cf. Pillay (2020:2). voices of men of colour, as these men remain beholden “to the supremacist ideas when
it comes to gender relations” (Maluleke 2009:33; cf. Pillay 2015:70).
26 See Levine and D’Agostino (2022).
27 On the contrary, oftentimes women are co-opted to uphold and hold the patriarchal
space (See Jacobson and Pillay 2022:9)
28 Moreover, African women theologians have, for many years, articulated their concerns
about the “dehumanizing effects of patriarchy on women and men” (Pillay 2017:8;
2020:2–3). This is with particular reference to the work of the Circle of African Women
Theologians. A vision and initiative of Mercy Amba Oduyoye, The Circle, as it has come
to be known, was inaugurated in 1989. Conversations had already begun in 1980 at a
meeting of African women theologians in Ibadan. For a brief overview of the history of
The Circle, see Oduyoye (1997:1–6); cf. Pillay (2020:2). 26 See Levine and D’Agostino (2022). t 27 On the contrary, oftentimes women are co-opted to uphold and hold the patriarchal
space (See Jacobson and Pillay 2022:9) e Levine and D Agostino (2022).
n the contrary, oftentimes women are co-opted to uphold and hold the patriarchal
ace (See Jacobson and Pillay 2022:9) 29 See Cannon (1995) and Pillay (2021:74–92).
30 For the notion on “palatable patriarchy” see Nadar (2009:554).
31 See Merriam Webster, “Unctuousness.” https://www.merriam-webster.com/thesaurus/
unctuous; https://www.thesaurus.com/browse/unctuous
32 Much of what I write here on the subject unctuousness is published in: The Virtue of
Unctuousness? Toward the Moral Agency of Women in Patriarchal Normative Contexts
(Pillay 2021:74-92). Also see Jakobson and Pillay (2022). Pillay • STJ 2023, Vol 9, No 1, 1–18 11 Cannon’s call to “forge “new patterns of ethical discourse that reject the
pernicious impact of sexism and racism, whilst attempting to shift the
balance of power towards alternative values” (1988:38–40). It is at this point
that I turn to unctuousness as a virtue as espoused by womanist theologian
Katie Cannon to push forward against second-generation gender bias.29 Cannon’s call to “forge “new patterns of ethical discourse that reject the
pernicious impact of sexism and racism, whilst attempting to shift the
balance of power towards alternative values” (1988:38–40). It is at this point
that I turn to unctuousness as a virtue as espoused by womanist theologian
Katie Cannon to push forward against second-generation gender bias.29 Women’s leadership trail: on debunking second-generation
gender bias I have, on many previous occasions, pointed out that patriarchal power and
privilege, so deeply entrenched in the psyche of women and men, continue
to be justified and reinscribed in ecclesial spaces and places. Therefore,
despite the presence of women leaders in previously male dominated
ecclesial spaces, “patriarchy continually raises its chauvinistic head”.25
Thus, it is what some feminists refer to as second-generation gender bias Pillay • STJ 2023, Vol 9, No 1, 1–18 10 that ought to be exposed and debunked by both women and men in
leadership. Already pointed out by Plaatjies-van Huffel, “Despite having
a constitution that entrenches equal rights, discriminatory practices,
structural inequalities, cultural factors, prejudices, patriarchy and sexism
are still prevalent in South Africa”, she calls for “the deconstruction of the
sexist and racist bias of our society” (2011b:6). Second-generation gender bias is the “more subtle, less visible, oftentimes
unintentional” and sometimes patronizing forms of discrimination. 26 It includes what Nadar (2009) calls “palatable patriarchy”. Second-
generation gender bias is often regarded and defended as unconscious,
normal behaviour. Moreover, as already pointed out, the mere presence
of women in leadership positions, previously reserved for men, does not
automatically transform the patriarchal normative culture of ecclesial and
academic spaces. Elsewhere, I’ve pointed out that “representation ≠ equal
transformation”.27 In similar vein Plaatjies van Huffel, with reference to
Schüssler Fiorenza’s argument on the “dehumanizing effects of patriarchal
structures” (Schüssler Fiorenza 1993:230), argues that “the presence
of women in the ecclesial and societal structures is not an indicator of
transformation” (2011b:7). In her use of “discipleship of equals” as a theoretical framing in the book
with the same title, Schüssler Fiorenza argues that “an impetus to overcome
the death-dealing powers of patriarchy” derives from the recognition of
the struggle that the dehumanizing effects of patriarchal structures have
on women’s lives as well as well as a “systemic analysis of dehumanizing
patriarchal structures” (1993:211–231).28 Thus, we have to take seriously Pillay • STJ 2023, Vol 9, No 1, 1–18 33 33 See Cannon (1995: 77-90). All quotations in this section are form chapter six in
Cannon, Katie’s Canon. Here, I am deliberate in my use of direct quotations and
capture Cannon’s own words in her analysis of Hurston’s writings as she (Cannon)
identifies unctuousness as a virtue in Hurston’s life and work. On women “talking back” and the virtue of Unctuousness Not being “allowed to talk back “amid the brutality of white supremacy
and patriarchy” is all too familiar to those who were raised, educated,
and socialized during apartheid and other systems of patriarchal and
paternalistic oppression. As Thyssen and Davis (2021:5) note with reference
to the feminist/womanist work of bell hooks, talking back “is a courageous
act – an act of risk and daring” (2015:22–29). Thus, “talking back” means
speaking back to a person of authority in a way that signals defiance and
resistance. In this regard I find Katie Cannon’s idea of a virtue of unctuousness
helpful in resisting second-generation gender bias – which, as previously
mentioned, is the subtle, less visible, oftentimes unintentional, and
sometimes patronising, “palatable” forms of patriarchy.30 Initially, I
found Cannon’s idea surprising and confusing, especially when I saw that
“unctuous” has many negative connotations, such as backhanded, double-
dealing, hypocritical, insincere, two-faced, fraudulent and pharisaic. How
then, I thought, could such an idea be considered a virtue? But upon further
investigation I saw that alternative “words related to unctuous” meant
uninhibited, unrestrained, and disarming. 31 That’s when the proverbial
penny dropped.32 It is precisely the idea of what is expected as “normal,
good Christian behaviour” that has to come under scrutiny as it serves
second-generation gender bias. Pillay • STJ 2023, Vol 9, No 1, 1–18 12 Unctuousness as a virtue is the subject of chapter seven of Katie’s Canon:
Womanism and the Soul of the Black community.33 Reflecting on the life
and writings of Zora Hurston, Cannon espouses “unctuousness as a virtue”
saying that: In both her life and work Hurston embodied a sensitized candour in
relation to the subtle, invisible ethos as well as the expressed moral
values emanating from within the cultural institutions in the Black
community. She maintained that Black life was more than defensive
reactions to the oppressive Western system of White male patriarchy. As a Black woman artist, subjected to the violence of Whites, of
male superiority, and of poverty, Zora Hurston offered an especially
concrete frame of reference for understanding the Black woman as a
moral agent (1995:91). On women “talking back” and the virtue of Unctuousness Cannon describes Hurston and her fictional counterparts as moral agents
who, in their struggle “to avoid the devastating effects of structural
oppression, create various coping mechanisms that free them from imposed
norms and expectations.” Though it must be noted that while Huston’s
struggle, as was the struggle against apartheid was politically ideological
and structural in terms of policies and laws, second-generation gender bias
is ideologically systemic. In case of the latter, Cannon highlights the fact
that Hurston “fully delineates the propositions, attitudes, and behaviours
that men exhibit to support their belief in the inherent inferiority of women
and their right to dominate them.” According to Cannon, Hurston understood suffering imposed by dominant
cultures, “not as a moral norm or as a desirable ethical quality, [as often
espoused in Christian circles] but rather as a typical state of affairs” which
results from the prevailing dominant ethos. In essence, unctuousness as a
virtue is the creative tension between resistance and endurance. Perhaps
this is why women of faith can publicly declare that they are “Gatvol yet Pillay • STJ 2023, Vol 9, No 1, 1–18 13 Hopeful” – in which case endurance is an ethical principle through which
the virtue of unctuousness is embodied.34 Thus, as an ethical principle, endurance means that women leaders would
claim their agency and risk going off the patriarchal beaten track to expose,
debunk and resist second-generation gender bias. As an ethical principle of
the virtue of unctuousness, endurance does not mean passive acceptance
but rather it points to the intentionality necessary to resist the subtle, less
visible, oftentimes unintentional, and sometimes patronising forms of
what is referred to here, as second-generation gender and racial bias. However, it is highly likely that being unctuous may be regarded as a vice and
not a virtue by both, men, and women, in patriarchal normative contexts. This was, as mentioned in a previous section of this article, the case with
the counter-response to the statement, Gatvol yet Hopeful! Women call
the Anglican Church of Southern Africa to Action. Moreover, anyone who
embodies unctuousness in resistance and defiance to patriarchal norms,
risks being branded as a bad, insincere, false, and deviant because the value
system of the dominant group is justified and understood to be beneficial
to the oppressed. Second, the oppressed group could brand anyone who
exercises endurance in the face of oppression as a sell-out (Pillay 2021:85). On women “talking back” and the virtue of Unctuousness This is why Cannon says that there has to be communal “recognition that
unctuousness is a virtue” so as to recognise the moral agency and legitimate
presence of women leaders in “previously” male-dominated ecclesial spaces
and places (Cannon 1995:91–94). If not, it is likely that women who do
embody unctuousness in patriarchal normative contexts are silenced, side-
lined, dismissed, ridiculed, and violated by dominant, oppressive systems
operative in racist, sexist and classist societies. 34 See Pillay and Jacobsen (2022). 35 See https://www.sun.ac.za/english/Lists/news/DispForm.aspx?ID=9456 [Accessed: 30
July 2023]. Concluding remarks: Toward the legitimate presence of
women leaders As the lived experiences of women like Plaatjies-van Huffel reveal, women
who are celebrated as being “the first of firsts” also carry the burden of
having the legitimacy of their presence questioned in patriarchal normative Pillay • STJ 2023, Vol 9, No 1, 1–18 14 communities. In reflecting on the trail left by Plaatjies-van Huffel’s “first
woman to …” achievements in contexts where gender discrimination is
unconstitutional, the reality is that second-generation gender bias continues
to thwart transformation of “previously” male dominated ecclesial spaces. Worse still, the presence of women leaders in positions previously reserved
for men is, often, dismissed as being irrelevant and of no consequence in
patriarchal normative institutional culture of the church. In this article, I have argued that embodying a virtue of unctuousness
means being intentional about going against norms and expectations
imposed by, for example, the patriarchal institutional culture of the
church. In this regard talking back against institutional authority is,
as Cannon says, forging “new patterns of ethical discourse”. Far from
being discounted from our understanding of moral agency, the virtue of
unctuousness goes beyond resistance to include endurance. To embody a
virtue of unctuousness is to be “Gatvol! Yet Hopeful”. Plaatjies-van Huffel pushed defiantly forward against patriarchal
pushback – albeit that patriarchal pushback remains camouflaged by
second-generation gender bias. Thus, I echo Nadine Bowers-du Toit’s
words of hope that, in celebrating the life and legacy of Plaatjies-van Huffel
we will in the years to come, “hold high the values that she [Plaatjies-van
Huffel] embodied – of justice, freedom, equity and liberation for all”.35 Bibliography Cannon, K.G. 1988. Black Womanist Ethics. Altanta: Scholars Press. Cannon, K.G. 1988. Black Womanist Ethics. Altanta: Scholars Press. Cannon, K.G. 1995. Katie’s Canon: Womanist and the Soul of the Black
Community. New York: Continuum. Dictionary, O.L. n.d. Oxford Learner’s Dictionaries. [Online]. Available:
https://www.oxfordlearnersdictionaries.com/definition/english/
gatvol. Dictionary, O.L. n.d. Oxford Learner’s Dictionaries. [Online]. Available:
https://www.oxfordlearnersdictionaries.com/definition/english/
gatvol. Pillay • STJ 2023, Vol 9, No 1, 1–18 15 Dottolo, L. & Tillery, S. 2015. Reflexivity and research: Feminist
interventions and their practical implications. In J. Amoreaux, & B. Steele. Reflexivity in international relations: Positionality, critique and
practice. New York: Routledge. 123–141. Felski, R. 2011. Critique and the Hermeneutics of Suspicion. M/C Journal
15(1). https://doi.org/10.5204/mcj.431https://doi.org/10.5204/mcj.431. Flaendorp, C. 2014. The life and times of Professor Mary-Anne Plaatjies
Van Huffel: A transformative church leader in Sub-Saharan Africa. Studia Historiae Ecclesiasticae 40(1):53–63. Graff, A. 1995. In Embrace of God: Feminist approaches to theological
anthropology . New York: Orbis Books. Graham, E. 2003. Different forms of feminist ethics. In C.H. Grenholm,
& K. (eds). Feminist Ethics: Perspectives, problems and possibilities. Stockholm: Eleandrs Gotab. 15–30. hooks, b. 2015. Talking Black: Thinking Feminist, Thinking Black. New
York: Routledge. Jakobson, W. & Pillay, M. 2022. Re-membering Tutu’s liberation theology:
toward gender justice from a theo-ethical feminist perspective. Anglican Theological Review 1–11. Kappeler, S. 1995. The Will To Violence: The Politics of Personal Behaviour. New York: Teachers College Press. Kgatla, S.T. 2021. Born into a World of Hostility and Contradiction: The
Role of Mary-Anne Elizabeth Plaatjies van Huffel in URCSA. UNISA
Press Journals 47(2):1–14. Landman, C. (2021). Mary-Anne Elizabeth Plaatjies-Van Huffel: A First
Voice on Gender Equity in South Africa. [Online]. Available: Historiae
Ecclesiasticae (2021) 47(2):1–15: https://dx.doi.org/10.25159/2412-
4265/9137 Levine, H. & D’Agostino. 2022. Making the case for addressing second-
generation gender bias in public administration. New York: John Jay
College Publications. Pillay • STJ 2023, Vol 9, No 1, 1–18 16 Maluleke, T. 2009. An African Perspective on Patriarchy. The Evil of
Patriarchy. Stellenbosch: IAM. 31–34. Marais, F. 2023. Program vir AMSS-Verwelkomingsdinee. In E. Mouton. Thin space:tussen hemel en harde grond. Wellington: Clair Vaux
Writer’s House. 130–135. Mulder, S. 2022. [Online]. Avaiable: https://www.sun.ac.za/english/Lists/
news/DispForm.aspx?ID=9456,w Nadar, S. 2009. Palatable patriarchy and violence against women in South
Africa . Scriptura 102:551–561. Nadar, S. & Robertson, M. 2012. Recognition, Resistance and Rest:
Drawing from the Womanist Wells of Katie Geneva Cannon. Journal
of Theology for Southern Africa 170:7–18. Nel, R. 2021. Bibliography Editorial: A Woman of Voice: Mary-Anne Elizabeth
Plaatjies-Van Huffel(15 December 1959–19 May2020). Studia Historiae
Ecclesiasticae 47(2):1–4. Oduyoye, M.A. 1997. Introduction. In M. A. Oduyoye. Transforming
power: Women in the household of God. Accra: Sam Woode Limited. 1–6. Pillay, M. 2012. The Anglican Church and Feminism: Challenging “the
Patriarchy of our Faith”. Journal of Gender and Religion in Africa
19(2):53–71. Pillay, M. 2015. Mighty men, mighty families: A pro-family Christian
movement to (re)enforce Patriarchal control? In E. Conradie, & M. Pillay. Ecclesial Reform and Deform Movements in the South African
Context. Stellenbosch: Sen Media. 61–77. Pillay, M. 2021. The Virtue of Unctuousness? Toward the moral agency
of women in patriarchal normative contexts. Journal of Theology for
Southern Africa 170:74–92. Pillay, M. 2008. Revisioning Stigma: A Socio-rhetorical reading of Luke
10:25–37 in the Context of HIV and AIDS in South Africa. [Online]. Available: https://etd.uwc.ac.za/xmlui/handle/11394/2242 Pillay • STJ 2023, Vol 9, No 1, 1–18 17 Pillay, M. 2020. Women, priests and patriarchal ecclesial spaces in
the Anglican Church of Southern Africa: On “interruption” as a
transformative rhetorical strategy. [Online]. Available: https://doi. org/10.4102/hts.v76i1.5820; https://hts.org.za/index.php/HTS/article/
view/5820 Plaaties van Huffel, M.-A. 2011b. Patriarchy as empire: a Theological
reflection. Studia Historiae Ecclesiasticae 37(1): 259–270. Plaatjies van Huffel, M.-A. 2008. About the Empowerment of Women
in the Church in Post-Apartheid South Africa: A Post-Structural
Approach. In S. d. Gruchy, N. Koopman, & S. Strijbos. From Our
Side: Emerging Perspectives on Development and Ethics. Amsterdam:
Rezenberg Publishers. 87–97. Plaatjies van Huffel, M.-A. 2011a. The Institutionalisation of Christian
women’s organisations: from docile recipients to agents of change. Studia Historiae Ecclesiasticae 37(1):105–119. rememr.com. 2020. Prof
Mary-Anne Plaatjies van Huffel. [Online]. Available: https://www. remembr.com/en/revprof.mary-anne.plaatjiesvanhuffel; https://www. remembr.com/en/revprof.mary-anne.plaatjiesvanhuffel Schüssler Fiorenza, E. 1993. Discipleship of Equals: A Critical Feminist
Ekklesia-ology of Liberation. New York: The Crossroad Publishing
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The*logy. Philadelphia: Fortess Press. Schüssler Fiorenza, E. 2014. Between Movement and Academy: Feminist
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in the Twentieth Century: Scholarship and movement. Atlanta: Society
of Biblical Literature. 1–20. Smit, D. (1996). Oor die kerk as n unieke samelewingsverband. Tydskrif
vir Geesteswetenskappe 36(2):119–129. The 1956 Women’s Marchm Pretoria 9 August n.d. [Online]. Available:
https://www.sahistory.org.za/article/1956-womens-march-pretoria-9-
august Pillay • STJ 2023, Vol 9, No 1, 1–18 18 Thyssen, A. & Davis, S. 2021. In Search of the Public Theologian: Mary-
Anne Elizabeth Plaatjies-Van Huffel’s Womanist Public Engagement. Studia Historiae Ecclesiasticae 47(2):1–17. Volmoed. (n.d.). Bibliography [Online]. Available: //volmoedyouth.files.wordpress. com/2020/08/final-acsa-call-to-action-1.pd. Zeze, W. 2021. What Does Mary-Anne Elizabeth Plaatjies Van Huffel
Have to Say to Silent Partners of the Reformed World? Studia
Historiae Ecclesiasticae 47(2):1–16.
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RNA-Sequencing Based microRNA Expression Signature of Colorectal Cancer: The Impact of Oncogenic Targets Regulated by miR-490-3p
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*
Correspondence: naoseki@faculty.chiba-u.jp; Tel.: +81-43-226-2971 Citation: Hozaka, Y.; Kita, Y.;
Yasudome, R.; Tanaka, T.; Wada, M.;
Idichi, T.; Tanabe, K.; Asai, S.; Moriya,
S.; Toda, H.; et al. RNA-Sequencing
Based microRNA Expression
Signature of Colorectal Cancer: The
Impact of Oncogenic Targets
Regulated by miR-490-3p. Int. J. Mol. Sci. 2021, 22, 9876. https://doi.org/
10.3390/ijms22189876 Abstract: To elucidate novel aspects of the molecular pathogenesis of colorectal cancer (CRC), we
have created a new microRNA (miRNA) expression signature based on RNA-sequencing. Analysis of
the signature showed that 84 miRNAs were upregulated, and 70 were downregulated in CRC tissues. Interestingly, our signature indicated that both guide and passenger strands of some miRNAs were
significantly dysregulated in CRC tissues. These findings support our earlier data demonstrating
the involvement of miRNA passenger strands in cancer pathogenesis. Our study focused on down-
regulated miR-490-3p and investigated its tumor-suppressive function in CRC cells. We successfully
identified a total of 38 putative oncogenic targets regulated by miR-490-3p in CRC cells. Among these
targets, the expression of three genes (IRAK1: p = 0.0427, FUT1: p = 0.0468, and GPRIN2: p = 0.0080)
significantly predicted 5-year overall survival of CRC patients. Moreover, we analyzed the direct
regulation of IRAK1 by miR-490-3p, and its resultant oncogenic function in CRC cells. Thus, we
have clarified a part of the molecular pathway of CRC based on the action of tumor-suppressive
miR-490-3p. This new miRNA expression signature of CRC will be a useful tool for elucidating new
molecular pathogenesis in this disease. Academic Editors: Asahiro Morishita,
Tomoyuki Okumura and
Yohei Shirakami Received: 23 August 2021
Accepted: 10 September 2021
Published: 13 September 2021 Received: 23 August 2021
Accepted: 10 September 2021
Published: 13 September 2021 Keywords: microRNA; colorectal cancer; RNA-sequencing; expression signature; tumor-suppressor;
miR-490-3p; IRAK1 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Article
RNA-Sequencing Based microRNA Expression Signature of
Colorectal Cancer: The Impact of Oncogenic Targets Regulated
by miR-490-3p Yuto Hozaka 1
, Yoshiaki Kita 1, Ryutaro Yasudome 1, Takako Tanaka 1, Masumi Wada 1, Tetsuya Idichi 1,
Kan Tanabe 1, Shunichi Asai 2, Shogo Moriya 3, Hiroko Toda 1, Shinichiro Mori 1, Hiroshi Kurahara 1
,
Takao Ohtsuka 1 and Naohiko Seki 2,* 1
Department of Digestive Surgery, Breast and Thyroid Surgery, Graduate School of Medical and
Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan; k6958371@kadai.jp (Y.H.);
north-y@m.kufm.kagoshima-u.ac.jp (Y.K.); k7682205@kadai.jp (R.Y.); k5495007@kadai.jp (T.T.);
k8911571@kadai.jp (M.W.); k3352693@kadai.jp (T.I.); k3113670@kadai.jp (K.T.); k4900207@kadai.jp (H.T.);
morishin@m3.kufm.kagoshima-u.ac.jp (S.M.); h-krhr@m3.kufm.kagoshima-u.ac.jp (H.K.);
takao-o@kufm.kagoshima-u.ac.jp (T.O.) 1
Department of Digestive Surgery, Breast and Thyroid Surgery, Graduate School of Medical and
Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan; k6958371@kadai.jp (Y.H.);
north-y@m.kufm.kagoshima-u.ac.jp (Y.K.); k7682205@kadai.jp (R.Y.); k5495007@kadai.jp (T.T.);
k8911571@kadai.jp (M.W.); k3352693@kadai.jp (T.I.); k3113670@kadai.jp (K.T.); k4900207@kadai.jp (H.T.)
morishin@m3.kufm.kagoshima-u.ac.jp (S.M.); h-krhr@m3.kufm.kagoshima-u.ac.jp (H.K.);
takao-o@kufm.kagoshima-u.ac.jp (T.O.) 2
Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan;
cada5015@chiba-u.jp 2
Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan;
cada5015@chiba-u.jp International Journal of
Molecular Sciences 1. Introduction Colorectal cancer (CRC) is one of the most common cancers of the digestive system,
as it constitutes approximately 10% of all diagnosed cancers, with almost 900,000 deaths
annually [1,2]. The expansion of treatment options and the spread of diagnostic systems
have greatly contributed to improving the prognosis of patients with CRC [2–4]. However,
for patients with advanced cancer with metastasis, the 5-year survival rate is approximately
14%, and improving the prognosis of these patients is an important clinical issue [3]. Furthermore, the number of young people (under the age of 50) with colorectal cancer is
increasing, especially rectal cancer and left-sided colon cancer [2]. 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/). Over the past few decades, the mechanism of CRC oncogenesis has been revealed
by molecular genetic analyses [5–7]. A previous study demonstrated that several genes https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9876. https://doi.org/10.3390/ijms22189876 Int. J. Mol. Sci. 2021, 22, 9876 2 of 17 are frequently mutated, e.g., APC, TP53, SMAD4, KRAS, PIK3CA, ARID1A, SOX9, and
FAM123B, and pivotal oncogenic pathways, e.g., WNT, RAS-MAPK, PI3K, TGF-β, P53, and
DNA mismatch-repair are closely associated with this disease [7]. Notably, for younger
CRC patients, the mutation rate of these genes is low, and it is expected that there are other
factors involved [8]. The Human Genome Project revealed that a large number of non-protein-coding
RNA molecules (ncRNAs) are transcribed from the human genome [9]. Current studies
indicate that a large number of ncRNAs play critical roles in various biological processes,
e.g., stabilization of RNA molecules, regulation of gene expression, and control of the
cell cycle [10,11]. Notably, it is evident that dysregulation of ncRNA is involved in the
enhancement of human diseases, including cancers [12]. g
Functional ncRNAs are classified by their nucleotide length: short ncRNAs (19–30 nu-
cleotides), medium ncRNAs (50–200 nucleotides), and long ncRNAs (>200 nucleotides) [13]. MicroRNAs (miRNAs) constitute the most commonly analyzed class of short ncRNAs. They
function as fine-tuners of gene expression, working in a sequence-dependent manner [14]. One of the hallmarks of miRNAs is that a single miRNA regulates multiple RNA transcripts. Moreover, they contribute to various cellular signaling pathways in both physiological
and pathological conditions [14,15]. 1. Introduction A vast number of studies have shown that ectopic
expression of miRNAs modulates oncogenes and/or tumor-suppressors in human cancer
cells, including CRC [16–18]. We have identified tumor-suppressive miRNAs, and their regulated oncogenes, in
several types of cancers [19–21]. Our previous studies revealed that the expression of
tumor-suppressive miRNAs and regulated oncogenes are closely related to the molecular
pathogenesis of cancers. Our miRNA-based strategy is an attractive approach to the
identification of novel oncogenic genes/pathways in cancer cells. In this study, we attempted to identify novel dysregulated miRNAs in CRC tissues. Thus, we created a new miRNA expression signature using next-generation sequencing
technology. Currently available RNA sequencing technologies are suitable for rapidly,
and accurately, generating miRNA signatures [22]. Our new CRC signature showed
that a total of 154 miRNAs (84 upregulated and 70 downregulated) were significantly
dysregulated in CRC tissues. Here, we focused on miR-490-3p (the most downregulated
miRNA in this signature), and we investigated its tumor-suppressive roles and its targeted
oncogenes in CRC cells. Finally, we identified IRAK1 as an oncogenic miR-490-3p target
and demonstrated that its expression is closely involved in CRC molecular pathogenesis. Using tumor-suppressive miR-490-3p as a starting point, we have succeeded in iden-
tifying molecules involved in the oncogenesis of CRC. The CRC miRNA signature we
provide in this study will contribute to the search for regulatory molecular networks
in CRC. 2.1. Creating miRNA Expression Signature in CRC A total of 10 samples (5 CRC tissues and 5 noncancerous tumor-adjacent tissues)
were analyzed using RNA sequencing techniques to generate a CRC miRNA signature. The characteristics of five patients with CRC are summarized in Table 1. In this analysis,
we obtained between 11,432,618 and 19,286,983 total sequence reads (Table S1). After
the trimming procedure, we mapped them to the human genome and determined that
3,018,259 to 16,436,280 sequences were small human RNAs (Table S1). Our RNA sequence
data was deposited in the Gene Expression Omnibus (GEO) database (GSE183437). Int. J. Mol. Sci. 2021, 22, 9876 3 of 17 Table 1. Clinicopathological features of five colorectal cancer (CRC) patients whose CRC tissue and normal colorectal tissue
were analyzed to generate the CRC miRNA signature. . Age
(Years)
Sex
Location
Differentiation
T
N
M
Stage
ly
v
Recurren Table 1. Clinicopathological features of five colorectal cancer (CRC) patients whose CRC tissue and normal colorectal tissue
were analyzed to generate the CRC miRNA signature. No. Age (Years)
Sex
Location
Differentiation
T
N
M
Stage
ly
v
Recurrence
1
66
Male
Rectum
Moderate
3
1a
0
IIIB
0
1
—
2
66
Male
Colon(S)
Moderate
3
1a
0
IIIB
1
1
—
3
79
Male
Rectum
Moderate
3
2a
0
IIIB
1
1
—
4
78
Female
Colon(S)
Moderate
3
0
0
IIA
0
1
—
5
83
Female
Colon(S)
Moderate
3
1a
0
IIIB
1
0
—
ly, lymphatic invasion; M, metastasis; N, nodes; T, tumor; v, venous invasion; colon (S), sigmoid colon. No. Age
(Years)
Sex
Location
Differentiation
T
N
M
Stage
ly
v
Recurrence
1
66
Male
Rectum
Moderate
3
1a
0
IIIB
0
1
—
2
66
Male
Colon(S)
Moderate
3
1a
0
IIIB
1
1
—
3
79
Male
Rectum
Moderate
3
2a
0
IIIB
1
1
—
4
78
Female
Colon(S)
Moderate
3
0
0
IIA
0
1
—
5
83
Female
Colon(S)
Moderate
3
1a
0
IIIB
1
0
—
ly, lymphatic invasion; M, metastasis; N, nodes; T, tumor; v, venous invasion; colon (S), sigmoid colon. Differentially expressed miRNAs were mapped on human chromosomes (Figures 1 and 2). Upregulated and downregulated miRNAs (84 and 70, respectively) were aberrantly dys-
regulated in CRC tissues (Tables S2 and S3). 2.1. Creating miRNA Expression Signature in CRC Interestingly, among the abnormally down-
regulated miRNAs in CRC tissue, 16 pairs of miRNAs (i.e., miR-490, miR-133a, miR-145,
miR-129, miR-143, miR-497, miR-9, miR-139, miR-125b, miR-100, miR-30a, miR-218, miR-195,
miR-99a, miR-29c, and miR-28) represented both miR-5p and -3p sequences. Differentially expressed miRNAs were mapped on human chromosomes (Figures
and 2). Upregulated and downregulated miRNAs (84 and 70, respectively) were aber
rantly dysregulated in CRC tissues (Tables S2 and S3). Interestingly, among the abnor
mally downregulated miRNAs in CRC tissue, 16 pairs of miRNAs (i.e., miR-490, miR-133a
miR-145, miR-129, miR-143, miR-497, miR-9, miR-139, miR-125b, miR-100, miR-30a, miR-218
miR-195, miR-99a, miR-29c, and miR-28) represented both miR-5p and -3p sequences. Figure 1. Chromosome mapping of aberrantly upregulated miRNAs in colorectal cancer (CRC). Up
regulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 84 miRNAs ar
identified by our RNA sequence based signature. Figure 1. Chromosome mapping of aberrantly upregulated miRNAs in colorectal cancer (CRC). Upregulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 84 miRNAs
are identified by our RNA sequence based signature. igure 1. Chromosome mapping of aberrantly upregulated miRNAs in colorectal cancer (CRC). Up
egulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 84 miRNAs ar
dentified by our RNA sequence based signature. Figure 1. Chromosome mapping of aberrantly upregulated miRNAs in colorectal cancer (CRC). Upregulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 84 miRNAs
are identified by our RNA sequence based signature. 4 of 17
4 of 17 Int. J. Mol. Sci. 2021, 22, 9876
J. Mol. Sci. 2021, 22, x FOR PEE Figure 2. Chromosome mapping of aberrantly downregulated miRNAs in colorectal cancer (CRC
Downregulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 70 miR
NAs are identified by our RNA sequence based signature. In order to ensure the validity of ou
signature created in this study, we compared to previous miRNA signatures of CRC [23–25]. Tw
miRNAs, miR-592 (upregulated miRNA) and miR-139-5p (downregulated miRNA) were commo
miRNAs to all four independent signatures (Table S4). Eight miRNAs, miR-335-3p, miR-552-5p, miR
3180-5p, miR-301b and miR-3144-3p (upregulated miRNA), and miR-378a-5p, miR-490-3p, and miR
422a were common to multiple independent signatures (Table S4). These signatures suggest tha
aberrant expressed miRNAs, listed in multiple studies, play a vital role in CRC tumorigenesis. Figure 2. 2.1. Creating miRNA Expression Signature in CRC Chromosome mapping of aberrantly downregulated miRNAs in colorectal cancer (CRC). Downregulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 70 miRNAs
are identified by our RNA sequence based signature. In order to ensure the validity of our signature
created in this study, we compared to previous miRNA signatures of CRC [23–25]. Two miRNAs,
miR-592 (upregulated miRNA) and miR-139-5p (downregulated miRNA) were common miRNAs
to all four independent signatures (Table S4). Eight miRNAs, miR-335-3p, miR-552-5p, miR-3180-5p,
miR-301b and miR-3144-3p (upregulated miRNA), and miR-378a-5p, miR-490-3p, and miR-422a were
common to multiple independent signatures (Table S4). These signatures suggest that aberrant
expressed miRNAs, listed in multiple studies, play a vital role in CRC tumorigenesis. gure 2. Chromosome mapping of aberrantly downregulated miRNAs in colorectal cancer (CRC)
Figure 2. Chromosome mapping of aberrantly downregulated miRNAs in colorectal cancer (CRC). gure 2. Chromosome mapping of aberrantly downregulated miRNAs in colorectal cancer (CRC)
ownregulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 70 miR
As are identified by our RNA sequence based signature. In order to ensure the validity of ou
gnature created in this study we compared to previous miRNA signatures of CRC [23 25] Two
Figure 2. Chromosome mapping of aberrantly downregulated miRNAs in colorectal cancer (CRC). Downregulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 70 miRNAs
are identified by our RNA sequence based signature. In order to ensure the validity of our signature
created in this study we compared to previous miRNA signatures of CRC [23 25] Two miRNAs g
pp
g
y
g
(
)
ownregulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 70 miR-
As are identified by our RNA sequence based signature. In order to ensure the validity of our
gnature created in this study, we compared to previous miRNA signatures of CRC [23–25]. Two
iRNAs, miR-592 (upregulated miRNA) and miR-139-5p (downregulated miRNA) were common
iRNAs to all four independent signatures (Table S4). Eight miRNAs, miR-335-3p, miR-552-5p, miR
80-5p, miR-301b and miR-3144-3p (upregulated miRNA), and miR-378a-5p, miR-490-3p, and miR
2a were common to multiple independent signatures (Table S4). These signatures suggest tha
errant expressed miRNAs, listed in multiple studies, play a vital role in CRC tumorigenesis. Downregulated miRNAs in CRC tissues are mapped on human chromosomes. A total of 70 miRNAs
are identified by our RNA sequence based signature. 2.1. Creating miRNA Expression Signature in CRC In order to ensure the validity of our signature
created in this study, we compared to previous miRNA signatures of CRC [23–25]. Two miRNAs,
miR-592 (upregulated miRNA) and miR-139-5p (downregulated miRNA) were common miRNAs
to all four independent signatures (Table S4). Eight miRNAs, miR-335-3p, miR-552-5p, miR-3180-5p,
miR-301b and miR-3144-3p (upregulated miRNA), and miR-378a-5p, miR-490-3p, and miR-422a were
common to multiple independent signatures (Table S4). These signatures suggest that aberrant
expressed miRNAs, listed in multiple studies, play a vital role in CRC tumorigenesis. 2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells
2.2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells (C) Spearman’s rank test showed positive correlations between the expression levels of miR-490-5p and miR-490-3p in clinical
specimens (r = 0.957, p < 0.001). Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-
3p in colorectal cancer (CRC) tissues and cell lines. (A) Volcano plot of the miRNA expression signature determined by
RNA sequencing. The log2 fold-change (FC) is plotted on the x-axis, and the −log10 (p-value) is plotted on the y-axis. The
blue points represent the downregulated miRNAs with an absolute −log2 FC ≥ 1 (FC = 2) and p-value < 0.05. (B) The
expression levels of miR-490-5p and miR-490-3p, by quantitative polymerase chain reaction, were evaluated in CRC clinical
tissues and cell lines (HCT116 and DLD-1). Expression levels of these miRNAs were significantly reduced in cancer tissues
(p < 0.001). (C) Spearman’s rank test showed positive correlations between the expression levels of miR-490-5p and miR-
490-3p in clinical specimens (r = 0.957, p < 0.001). To investigate the antitumor functions of miR-490-3p and miR-490-5p in CRC cells,
we performed ectopic expression assays. The experiments were performed by a transient
transfection method using mature miRNAs, miR-490-3p (caaccuggaggacuccaugcug), or
miR-490-5p (ccauggaucuccaggugggu). Cell proliferation, migration and invasive ability
were significantly attenuated by miR-490-3p transfection into CRC cell lines (Figure 4A–
C). In contrast, cell migration abilities were not blocked by miR-490-5p transfection into
CRC cell lines (Figure 4C). To investigate the antitumor functions of miR-490-3p and miR-490-5p in CRC cells,
we performed ectopic expression assays. The experiments were performed by a transient
transfection method using mature miRNAs, miR-490-3p (caaccuggaggacuccaugcug), or
miR-490-5p (ccauggaucuccaggugggu). Cell proliferation, migration and invasive ability
were significantly attenuated by miR-490-3p transfection into CRC cell lines (Figure 4A–C). In contrast, cell migration abilities were not blocked by miR-490-5p transfection into CRC
cell lines (Figure 4C). To investigate the antitumor functions of miR-490-3p and miR-490-5p in CRC cells
we performed ectopic expression assays. The experiments were performed by a transien
transfection method using mature miRNAs, miR-490-3p (caaccuggaggacuccaugcug), o
miR-490-5p (ccauggaucuccaggugggu). Cell proliferation, migration and invasive abilit
were significantly attenuated by miR-490-3p transfection into CRC cell lines (Figure 4A
C). In contrast, cell migration abilities were not blocked by miR-490-5p transfection int
CRC cell lines (Figure 4C). Figure 4. Tumor-suppressive roles of miR-490-5p and miR-490-3p in colorectal cancer (CRC) cells (HCT116 and DLD-1). 2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells
2.2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells pp
f
p
p
The miR-490-duplex was the most downregulated in this miRNA expression signa-
ure (Figure 3A). In this study, we investigated the functional and oncogenic significance
f both strands of the miR-490-duplex in CRC cells. To confirm the validity of the CRC
gnature, we measured the expression levels of miR-490-3p and miR-490-5p in clinical
pecimens (27 CRC specimens and 27 tumor-adjacent normal colorectal epithelial speci-
ens). The characteristics of 27 patients with CRC are summarized in Table S5. Expression
vels of both miR-490-3p and miR-490-5p were significantly reduced (p < 0.0001) in CRC
ssues compared with those in normal colorectal epithelial tissues, assessed by quantita-
ve polymerase chain reaction (qPCR). (Figure 3B). The expression levels of these miR-
As in two cell lines (HCT116 and DLD-1) were lower than those in normal colorectal
pithelial tissues (Figure 3B). There was a positive correlation between the expression lev-
The miR-490-duplex was the most downregulated in this miRNA expression signature
(Figure 3A). In this study, we investigated the functional and oncogenic significance of both
strands of the miR-490-duplex in CRC cells. To confirm the validity of the CRC signature,
we measured the expression levels of miR-490-3p and miR-490-5p in clinical specimens
(27 CRC specimens and 27 tumor-adjacent normal colorectal epithelial specimens). The
characteristics of 27 patients with CRC are summarized in Table S5. Expression levels of
both miR-490-3p and miR-490-5p were significantly reduced (p < 0.0001) in CRC tissues
compared with those in normal colorectal epithelial tissues, assessed by quantitative
polymerase chain reaction (qPCR). (Figure 3B). The expression levels of these miRNAs in
two cell lines (HCT116 and DLD-1) were lower than those in normal colorectal epithelial
tissues (Figure 3B). There was a positive correlation between the expression levels of the
two miRNAs by Spearman’s rank analysis (r = 0.957, p < 0.0001; Figure 3C). Int. J. Mol. Sci. 2021, 22, 9876
Int. J. Mol. Sci. 2021, 22, x FOR P
Int. J. Mol. Sci. 2021, 22, x FO 5 of 17
5 of 17
5 of 1 Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-
3p in colorectal cancer (CRC) tissues and cell lines. (A) Volcano plot of the miRNA expression signature determined by
RNA sequencing. The log2 fold-change (FC) is plotted on the x-axis, and the −log10 (p-value) is plotted on the y-axis. 2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells
2.2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells Functional assays of miR-490-5p and miR-490-3p in CRC cells using miRNAs precursors. (A) Cell proliferation, assessed
by XTT assay, 72 h after transfection of mature miRNAs (B) Cell invasion, determined by Matrigel invasion assay, 48 h
after seeding inhibitor-transfected cells into the chambers. (C) Cell migration assessed, using a membrane culture system,
48 h after seeding inhibitor-transfected cells into the chambers. Figure 4. Tumor-suppressive roles of miR-490-5p and miR-490-3p in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of miR-490-5p and miR-490-3p in CRC cells using miRNAs precursors. (A) Cell proliferation, assessed
by XTT assay, 72 h after transfection of mature miRNAs (B) Cell invasion, determined by Matrigel invasion assay, 48 h
after seeding inhibitor-transfected cells into the chambers. (C) Cell migration assessed, using a membrane culture system,
48 h after seeding inhibitor-transfected cells into the chambers. Figure 4. Tumor-suppressive roles of miR-490-5p and miR-490-3p in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of miR-490-5p and miR-490-3p in CRC cells using miRNAs precursors. (A) Cell proliferation, assessed by
XTT assay, 72 h after transfection of mature miRNAs (B) Cell invasion, determined by Matrigel invasion assay, 48 h after
seeding inhibitor-transfected cells into the chambers. (C) Cell migration assessed, using a membrane culture system, 48 h
after seeding inhibitor-transfected cells into the chambers. Figure 4. Tumor-suppressive roles of miR-490-5p and miR-490-3p in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of miR-490-5p and miR-490-3p in CRC cells using miRNAs precursors. (A) Cell proliferation, assessed
by XTT assay, 72 h after transfection of mature miRNAs (B) Cell invasion, determined by Matrigel invasion assay, 48 h
after seeding inhibitor-transfected cells into the chambers. (C) Cell migration assessed, using a membrane culture system,
48 h after seeding inhibitor-transfected cells into the chambers. Figure 4. Tumor-suppressive roles of miR-490-5p and miR-490-3p in colorectal cancer (CRC) cells (HCT116 and DLD-1)
Functional assays of miR-490-5p and miR-490-3p in CRC cells using miRNAs precursors. (A) Cell proliferation, assessed
by XTT assay, 72 h after transfection of mature miRNAs (B) Cell invasion, determined by Matrigel invasion assay, 48 h
after seeding inhibitor-transfected cells into the chambers. (C) Cell migration assessed, using a membrane culture system
48 h after seeding inhibitor-transfected cells into the chambers. Figure 4. Tumor-suppressive roles of miR-490-5p and miR-490-3p in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of miR-490-5p and miR-490-3p in CRC cells using miRNAs precursors. 2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells
2.2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells The
blue points represent the downregulated miRNAs with an absolute −log2 FC ≥ 1 (FC = 2) and p-value < 0.05. (B) The
expression levels of miR-490-5p and miR-490-3p, by quantitative polymerase chain reaction, were evaluated in CRC clinical
tissues and cell lines (HCT116 and DLD-1). Expression levels of these miRNAs were significantly reduced in cancer tissues
(p < 0.001). (C) Spearman’s rank test showed positive correlations between the expression levels of miR-490-5p and miR-
490-3p in clinical specimens (r = 0.957, p < 0.001). Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5
Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p
Fi
3 E
i
f
iR 490 5
d
iR 490 3 b
ll RNA
i
d
i
f
iR 49 Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-
3p in colorectal cancer (CRC) tissues and cell lines. (A) Volcano plot of the miRNA expression signature determined by
RNA sequencing. The log2 fold-change (FC) is plotted on the x-axis, and the −log10 (p-value) is plotted on the y-axis. The
blue points represent the downregulated miRNAs with an absolute −log2 FC ≥ 1 (FC = 2) and p-value < 0.05. (B) The
expression levels of miR-490-5p and miR-490-3p, by quantitative polymerase chain reaction, were evaluated in CRC clinical
tissues and cell lines (HCT116 and DLD-1). Expression levels of these miRNAs were significantly reduced in cancer tissues
(p < 0.001). (C) Spearman’s rank test showed positive correlations between the expression levels of miR-490-5p and miR-
490-3p in clinical specimens (r = 0.957, p < 0.001). Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-3p
in colorectal cancer (CRC) tissues and cell lines. (A) Volcano plot of the miRNA expression signature determined by RNA
sequencing. The log2 fold-change (FC) is plotted on the x-axis, and the −log10 (p-value) is plotted on the y-axis. The blue
points represent the downregulated miRNAs with an absolute −log2 FC ≥1 (FC = 2) and p-value < 0.05. (B) The expression
levels of miR-490-5p and miR-490-3p, by quantitative polymerase chain reaction, were evaluated in CRC clinical tissues and
cell lines (HCT116 and DLD-1). Expression levels of these miRNAs were significantly reduced in cancer tissues (p < 0.001). 2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells
2.2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells The
blue points represent the downregulated miRNAs with an absolute −log2 FC ≥ 1 (FC = 2) and p-value < 0.05. (B) The
expression levels of miR-490-5p and miR-490-3p, by quantitative polymerase chain reaction, were evaluated in CRC clinical
tissues and cell lines (HCT116 and DLD-1). Expression levels of these miRNAs were significantly reduced in cancer tissues
(p < 0.001). (C) Spearman’s rank test showed positive correlations between the expression levels of miR-490-5p and miR-
490-3p in clinical specimens (r = 0.957, p < 0.001). Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-3p
in colorectal cancer (CRC) tissues and cell lines. (A) Volcano plot of the miRNA expression signature determined by RNA
sequencing. The log2 fold-change (FC) is plotted on the x-axis, and the −log10 (p-value) is plotted on the y-axis. The blue
points represent the downregulated miRNAs with an absolute −log2 FC ≥1 (FC = 2) and p-value < 0.05. (B) The expression
levels of miR-490-5p and miR-490-3p, by quantitative polymerase chain reaction, were evaluated in CRC clinical tissues and
cell lines (HCT116 and DLD-1). Expression levels of these miRNAs were significantly reduced in cancer tissues (p < 0.001). (C) Spearman’s rank test showed positive correlations between the expression levels of miR-490-5p and miR-490-3p in clinical
specimens (r = 0.957, p < 0.001). Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-
3p in colorectal cancer (CRC) tissues and cell lines. (A) Volcano plot of the miRNA expression signature determined by
RNA sequencing. The log2 fold-change (FC) is plotted on the x-axis, and the −log10 (p-value) is plotted on the y-axis. The
blue points represent the downregulated miRNAs with an absolute −log2 FC ≥ 1 (FC = 2) and p-value < 0.05. (B) The
expression levels of miR-490-5p and miR-490-3p, by quantitative polymerase chain reaction, were evaluated in CRC clinical
tissues and cell lines (HCT116 and DLD-1). Expression levels of these miRNAs were significantly reduced in cancer tissues
(p < 0.001). (C) Spearman’s rank test showed positive correlations between the expression levels of miR-490-5p and miR-
490-3p in clinical specimens (r = 0.957, p < 0.001). 2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells
2.2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells ion of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-
sion of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-3p
i
f
iR 490 5
d
iR 490 3 b
ll RNA
i
d
i
f
iR 490 5
d
iR 49 Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-
3p in colorectal cancer (CRC) tissues and cell lines. (A) Volcano plot of the miRNA expression signature determined by
RNA sequencing. The log2 fold-change (FC) is plotted on the x-axis, and the −log10 (p-value) is plotted on the y-axis. The
blue points represent the downregulated miRNAs with an absolute −log2 FC ≥ 1 (FC = 2) and p-value < 0.05. (B) The
expression levels of miR-490-5p and miR-490-3p, by quantitative polymerase chain reaction, were evaluated in CRC clinical
tissues and cell lines (HCT116 and DLD-1). Expression levels of these miRNAs were significantly reduced in cancer tissues
(p < 0.001). (C) Spearman’s rank test showed positive correlations between the expression levels of miR-490-5p and miR-
490-3p in clinical specimens (r = 0.957, p < 0.001). Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-3p
in colorectal cancer (CRC) tissues and cell lines. (A) Volcano plot of the miRNA expression signature determined by RNA
sequencing. The log2 fold-change (FC) is plotted on the x-axis, and the −log10 (p-value) is plotted on the y-axis. The blue
points represent the downregulated miRNAs with an absolute −log2 FC ≥1 (FC = 2) and p-value < 0.05. (B) The expression
levels of miR-490-5p and miR-490-3p, by quantitative polymerase chain reaction, were evaluated in CRC clinical tissues and
cell lines (HCT116 and DLD-1). Expression levels of these miRNAs were significantly reduced in cancer tissues (p < 0.001). (C) Spearman’s rank test showed positive correlations between the expression levels of miR-490-5p and miR-490-3p in clinical
specimens (r = 0.957, p < 0.001). Figure 3. Expression of miR-490-5p and miR-490-3p by small RNA sequencing and expression of miR-490-5p and miR-490-
3p in colorectal cancer (CRC) tissues and cell lines. (A) Volcano plot of the miRNA expression signature determined by
RNA sequencing. The log2 fold-change (FC) is plotted on the x-axis, and the −log10 (p-value) is plotted on the y-axis. 2.4. Screening of Putative Oncogenic Targets by miR-490-3p Regulation in CRC Cells
ve oncogenic targets of miR 490 3p regulation in CRC
i e TargetScan database the GEO database (genes do 2.4. Screening of Putative Oncogenic Targets by miR-490-3p Regulation in CRC Cells
ve o coge ic ta gets o mi
90 3p egu atio
i
C C
i e TargetScan database the GEO database (genes do Our screening strategy of miR-490-3p targets is shown in Figure 5. To identify the puta-
tive oncogenic targets of miR-490-3p regulation in CRC cells, we assessed three datasets, i.e.,
TargetScan database, the GEO database (genes downregulated in miR-490-3p-transfected
HCT116), and TCGA-COAD database through the GEPIA2 platform (genes upregulated in
CRC tissues). , i.e., TargetScan database, the GEO database (genes downreg
ected HCT116), and TCGA-COAD database through the GE
ulated in CRC tissues). e 5. Flowchart for searching for oncogenic targets subject to miR-490-3
r (CRC) cells. To identify genes controlled by miR-490-3p in CRC cells
ase and gene expression profile of miR-490-3p transfected HCT116 ce
Figure 5. Flowchart for searching for oncogenic targets subject to miR-490-3p regulation in colorectal
cancer (CRC) cells. To identify genes controlled by miR-490-3p in CRC cells, we used the TargetScan
database and gene expression profile of miR-490-3p transfected HCT116 cells (GEO accession number:
GSE129043). Moreover, GEPIA2 database analysis was used to identify upregulated genes in CRC
tissues. A total of 38 genes were identified as possibly controlled by miR-490-3p in CRC cells. e 5. Flowchart for searching for oncogenic targets subject to miR-490-3
(CRC) cells. To identify genes controlled by miR-490-3p in CRC cells
ase and gene expression profile of miR-490-3p transfected HCT116 ce
Figure 5. Flowchart for searching for oncogenic targets subject to miR-490-3p regulation in colorectal
cancer (CRC) cells. To identify genes controlled by miR-490-3p in CRC cells, we used the TargetScan
database and gene expression profile of miR-490-3p transfected HCT116 cells (GEO accession number:
GSE129043). Moreover, GEPIA2 database analysis was used to identify upregulated genes in CRC
tissues. A total of 38 genes were identified as possibly controlled by miR-490-3p in CRC cells. 5. Flowchart for searching for oncogenic targets subject to miR-490-3
(CRC) cells. To identify genes controlled by miR-490-3p in CRC cells
se and gene expression profile of miR 490 3p transfected HCT116 ce
Figure 5. Flowchart for searching for oncogenic targets subject to miR-490-3p regulation in colorectal
cancer (CRC) cells. To identify genes controlled by miR-490-3p in CRC cells, we used the TargetScan
database and gene expression profile of miR-490-3p transfected HCT116 cells (GEO accession number:
GSE129043). 2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells
2.2. Tumor-Suppressive Roles of miR-490-5p and miR-490-3p in CRC Cells (A) Cell proliferation, assessed by
XTT assay, 72 h after transfection of mature miRNAs (B) Cell invasion, determined by Matrigel invasion assay, 48 h after
seeding inhibitor-transfected cells into the chambers. (C) Cell migration assessed, using a membrane culture system, 48 h
after seeding inhibitor-transfected cells into the chambers. Int. J. Mol. Sci. 2021, 22, 9876
ing 6 of 17
n into 2.3. MiR-490-3p and miR-490-5p Was Incorporated into the RNA-Induced Silencing Complex
(RISC) in CRC Cells
he RISC. Levels of miR-490-3p were significantly higher than
mock, miR-control, and miR-490-5p (Figure S1). Similarly, wh 2.3. MiR-490-3p and miR-490-5p Was Incorporated into the RNA-Induced Silencing Complex
(RISC) in CRC Cells
e RISC. Levels of miR-490-3p were significantly higher than
mock, miR-control, and miR-490-5p (Figure S1). Similarly, wh In miRNA biogenesis, it is essential that miRNAs are incorporated into the RISC to
regulate target genes. Ago2 is a fundamental component of the RISC. We investigated that
miR-490-3p and miR-490-5p actually functioned in CRC cells by immunoprecipitation using
an anti-Ago2 antibody. Levels of the miRNA incorporation into Ago2 were quantified
with qPCR. Mature miR-490-3p was transfected into CRC cells, and it was incorporated
into the RISC. Levels of miR-490-3p were significantly higher than that in cells transfected
with mock, miR-control, and miR-490-5p (Figure S1). Similarly, when transfected with
mature miR-490-5p, the levels of miR-490-5p were significantly higher than those of cells
transfected with mock, miR-control, and miR-490-3p (Figure S1). These results indicated
that miR-490-3p and miR-490-5p were incorporated into RISC separately. ,
,
p (
g
)
y,
miR-490-5p, the levels of miR-490-5p were significantly high
ected with mock, miR-control, and miR-490-3p (Figure S1). T
miR-490-3p and miR-490-5p were incorporated into RISC sepa
creening of Putative Oncogenic Targets by miR-490-3p Regulation
Our screening strategy of miR-490-3p targets is shown in Fig
i e oncogenic targets of miR 490 3p regulation in CRC cells 2.4. Screening of Putative Oncogenic Targets by miR-490-3p Regulation in CRC Cells
ve oncogenic targets of miR 490 3p regulation in CRC
i e TargetScan database the GEO database (genes do Moreover, GEPIA2 database analysis was used to identify upregulated genes in CRC
tissues. A total of 38 genes were identified as possibly controlled by miR-490-3p in CRC cells. g
p
p
p
SE129043). Moreover, GEPIA2 database analysis was used to ident
issues. A total of 38 genes were identified as possibly controlled by m
There were 38 candidate targets for miR-490-3p regulation in CRC cells (Table 2),
of which three genes (IRAK1, FUT1, and GPRIN2) were associated with poor prognosis
(Figure 6). Among these targets, we focused on IRAK1 as an oncogenic target of miR-490-3p
regulation in CRC cells. Int. J. Mol. Sci. 2021, 22, 9876 7 of 17 Table 2. Candidate genes of miR-490-3p targets in CRC cells. Table 2. Candidate genes of miR-490-3p targets in CRC cells. Table 2. Candidate genes of miR-490-3p targets in CRC cells. 2.4. Screening of Putative Oncogenic Targets by miR-490-3p Regulation in CRC Cells
ve oncogenic targets of miR 490 3p regulation in CRC
i e TargetScan database the GEO database (genes do Entrez
Gene ID
Gene
Symbol
Gene Name
Binding
Sites
HCT116
miR-490-3p
Transfectant
log2 FC ≤1
OncoLnc 5 yrs
(p-Value *)
345079
SOWAHB
sosondowah ankyrin repeat domain family
member B
1
−1.8673139
0.0519
* Kaplan-Meier analysis log-rank p-value < 0.05. Bold values, Kaplan-Meier analysis log-rank p-value < 0.05, poor prognosis with a high
expression. Italic value, Kaplan-Meier analysis log-rank p-value < 0.05, poor prognosis with a low expression. 201595
STT3B
STT3B, subunit of the oligosaccharyltransferase
complex (catalytic)
1
−2.0069218
0.0046
345079
SOWAHB
sosondowah ankyrin repeat domain family
member B
1
−1.8673139
0.0519
* Kaplan-Meier analysis log-rank p-value < 0.05. Bold values, Kaplan-Meier analysis log-rank p-value < 0.05, poor progno-
sis with a high expression. Italic value, Kaplan-Meier analysis log-rank p-value < 0.05, poor prognosis with a low expres-
sion. Table 2. Cont. Entrez
Gene ID
Gene
Symbol
Gene Name
Binding
Sites
HCT116
miR-490-3p
Transfectant
log2 FC ≤1
OncoLnc 5 yrs
(p-Value *)
345079
SOWAHB
sosondowah ankyrin repeat domain family
member B
1
−1.8673139
0.0519
* Kaplan-Meier analysis log-rank p-value < 0.05. Bold values, Kaplan-Meier analysis log-rank p-value < 0.05, poor prognosis with a high
expression. Italic value, Kaplan-Meier analysis log-rank p-value < 0.05, poor prognosis with a low expression. 201595
STT3B
STT3B, subunit of the oligosaccharyltransferase
complex (catalytic)
1
−2.0069218
0.0046
345079
SOWAHB
sosondowah ankyrin repeat domain family
member B
1
−1.8673139
0.0519
* Kaplan-Meier analysis log-rank p-value < 0.05. Bold values, Kaplan-Meier analysis log-rank p-value < 0.05, poor progno-
sis with a high expression. Italic value, Kaplan-Meier analysis log-rank p-value < 0.05, poor prognosis with a low expres-
sion. Table 2. Cont. Entrez
Gene ID
Gene
Symbol
Gene Name
Binding
Sites
HCT116
miR-490-3p
Transfectant
log2 FC ≤1
OncoLnc 5 yrs
(p-Value *)
345079
SOWAHB
sosondowah ankyrin repeat domain family
member B
1
−1.8673139
0.0519
* Kaplan-Meier analysis log-rank p-value < 0.05. Bold values, Kaplan-Meier analysis log-rank p-value < 0.05, poor prognosis with a high
expression. Italic value, Kaplan-Meier analysis log-rank p-value < 0.05, poor prognosis with a low expression. 201595
STT3B
STT3B, subunit of the oligosaccharyltransferase
complex (catalytic)
1
−2.0069218
0.0046
345079
SOWAHB
sosondowah ankyrin repeat domain family
member B
1
−1.8673139
0.0519
* Kaplan-Meier analysis log-rank p-value < 0.05. Bold values, Kaplan-Meier analysis log-rank p-value < 0.05, poor progno-
sis with a high expression. Italic value, Kaplan-Meier analysis log-rank p-value < 0.05, poor prognosis with a low expres-
sion. Table 2. Cont. 2.4. Screening of Putative Oncogenic Targets by miR-490-3p Regulation in CRC Cells
ve oncogenic targets of miR 490 3p regulation in CRC
i e TargetScan database the GEO database (genes do Entrez
Gene ID
Gene
Symbol
Gene Name
Binding
Sites
HCT116
miR-490-3p
Transfectant
log2 FC ≤1
OncoLnc 5 yrs
(p-Value *)
9721
GPRIN2
G protein regulated inducer of neurite
outgrowth 2
1
−2.3130598
0.008
3654
IRAK1
interleukin-1 receptor-associated kinase 1
1
−1.0598125
0.0427
2523
FUT1
fucosyltransferase 1 (galactoside
2-alpha-L-fucosyltransferase, H blood
group)
1
−1.5235968
0.0468
135112
NCOA7
nuclear receptor coactivator 7
1
−1.177208
0.0585
84061
MAGT1
magnesium transporter 1
1
−1.5050192
0.0799
6382
SDC1
syndecan 1
1
−1.0336791
0.088
3635
INPP5D
inositol polyphosphate-5-phosphatase,
145 kDa
1
−1.2042127
0.1059
3178
HNRNPA1
heterogeneous nuclear
ribonucleoprotein A1
3
−1.6466646
0.1496
84152
PPP1R1B
protein phosphatase 1, regulatory
(inhibitor) subunit 1B
1
−2.459818
0.163
56886
UGGT1
UDP-glucose glycoprotein
glucosyltransferase 1
1
−1.0872145
0.2326
8529
CYP4F2
cytochrome P450, family 4, subfamily F,
polypeptide 2
1
−2.5992675
0.2469
647024
C6orf132
chromosome 6 open reading frame 132
2
−1.2631998
0.3329
10525
HYOU1
hypoxia up-regulated 1
2
−1.6224588
0.3420
2444
FRK
fyn-related kinase
2
−1.2127504
0.3546
154796
AMOT
angiomotin
1
−1.0376037
0.3709
23446
SLC44A1
solute carrier family 44 (choline
transporter), member 1
2
−1.1456499
0.4167
440145
MZT1
mitotic spindle organizing protein 1
1
−1.0596924
0.4636
28985
MCTS1
malignant T cell amplified sequence 1
1
−1.0754105
0.4927
4642
MYO1D
myosin ID
1
−1.1291242
0.5085
84187
TMEM164
transmembrane protein 164
1
−1.0723546
0.5172
80223
RAB11FIP1
RAB11 family interacting protein 1 (class I)
2
−1.374427
0.5477
8635
RNASET2
ribonuclease T2
1
−1.4826338
0.5618
3146
HMGB1
high mobility group box 1
1
−1.8401318
0.5719
340706
VWA2
von Willebrand factor A domain
containing 2
1
−1.3371019
0.6107
51561
IL23A
interleukin 23, alpha subunit p19
1
−1.1564418
0.6474
6850
SYK
spleen tyrosine kinase
1
−1.065637
0.6535
2232
FDXR
ferredoxin reductase
1
−1.0080034
0.7008
2525
FUT3
fucosyltransferase 3 (galactoside
3(4)-L-fucosyltransferase, Lewis
blood group)
1
−2.2499368
0.7117
7416
VDAC1
voltage-dependent anion channel 1
2
−1.3866558
0.7136
80201
HKDC1
hexokinase domain containing 1
1
−2.3432722
0.7136
196264
MPZL3
myelin protein zero-like 3
1
−1.0834669
0.7250
3703
STT3A
STT3A, subunit of the
oligosaccharyltransferase complex
(catalytic)
1
−1.6853191
0.7465
7039
TGFA
transforming growth factor, alpha
2
−1.0281087
0.7569
4494
MT1F
metallothionein 1F
1
−1.7403115
0.7756
6523
SLC5A1
solute carrier family 5 (sodium/glucose
cotransporter), member 1
1
−2.3984282
0.8507
6653
SORL1
sortilin-related receptor, L (DLR class) A
repeats containing
1
−1.0742446
0.853
201595
STT3B
STT3B, subunit of the
oligosaccharyltransferase complex
(catalytic)
1
−2.0069218
0.0046 Int. J. Mol. Sci. 2021, 22, 9876 8 of 17 Table 2. Cont. 2.4. Screening of Putative Oncogenic Targets by miR-490-3p Regulation in CRC Cells
ve oncogenic targets of miR 490 3p regulation in CRC
i e TargetScan database the GEO database (genes do Gene
Symbol
STT3B Gene Name
complex (cata Clinical significance of miR-490-3p target genes (IRAK1, FUT1, and GPRIN2) in CRC cohort data analysis. Among
ve target genes, 3 genes (IRAK1, FUT1 and GPRIN2) were significantly associated with poor prognosis in patients
rectal cancer (p < 0.05). Kaplan–Meier curves for 5-year overall survivals of 3 genes are shown at left side. Patients
ded into high and low groups (relative to median expression) according to miRNA expression. The red line
e high expression group, and the blue line shows the low expression group. Expression levels of three genes
UT1, and GPRIN2) in CRC tissues and normal tissues obtained from TCGA-COAD, based on the GEPIA2 plat-
shown at the right side. Figure 6. Clinical significance of miR-490-3p target genes (IRAK1, FUT1, and GPRIN2) in CRC cohort
data analysis. Among 38 putative target genes, 3 genes (IRAK1, FUT1 and GPRIN2) were significantly
associated with poor prognosis in patients with colorectal cancer (p < 0.05). Kaplan–Meier curves for
5-year overall survivals of 3 genes are shown at left side. Patients were divided into high and low
groups (relative to median expression) according to miRNA expression. The red line shows the high
expression group, and the blue line shows the low expression group. Expression levels of three genes
(IRAK1, FUT1, and GPRIN2) in CRC tissues and normal tissues obtained from TCGA-COAD, based
on the GEPIA2 platform, are shown at the right side. Figure 6. Clinical significance of miR-490-3p target genes (IRAK1, FUT1, and GPRIN2) in CRC cohort da
38 putative target genes, 3 genes (IRAK1, FUT1 and GPRIN2) were significantly associated with poor p
with colorectal cancer (p < 0.05). Kaplan–Meier curves for 5-year overall survivals of 3 genes are shown
were divided into high and low groups (relative to median expression) according to miRNA expre
shows the high expression group, and the blue line shows the low expression group. Expression le
(IRAK1, FUT1, and GPRIN2) in CRC tissues and normal tissues obtained from TCGA-COAD, based o
form, are shown at the right side. Figure 6. Clinical significance of miR-490-3p target genes (IRAK1, FUT1, and GP
data analysis. Among 38 putative target genes, 3 genes (IRAK1, FUT1 and GPRIN
associated with poor prognosis in patients with colorectal cancer (p < 0.05). Kap
5-year overall survivals of 3 genes are shown at left side. Patients were divid
groups (relative to median expression) according to miRNA expression. 2.4. Screening of Putative Oncogenic Targets by miR-490-3p Regulation in CRC Cells
ve oncogenic targets of miR 490 3p regulation in CRC
i e TargetScan database the GEO database (genes do Luciferase activity was significantly reduced after
co-transfection of miR-490-3p and a vector carrying the wild-type miR-490-3p target site. In
contrast, luciferase activity was not changed following co-transfection of miR-490-3p and a
vector carrying the deletion-type miR-490-3p target site (Figure 7C). These results showed
that IRAK1 was directly regulated by miR-490-3p in CRC cells. Next, a dual luciferase reporter assay was performed to assess whether miR
bound directly to the IRAK1 target site. Luciferase activity was significantly reduce
co-transfection of miR-490-3p and a vector carrying the wild-type miR-490-3p targ
In contrast, luciferase activity was not changed following co-transfection of miR
and a vector carrying the deletion-type miR-490-3p target site (Figure 7C). These
showed that IRAK1 was directly regulated by miR-490-3p in CRC cells. regulation of IRAK1 expression by miR-490-3p in colorectal cancer (CRC) cells (HCT116 and DLD-1). showing significantly reduced expression of IRAK1 mRNA at 72 h after miR-490-3p transfection in C
n of GAPDH was used as an internal control. (B) Western blot showing reduced expression of IRAK1 p
r miR-490-3p transfection in CRC cells. Expression of GAPDH was used as an internal control. (C) O
0-3p binding site predicted within the 3′-UTR of IRAK1 by TargetScanHuman analyses (upper panel). D
ter assays showed reduced luminescence activity after cotransfection of the wild-type IRAK1 3′-UTR
ing the miR-490-3p binding site) with miR-490-3p in CRC cells (lower panel). Normalized data were cal
illa/firefly luciferase activity ratio (N.S., not significant). 2.6. Effects of IRAK1 Knockdown in CRC Cells
To investigate the expression of IRAK1 mRNA/IRAK1 protein in CRC cells,
formed CRC knockdown assays using two different siRNAs. The expression levels
Figure 7. Direct regulation of IRAK1 expression by miR-490-3p in colorectal cancer (CRC) cells
(HCT116 and DLD-1). (A) Real-time PCR showing significantly reduced expression of IRAK1 mRNA
at 72 h after miR-490-3p transfection in CRC cells. Expression of GAPDH was used as an internal
control. (B) Western blot showing reduced expression of IRAK1 protein at 72 h after miR-490-3p
transfection in CRC cells. Expression of GAPDH was used as an internal control. (C) One putative
miR-490-3p binding site predicted within the 3′-UTR of IRAK1 by TargetScanHuman analyses (upper
panel). Dual luciferase reporter assays showed reduced luminescence activity after cotransfection of
the wild-type IRAK1 3′-UTR sequence (containing the miR-490-3p binding site) with miR-490-3p in
CRC cells (lower panel). Normalized data were calculated as the Renilla/firefly luciferase activity
ratio (N.S., not significant). 2.4. Screening of Putative Oncogenic Targets by miR-490-3p Regulation in CRC Cells
ve oncogenic targets of miR 490 3p regulation in CRC
i e TargetScan database the GEO database (genes do The re
expression group, and the blue line shows the low expression group. Expression
(IRAK1, FUT1, and GPRIN2) in CRC tissues and normal tissues obtained from
on the GEPIA2 platform, are shown at the right side. ificance of miR-490-3p target genes (IRAK1, FUT1, and GPRIN2) in CRC cohort data analysis. Among
es, 3 genes (IRAK1, FUT1 and GPRIN2) were significantly associated with poor prognosis in patients
(p < 0.05). Kaplan–Meier curves for 5-year overall survivals of 3 genes are shown at left side. Patients
gh and low groups (relative to median expression) according to miRNA expression. The red line
ession group, and the blue line shows the low expression group. Expression levels of three genes
PRIN2) in CRC tissues and normal tissues obtained from TCGA-COAD, based on the GEPIA2 plat-
e right side. Figure 6. Clinical significance of miR-490-3p target genes (IRAK1, FUT1, and GPRIN2) in CRC cohort
data analysis. Among 38 putative target genes, 3 genes (IRAK1, FUT1 and GPRIN2) were significantly
associated with poor prognosis in patients with colorectal cancer (p < 0.05). Kaplan–Meier curves for
5-year overall survivals of 3 genes are shown at left side. Patients were divided into high and low
groups (relative to median expression) according to miRNA expression. The red line shows the high
expression group, and the blue line shows the low expression group. Expression levels of three genes
(IRAK1, FUT1, and GPRIN2) in CRC tissues and normal tissues obtained from TCGA-COAD, based
on the GEPIA2 platform, are shown at the right side. Int. J. Mol. Sci. 2021, 22, 9876
Int. J. Mol. Sci. 2021 9 of 17 2.5. Direct Regulation of IRAK1 by miR-490-3p in CRC Cells
2.5. Direct Regulation of IRAK1 by miR-490-3p in CR
W i
i
d di
l
i
f IRAK1 We investigated direct regulation of IRAK1 by miR-490-3p in CRC cells. Expression lev-
els of IRAK1 mRNA and IRAK1 protein were significantly reduced by aberrant expression
of miR-490-3p in CRC cells (HCT116 and DLD-1, Figure 7A,B). We investigated direct regulation of IRAK1 by miR-490-3p in CRC cells. Exp
levels of IRAK1 mRNA and IRAK1 protein were significantly reduced by aberrant
sion of miR-490-3p in CRC cells (HCT116 and DLD-1, Figure 7A,B). N
t
d
l l
if
t
f
d t
h th
iR Next, a dual luciferase reporter assay was performed to assess whether miR-490-3p
bound directly to the IRAK1 target site. 2.4. Screening of Putative Oncogenic Targets by miR-490-3p Regulation in CRC Cells
ve oncogenic targets of miR 490 3p regulation in CRC
i e TargetScan database the GEO database (genes do 2 6 Eff t
f IRAK1 K
kd
i
CRC C ll regulation of IRAK1 expression by miR-490-3p in colorectal cancer (CRC) cells (HCT116 and DLD-1). howing significantly reduced expression of IRAK1 mRNA at 72 h after miR-490-3p transfection in C
of GAPDH was used as an internal control. (B) Western blot showing reduced expression of IRAK1 p
miR-490-3p transfection in CRC cells. Expression of GAPDH was used as an internal control. (C) O
-3p binding site predicted within the 3′-UTR of IRAK1 by TargetScanHuman analyses (upper panel). D
er assays showed reduced luminescence activity after cotransfection of the wild-type IRAK1 3′-UTR
ng the miR-490-3p binding site) with miR-490-3p in CRC cells (lower panel). Normalized data were cal
lla/firefly luciferase activity ratio (N.S., not significant). 2.6. Effects of IRAK1 Knockdown in CRC Cells
To investigate the expression of IRAK1 mRNA/IRAK1 protein in CRC cells,
Figure 7. Direct regulation of IRAK1 expression by miR-490-3p in colorectal cancer (CRC) cells
(HCT116 and DLD-1). (A) Real-time PCR showing significantly reduced expression of IRAK1 mRNA
at 72 h after miR-490-3p transfection in CRC cells. Expression of GAPDH was used as an internal
control. (B) Western blot showing reduced expression of IRAK1 protein at 72 h after miR-490-3p
transfection in CRC cells. Expression of GAPDH was used as an internal control. (C) One putative
miR-490-3p binding site predicted within the 3′-UTR of IRAK1 by TargetScanHuman analyses (upper
panel). Dual luciferase reporter assays showed reduced luminescence activity after cotransfection of
the wild-type IRAK1 3′-UTR sequence (containing the miR-490-3p binding site) with miR-490-3p in
CRC cells (lower panel). Normalized data were calculated as the Renilla/firefly luciferase activity
ratio (N.S., not significant). both IRAK1 mRNA and IRAK1 protein were markedly reduced by siIRAK1-1 and siIRAK1-2
in the two cell lines (Figure 8A,B).
10 of 17
REVIEW
10 of 17 both IRAK1 mRNA and IRAK1 protein were markedly reduced by siIRAK1-1 and siIRAK1-2
in the two cell lines (Figure 8A,B). 10 of 17
REVIEW
10 of 17 Knockdown efficiencies of IRAK1 expression by siRNAs in colorectal cancer (CRC) cells. rent siRNAs, siIRAK1-1 and siIRAK1-2 were used. (A) Real-time PCR showing signifi-
duced expression of IRAK1 mRNA 72 h after either siIRAK1-1 or siIRAK1-2 transfection in
(HCT116 and DLD-1). Expression of GAPDH was used as an internal control. (B) Western
wing reduced expression of IRAK1 protein 72 h after siIRAK1-1 or siIRAK1-2 transfection
lls. Expression of GAPDH was used as an internal control. Figure 8. Knockdown efficiencies of IRAK1 expression by siRNAs in colorectal cancer (CRC) cells. Two different siRNAs, siIRAK1-1 and siIRAK1-2 were used. (A) Real-time PCR showing significantly
reduced expression of IRAK1 mRNA 72 h after either siIRAK1-1 or siIRAK1-2 transfection in CRC
cells (HCT116 and DLD-1). Expression of GAPDH was used as an internal control. (B) Western blots
showing reduced expression of IRAK1 protein 72 h after siIRAK1-1 or siIRAK1-2 transfection in CRC
cells. Expression of GAPDH was used as an internal control. Figure 8. Knockdown efficiencies of IRAK1 expression by siRNAs in colorectal cancer (CRC) cells. Two different siRNAs, siIRAK1-1 and siIRAK1-2 were used. (A) Real-time PCR showing signifi-
cantly reduced expression of IRAK1 mRNA 72 h after either siIRAK1-1 or siIRAK1-2 transfection in
CRC cells (HCT116 and DLD-1). Expression of GAPDH was used as an internal control. (B) Western
blots showing reduced expression of IRAK1 protein 72 h after siIRAK1-1 or siIRAK1-2 transfection
in CRC cells. Expression of GAPDH was used as an internal control. By suppressing IRAK1 expression, cell proliferation assays showed no significant ef- 8 K
kd
ffi i
i
f IR Knockdown efficiencies of IRAK1 expression by siRNAs in colorectal cancer (CRC) cells. rent siRNAs, siIRAK1-1 and siIRAK1-2 were used. (A) Real-time PCR showing signifi-
uced expression of IRAK1 mRNA 72 h after either siIRAK1-1 or siIRAK1-2 transfection in
(HCT116 and DLD-1). Expression of GAPDH was used as an internal control. (B) Western
wing reduced expression of IRAK1 protein 72 h after siIRAK1-1 or siIRAK1-2 transfection
ls. Expression of GAPDH was used as an internal control. Figure 8. Knockdown efficiencies of IRAK1 expression by siRNAs in colorectal cancer (CRC) cells. Two different siRNAs, siIRAK1-1 and siIRAK1-2 were used. formed CRC knockdown assays usin
IRAK1 mRNA and IRAK1 protein w
2.6. Effects of IRAK1 Knockdown in CRC Cells IRAK1 mRNA and IRAK1 protein were markedly reduced by siIRAK1 1 and siIRA
the two cell lines (Figure 8A,B). To investigate the expression of IRAK1 mRNA/IRAK1 protein in CRC cells, we
performed CRC knockdown assays using two different siRNAs. The expression levels of Int. J. Mol. Sci. 2021, 22, 9876 10 of 17 both IRAK1 mRNA and IRAK1 protein were markedly reduced by siIRAK1-1 and siIRAK1-2
in the two cell lines (Figure 8A,B).
10 of 17
REVIEW
10 of 17 (A) Real-time PCR showing significantly
reduced expression of IRAK1 mRNA 72 h after either siIRAK1-1 or siIRAK1-2 transfection in CRC
cells (HCT116 and DLD-1). Expression of GAPDH was used as an internal control. (B) Western blots
showing reduced expression of IRAK1 protein 72 h after siIRAK1-1 or siIRAK1-2 transfection in CRC
cells. Expression of GAPDH was used as an internal control. Figure 8. Knockdown efficiencies of IRAK1 expression by siRNAs in colorectal cancer (CRC) cells. Two different siRNAs, siIRAK1-1 and siIRAK1-2 were used. (A) Real-time PCR showing signifi-
cantly reduced expression of IRAK1 mRNA 72 h after either siIRAK1-1 or siIRAK1-2 transfection in
CRC cells (HCT116 and DLD-1). Expression of GAPDH was used as an internal control. (B) Western
blots showing reduced expression of IRAK1 protein 72 h after siIRAK1-1 or siIRAK1-2 transfection
in CRC cells. Expression of GAPDH was used as an internal control. By suppressing IRAK1 expression cell proliferation assays showed no significant ef uppressing IRAK1 expression, cell proliferation assays showed no significant ef-
hese siRNA transfected into the two cell lines (Figure 9A), but migration and
abilities were significantly blocked in CRC cells (Figure 9B,C). These data sug-
at aberrant expression of IRAK1 promoted cancer-related phenotypes in CRC
By suppressing IRAK1 expression, cell proliferation assays showed no significant
effects of these siRNA transfected into the two cell lines (Figure 9A), but migration and in-
vasive abilities were significantly blocked in CRC cells (Figure 9B,C). These data suggested
that aberrant expression of IRAK1 promoted cancer-related phenotypes in CRC cells. y
pp
g
p
p
y
g
fects of these siRNA transfected into the two cell lines (Figure 9A), but migration and
nvasive abilities were significantly blocked in CRC cells (Figure 9B,C). These data sug-
gested that aberrant expression of IRAK1 promoted cancer-related phenotypes in CRC
cells. Figure 9. Cont. Figure 9. Cont. Figure 9. Cont. 11 of 17
17 Int. J. Mol. Sci. 2021, 22, 9876
Mol. Sci. 2021, 22, x FOR PEER Figure 9. Effects of knockdown of IRAK1 in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of IRAK1
(siIRAK1-1 and siIRAK1-2 transfection) in CRC cells. (A) Cell proliferation assessed by XTT assay 72 h after transfection of
siRNA (B) Cell invasion determined by Matrigel invasion assay 48 h after seeding inhibitor-transfected cells into the cham-
bers. 2 7 Expression of IRAK1 in CRC Clinical Specimens
2.7. Expression of IRAK1 in CRC Clinical Specimens
Expression of IRAK1 in CRC Clinical Specimens 2.7. Expression of IRAK1 in CRC Clinical Specimens
We analyzed expression levels of IRAK1 (RNA-Sequence data in 275 colon cancer
tissues compared to 349 normal colon tissues) using TCGA-COAD database through the
GEPIA2 platform. Expression of IRAK1 was significantly upregulated (p < 0.01; Figure 6). In addition, immunohistochemistry was used to assess protein expression levels of IRAK1
in CRC clinical specimens, and high expression of IRAK1 were shown in cancer lesions
(Figure 10)
We analyzed expression levels of IRAK1 (RNA-Sequence data in 275 colon cancer
tissues compared to 349 normal colon tissues) using TCGA-COAD database through the
GEPIA2 platform. Expression of IRAK1 was significantly upregulated (p < 0.01; Figure 6). In addition, immunohistochemistry was used to assess protein expression levels of IRAK1
in CRC clinical specimens, and high expression of IRAK1 were shown in cancer lesions
(Figure 10). We analyzed expression levels of IRAK1 (RNA-Sequence data in 275 colon cancer
es compared to 349 normal colon tissues) using TCGA-COAD database through the
IA2 platform. Expression of IRAK1 was significantly upregulated (p < 0.01; Figure 6). ddition, immunohistochemistry was used to assess protein expression levels of IRAK1
RC clinical specimens, and high expression of IRAK1 were shown in cancer lesions
ure 10). (
g
)
Figure 10. IRAK1 was high expression in colorectal tissues compared to adjacent normal colon tis-
sues as demonstrated. The representative clinical sections of samples from case 1 (patient number
8) and case 2 (patient number 17) are shown. (A) Microscopic finding of immunohistochemical stain-
ing for IRAK1 on the left side and hematoxylin and eosin (H&E) staining on the right side were
shown (magnification, ×20). Areas in the boxes are shown magnified at colon cancer tissue (B) and
adjacent normal colon mucosa tissue (C). (B) Immunohistochemical staining for IRAK1 H&E stain-
ing in the colon cancer cell were shown (magnification, ×200). (C) Immunohistochemical staining
for IRAK1 and H&E staining and in the normal colon epithelial cells were shown (magnification,
×200)
ure 10. IRAK1 was high expression in colorectal tissues compared to adjacent normal colon tis-
as demonstrated. The representative clinical sections of samples from case 1 (patient number
nd case 2 (patient number 17) are shown. (A) Microscopic finding of immunohistochemical stain-
for IRAK1 on the left side and hematoxylin and eosin (H&E) staining on the right side were
wn (magnification, ×20). 2 7 Expression of IRAK1 in CRC Clinical Specimens
2.7. Expression of IRAK1 in CRC Clinical Specimens
Expression of IRAK1 in CRC Clinical Specimens Areas in the boxes are shown magnified at colon cancer tissue (B) and
cent normal colon mucosa tissue (C). (B) Immunohistochemical staining for IRAK1 H&E stain-
n the colon cancer cell were shown (magnification, ×200). (C) Immunohistochemical staining
RAK1 and H&E staining and in the normal colon epithelial cells were shown (magnification,
). Figure 10. IRAK1 was high expression in colorectal tissues compared to adjacent normal colon tissues
as demonstrated. The representative clinical sections of samples from case 1 (patient number 8) and
case 2 (patient number 17) are shown. (A) Microscopic finding of immunohistochemical staining
for IRAK1 on the left side and hematoxylin and eosin (H&E) staining on the right side were shown
(magnification, ×20). Areas in the boxes are shown magnified at colon cancer tissue (B) and adjacent
normal colon mucosa tissue (C). (B) Immunohistochemical staining for IRAK1 H&E staining in the
colon cancer cell were shown (magnification, ×200). (C) Immunohistochemical staining for IRAK1
and H&E staining and in the normal colon epithelial cells were shown (magnification, ×200). Figure 10. IRAK1 was high expression in colorectal tissues compared to adjacent normal colon tis-
sues as demonstrated. The representative clinical sections of samples from case 1 (patient number
8) and case 2 (patient number 17) are shown. (A) Microscopic finding of immunohistochemical stain-
ing for IRAK1 on the left side and hematoxylin and eosin (H&E) staining on the right side were
shown (magnification, ×20). Areas in the boxes are shown magnified at colon cancer tissue (B) and
adjacent normal colon mucosa tissue (C). (B) Immunohistochemical staining for IRAK1 H&E stain-
ing in the colon cancer cell were shown (magnification, ×200). (C) Immunohistochemical staining
for IRAK1 and H&E staining and in the normal colon epithelial cells were shown (magnification,
re 10. IRAK1 was high expression in colorectal tissues compared to adjacent normal colon tis-
as demonstrated. The representative clinical sections of samples from case 1 (patient number
d case 2 (patient number 17) are shown. (A) Microscopic finding of immunohistochemical stain-
or IRAK1 on the left side and hematoxylin and eosin (H&E) staining on the right side were
wn (magnification, ×20). Areas in the boxes are shown magnified at colon cancer tissue (B) and
cent normal colon mucosa tissue (C). (B) Immunohistochemical staining for IRAK1 H&E stain-
n the colon cancer cell were shown (magnification, ×200). both IRAK1 mRNA and IRAK1 protein were markedly reduced by siIRAK1-1 and siIRAK1-2
in the two cell lines (Figure 8A,B).
10 of 17
REVIEW
10 of 17 (C) Cell migration assessed using a membrane culture system 48 h after seeding inhibitor-transfected cells into the
chambers
Figure 9. Effects of knockdown of IRAK1 in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of IRAK1
(siIRAK1-1 and siIRAK1-2 transfection) in CRC cells. (A) Cell proliferation assessed by XTT assay 72 h after transfection
of siRNA (B) Cell invasion determined by Matrigel invasion assay 48 h after seeding inhibitor-transfected cells into the
chambers. (C) Cell migration assessed using a membrane culture system 48 h after seeding inhibitor-transfected cells into
the chambers. ure 9. Effects of knockdown of IRAK1 in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of IRAK1
RAK1-1 and siIRAK1-2 transfection) in CRC cells. (A) Cell proliferation assessed by XTT assay 72 h after transfection of
NA (B) Cell invasion determined by Matrigel invasion assay 48 h after seeding inhibitor-transfected cells into the cham-
. (C) Cell migration assessed using a membrane culture system 48 h after seeding inhibitor-transfected cells into the
mbers. Figure 9. Effects of knockdown of IRAK1 in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of IRAK1
(siIRAK1-1 and siIRAK1-2 transfection) in CRC cells. (A) Cell proliferation assessed by XTT assay 72 h after transfection of
siRNA (B) Cell invasion determined by Matrigel invasion assay 48 h after seeding inhibitor-transfected cells into the cham-
bers. (C) Cell migration assessed using a membrane culture system 48 h after seeding inhibitor-transfected cells into the
chambers
Figure 9. Effects of knockdown of IRAK1 in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of IRAK1
(siIRAK1-1 and siIRAK1-2 transfection) in CRC cells. (A) Cell proliferation assessed by XTT assay 72 h after transfection
of siRNA (B) Cell invasion determined by Matrigel invasion assay 48 h after seeding inhibitor-transfected cells into the
chambers. (C) Cell migration assessed using a membrane culture system 48 h after seeding inhibitor-transfected cells into
the chambers. re 9. Effects of knockdown of IRAK1 in colorectal cancer (CRC) cells (HCT116 and DLD-1). Functional assays of IRAK1
AK1-1 and siIRAK1-2 transfection) in CRC cells. (A) Cell proliferation assessed by XTT assay 72 h after transfection of
NA (B) Cell invasion determined by Matrigel invasion assay 48 h after seeding inhibitor-transfected cells into the cham-
(C) Cell migration assessed using a membrane culture system 48 h after seeding inhibitor-transfected cells into the
mbers. 3. Discussion Clarifying the molecular pathogenesis of CRC based on the latest genomic analy-
ses will contribute to the development of new treatment strategies for this disease. We
have elucidated novel molecular pathogenesis using a miRNA-based approach in vari-
ous carcinomas [26,27]. To date, high-throughput technologies (e.g., oligo-microarrays,
PCR-based arrays, and RNA sequences) have enabled the construction of CRC miRNA
expression signatures, revealing aberrant expression of many miRNAs [24,28–34]. Previous
studies have shown that miR-490-3p, miR-195-5p, and miR-30a-5p, which are frequently
downregulated in CRC, function as tumor-suppressive miRNA in CRC cells [25,35–39]. These miRNAs were included in the signature we created in this study. Importantly, for
16 pairs downregulated, both miRNA strands (−5p and −3p) were ectopically expressed
in cancer tissues. Recently, it was suggested that the initial hypothesis that one of the
two strands is degraded during miRNA biosynthesis may be incorrect [40]. Our recent
studies show that some passenger miRNAs have a tumor suppressor function in cancer
cells. (e.g., miR-148-5p, miR-145-3p, miR-143-5p, miR-30c-2-3p, and miR-30a-3p) [19,26,41,42]. Those miRNA duplexes and their target oncogenic genes are closely associated with cancer
pathogenesis [19,26,41,42]. Growing body of studies showed that downregulation of miR-490-5p and miR-490-3p
were closely associated with a wide range of human cancers [43]. Among them, we focused
on miR-490-3p, which was the most down-regulated in our newly created signature. Previ-
ous study showed that downregulation of miR-490-3p was reported by high-throughput
sequence-based analysis in CRC [24,25]. Moreover, several studies have shown that down-
regulation of miR-490-3p occurs frequently in several types of cancer (e.g., breast cancer,
lung adenocarcinoma) and that this miRNA acts as a tumor suppressor [44–46]. These
reports indicate that downregulation of miR-490-3p has a critical effect on human tumori-
genesis, and it is an important issue to identify for target molecules of miR-490-3p in each
cancer type. yp
Previous studies showed that tumor-suppressive function of miR-490-3p in CRC cells
through targeting several oncogenic genes [35,36,47,48]. For example, ectopic expression
of miR-490-3p blocked migration and invasion abilities in CRC cells [47]. Transfection of
miR-490-3p inhibited cancer cell malignant phenotypes, e.g., cells proliferation, metastasis,
invasion, and anti-apoptosis [36]. In addition, miR-490-3p directly regulated VDAC1
expression, and a negatively controlled VDAC1/AMPK/mTOR pathway [36]. Ectopic
expression of miR-490-3p attenuated to cancer cell malignant transformation both in vitro
and in vivo [48]. Additionally, miR-490-3p directly controlled RAB14 in CRC cells [48]. 2 7 Expression of IRAK1 in CRC Clinical Specimens
2.7. Expression of IRAK1 in CRC Clinical Specimens
Expression of IRAK1 in CRC Clinical Specimens (C) Immunohistochemical staining
RAK1 and H&E staining and in the normal colon epithelial cells were shown (magnification,
). Figure 10. IRAK1 was high expression in colorectal tissues compared to adjacent normal colon tissues
as demonstrated. The representative clinical sections of samples from case 1 (patient number 8) and
case 2 (patient number 17) are shown. (A) Microscopic finding of immunohistochemical staining
for IRAK1 on the left side and hematoxylin and eosin (H&E) staining on the right side were shown
(magnification, ×20). Areas in the boxes are shown magnified at colon cancer tissue (B) and adjacent
normal colon mucosa tissue (C). (B) Immunohistochemical staining for IRAK1 H&E staining in the
colon cancer cell were shown (magnification, ×200). (C) Immunohistochemical staining for IRAK1
and H&E staining and in the normal colon epithelial cells were shown (magnification, ×200). Int. J. Mol. Sci. 2021, 22, 9876 12 of 17 12 of 17 4.1. Patient Samples CRC tissues and noncancerous tumor-adjacent tissues (27 each) were used to verify the
expression status of miR-490-5p and miR-490-3p (Table S5). All samples were collected from
patients who underwent surgical resection at Kagoshima University Hospital between
2014 and 2017. Written informed consent for the use of their specimens was obtained
from all patients. The study was conducted according to the guidelines of the Declaration
of Helsinki and approved by Ethics Committee of Kagoshima University (approval no. 160,038 28-65, date of approval: 19 March 2021) 4.2. CRC Cell Lines and Cell Culture The two human CRC cell lines (HCT116 and DLD-1) were used in this study. HCT116
cells were obtained from the RIKEN Cell Bank (Tsukuba, Ibaraki, Japan) and DLD-1 cells
were obtained from the Cell Resource Center for Biomedical Research/Cell Bank (Sendai,
Miyagi, Japan). HCT116 was maintained in DMEM supplemented with 10% fetal bovine
serum (FBS). DLD-1 was cultured in RPMI-1640 medium with 10% FBS. 4.3. Small RNA Sequencing The small RNA sequencing and data mining process were performed as in previous
studies [19,21]. Briefly, flesh frozen on dry ice using 10 samples (5 CRC tissues, and RNA
was harvested using Trizol reagent). Illumina TruSeq Small RNA Sample Preparation Kit
was used for the construction of sequencing libraries. miRNA libraries were prepared for
sequencing using standard Illumina protocols. New miRNA expression signatures were
generated using a next-generation sequencer HiSeq 2500 (Illumina, San Diego, CA, USA). Sequenced reads were trimmed for adaptor sequence, and masked for low-quality sequence
using cutadapt v.1.2.1. A false discovery rate (FDR) less than 0.05 was considered significant. The present RNA sequencing data was deposited in GEO database (GSE183437). 4.5. Transfection of Mature miRNAs, Small-Interfering RNAs, and Plasmid Vectors into
CRC Cells The miRNA precursors and siRNAs were obtained by Invitrogen (Thermo Fisher
Scientific, Waltham, MA, USA). Transfection of miRNA precursors, siRNAs, or negative
control miRNA/siRNA was performed with Lipofectamine™RNAiMAX and that of
plasmid vectors was performed with Lipofectamine™2000 (Thermo Fisher Scientific,
Waltham, MA, USA). HCT116 and DLD-1 cells were transfected with 10 nM miRNA,
siRNA, or negative control miRNA/siRNA. The reagents used in the process are listed in
Table S6. 4.6. Cell Proliferation, Migration and Invasion Assays in CRC Cells 4.4. RNA Extraction and qPCR We performed RNA extraction from clinical samples, cell lines, and qRT-PCR using the
methods we described previously [16,21]. According to the manufacturer’s protocol, total
RNA was isolated from flesh frozen colorectal tissues and cell lines using TRIzol reagent. RNA quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies,
Santa Clara, CA, USA). Then, RNA sample reverse transcription was achieved with a High
Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). The
qPCR was performed with a PCR Master Mix (Applied Biosystems, Waltham, MA, USA)
real-time PC detection system (BioRad Laboratories, Hercules, CA, USA). RNU48 and
GUSB were used as normalized controls. The reagents used in the analysis are listed in
Table S6. 3. Discussion Expression of miR-490-3p affected cell viability and resistance to chemotherapy in CRC
cells through regulating TNKS2 [35]. Our data also showed that ectopic expression of
miR-490-3p significantly blocked malignant phenotypes of CRC cells, which is completely
consistent with previous reports. p
p
Next, we aimed to elucidate miR-490-3p-regulated oncogenes and oncogenic pathways
in CRC cells. Our in silico analysis revealed that three genes (IRAK1, FUT1, and GPRIN2)
were associated with poor prognosis. In this study, we focused on IRAK1 (interleukin-1
receptor-related kinase 1) because this kinase family is a target of drug discovery. IRAK1 is one of the serine-threonine kinases that mediates the signaling pathways of
toll-like receptors and the inflammatory mediator interleukin-1 [49,50]. Recent studies have
revealed that IRAK1 is involved not only in inflammatory diseases but also in progression
of several cancers [51–54]. In vivo, inhibition of IRAK1, in mice with colitis-induced tumori-
genesis, reduced the inflammatory response and inhibited the epithelial–mesenchymal
transition [55]. In the current study, we focused on IRAK1 and showed that its aberrant
expression was closely associated with CRC malignant phenotypes. Further functional
analyses of IRAK1 will possibly reveal the biological characteristics of CRC. Starting from
tumor-suppressive miRNA, we identified effective prognostic markers and therapeutic
targets for CRC, indicating that our miRNA-based strategy was feasible. Int. J. Mol. Sci. 2021, 22, 9876 13 of 17 13 of 17 4.8. Candidate Target Genes Controlled by miR-490-3p in CRC Cells To identify genes targeted by miR-490-3p, we obtained microarray data for HCT116
cells transfected with miR-490-3p. By combining these data with the Target Scan Human
7.2 database (http://www.targetscan.org/vert_71 (accessed on 12 May 2021)), we extracted
a total of 249 possible target genes with mi-490-3p binding sites. Next, we analyzed the
gene expression levels of 275 colon cancer tissues and 349 normal colon tissue samples
in the Cancer Genome Atlas (TCGA)-COAD database via the GEPIA2 platform (https:
//cancergenome.nih.gov/ (accessed on 2 May 2021)) and thereby identified 38 oncogenic
genes. Among those 38 genes, the expression of 3 genes showed statistically significant
correlations with the 5-year overall survival rates of patients with CRC obtained from
OncoLnc (http://www.oncolnc.org/ (accessed on 9 May 2021)). Our microarray data were
deposited in the GEO database (GSE129043). 4.10. Western Blotting and Immunohistochemistry The procedures for Western blotting and immunostaining were described
previously [16,21]. Briefly, 21 µg of protein lysates were separated on 4–20% SDS PAGE
Gel and transferred to PVDF membranes (Thermo Fisher Scientific). Membranes were
blocked with skim milk and incubated with the indicated primary antibodies overnight at
4 ◦C. The antibodies used in this study are shown in Table S6. GAPDH was used as the
internal control. We assessed expression of IRAK1 proteins by immunohistochemistry. The
procedure for immunostaining was described previously. 4.9. Dual-Luciferase Reporter Assays The predicted binding site sequence of miR-490-3p in the 3′-UTR of IRAK1 was ex-
tracted from the TargetScanHuman database (https://www.targetscan.org/, release 7.2
(accessed on 12 May 2021)). Based on those data, a PsiCHECK-2 plasmid vector (Promega,
Madison, Wisconsin, USA) containing wild-type was used. To generate the IRAK1 mutant
reporter, the seed region of the IRAK1 3′-UTR was mutated to remove all complementarity
to nucleotides of miR-490-3p. CRC cells were seeded into a 24-well plate. After being
cultured overnight, CRC cells were co-transfected with the indicated vectors and miR
precursor of miR-490-3p or negative control miRNA. Luciferase assays were performed 60 h
after transfection using the Dual Luciferase Reporter Assay System (Promega). Normalized
data were calculated as the Renilla/firefly luciferase activity ratio. 4.6. Cell Proliferation, Migration and Invasion Assays in CRC Cells The methods used for functional assessment of CRC cells (e.g., proliferation, invasion,
and migration) were outlined in previous studies [16,21]. In brief, for proliferation assays,
cells were transferred to 96-well plates. HCT116 or DLD-1 cells were plated at 1.0 × 104 cells Int. J. Mol. Sci. 2021, 22, 9876 14 of 17 14 of 17 per well. After 72 h, cell proliferation was evaluated using XTT assays. For migration and
invasion assays, HCT116 cells or DLD-1 cells at 1.2 × 105 were transfected in 6-well plates. After 72 h, HCT116 cells or DLD-1 cells were added into each chamber at 2.5 × 105 per
well. After 48 h, the cells on the lower surface were counted for analysis. All experiments
were performed in triplicate. per well. After 72 h, cell proliferation was evaluated using XTT assays. For migration and
invasion assays, HCT116 cells or DLD-1 cells at 1.2 × 105 were transfected in 6-well plates. After 72 h, HCT116 cells or DLD-1 cells were added into each chamber at 2.5 × 105 per
well. After 48 h, the cells on the lower surface were counted for analysis. All experiments
were performed in triplicate. 4.7. Assay of miR-490-3p Incorporation into the RNA-Induced Silencing Complex (RISC) Mature miR-490-3p and miR-490-5p were separately transfected into 1.2 × 105 CRC
cells (HCH116 and DLD-1) per ml. After 72 h, miRNA incorporated into the RISC were
isolated using a human AGO2 miRNA isolation kit (Wako Pure Chemical Industries,
Ltd., Osaka, Japan) according to the manufacturer’s protocol. Amount of incorporated
miRNA was evaluated by RT-qPCR as described previously [56]. miR-21 was used as the
internal control. 4.11. Statistical Analyses Differences between 2 groups were evaluated using Mann–Whitney U tests. Cor-
relation coefficients were evaluated using Spearman’s test. All statistical analyses were
performed using JMP Pro 15 (SAS Institute Inc., Cary, NC, USA). p-values < 0.05 were
considered statistically significant, and all data are presented as the mean ± standard
error (SE). Int. J. Mol. Sci. 2021, 22, 9876 15 of 17 15 of 17 Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/ijms22189876/s1. Author Contributions: Conceptualization, Y.H. and N.S.; data curation, Y.H., Y.K. and R.Y.; formal
analysis, Y.H. and T.I.; funding acquisition, N.S., T.T., T.I., M.W., H.T. and T.O.; investigation, Y.H., T.I. and K.T.; methodology, N.S.; project administration, N.S.; resources, T.I., H.K. and T.O.; supervision,
T.O.; validation, S.A. and S.M. (Shogo Moriya); visualization, Y.H. and Y.K.; writing—original draft
preparation, Y.H. and Y.K.; writing—review and editing, S.M. (Shinichiro Mori) and N.S. All authors
have read and agreed to the published version of the manuscript. Funding: The present study was supported by KAKENHI Grant Number JP18K08687, JP18K16322,
JP19K09077, JP19K09049, JP19K09050, JP21K08783, JP21K16426, JP21K15597 and JP21K09577. Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by Ethics Committee of Kagoshima University (approval no. 160038 28-65, date of approval: 19 March 2021). Informed Consent Statement: Written prior informed consent and approval were obtained from all
patients who were diagnosed with CRC and resected at Kagoshima University Hospital. Informed Consent Statement: Written prior informed consent and approval were obtained from all
patients who were diagnosed with CRC and resected at Kagoshima University Hospital. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Conflicts of Interest: The authors declare no conflict of interest. References 1. Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: Globocan estimates of
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Obstacles in the detection and reporting of tuberculosis cases: a discursive analysis
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ABSTRACT Objective: To analyze the discourses of health professionals about the obstacles in the process of detection and reporting
of tuberculosis cases in Mozambique. Method: Qualitative exploratory study with a theoretical-methodological approach of
Discourse Analysis of French matrix. The study was conducted in Mozambique in 2014 at three levels: central, provincial and
district. The study included 15 health professionals, 4 physicians, 6 technicians and 5 nursing professionals, who worked in the
National Tuberculosis Control Program, with more than 1 year of experience. Result: The following discursive blocks emerged:
Detection of tuberculosis cases in laboratories; Underreporting of tuberculosis cases; Obstacles to detect cases of tuberculosis:
long distances and lack of transport; and Reporting of cases for decision making. Final considerations: The discourses analyzed
point to the ideological affi liation that includes the lack of investment policies in the health sector and the political commitment
as basic obstacles in the detection and reporting of tuberculosis cases. p
g
Descriptors: Tuberculosis; Social Policy; Health Policy; Health Service; Nursing in Public Health. THEMATIC ISSUE:
CONTRIBUTIONS AND CHALLENGES OF NURSING
PRACTICES IN COLLECTIVE HEALTH RESEARCH Fernando MitanoI, Amélia Nunes SicsúI, Luciana de Oliveira SousaI, Rarianne Carvalho PeruhypeI,
Jaqueline Garcia de Almeida BallesteroI, Pedro Fredemir PalhaI I Universidade de São Paulo, Nursing School of Ribeirão Preto, Postgraduate Program in Nursing
Ribeirão Preto, São Paulo, Brazil. RESUMO Objetivo: Analisar os discursos dos profi ssionais de saúde acerca das barreiras no processo de detecção e notifi cação dos casos
de tuberculose em Moçambique. Método: Estudo exploratório qualitativo com abordagem teórico-metodológica da Análise de
Discurso de matriz francesa. O estudo foi realizado em Moçambique, no ano de 2014, em três níveis: central, provincial e distrital. Fizeram parte do estudo 15 profi ssionais de saúde, sendo 4 médicos, 6 técnicos e 5 profi ssionais de enfermagem, que atuavam
no Programa Nacional de Controle da Tuberculose, com mais de 1 ano de experiência. Resultado: Emergiram os seguintes blocos
discursivos: Detecção dos casos da tuberculose em laboratórios; Subnotifi cação dos casos da tuberculose; Barreiras para detecção
de casos de tuberculose: longas distâncias e falta de transporte; e Notifi cação dos casos para a tomada de decisão. Considerações
fi nais: Os discursos analisados apontam para a fi liação ideológica que compreende a falta de políticas de investimento no setor da
saúde e do comprometimento político como barreiras basilares na detecção e notifi cação dos casos da tuberculose. Descritores: Tuberculose; Política Social; Política de Saúde; Serviço de Saúde; Enfermagem em Saúde Pública. ose; Política Social; Política de Saúde; Serviço de Saúde; Enfermagem em Saúde Pública. How to cite this article: Mitano F, Sicsú AN, Sousa LO, Peruhype RC, Ballestero JGA, Palha PF. Obstacles in the detection and reporting
of tuberculosis cases: a discursive analysis. Rev Bras Enferm [Internet]. 2018;71(Suppl 1):523-30. [Thematic Issue:
Contributions and challenges of nursing practices in collective health] DOI: http://dx.doi.org/10.1590/0034-7167-2016-0673 Barreiras na detecção e notifi cação dos casos da tuberculose: uma análise discursiva
Barreras en la detección y notifi cación de los casos de tuberculosis: un análisis discursivo Fernando MitanoI, Amélia Nunes SicsúI, Luciana de Oliveira SousaI, Rarianne Carvalho PeruhypeI,
Jaqueline Garcia de Almeida BallesteroI, Pedro Fredemir PalhaI Obstacles in the detection and reporting of tuberculosis cases:
a discursive analysis Barreiras na detecção e notifi cação dos casos da tuberculose: uma análise discursiva
Barreras en la detección y notifi cación de los casos de tuberculosis: un análisis discursivo RESUMEN Objetivo: Analizar los discursos de los profesionales de salud acerca de las barreras en el proceso de detección y notifi cación
de los casos de tuberculosis en Mozambique. Método: Estudio exploratorio cualitativo con abordaje teórico-metodológico del
Análisis de Discurso de matriz francesa. El estudio fue realizado en Mozambique, en el año 2014, en tres niveles: central,
provincial y distrital. Quince profesionales de la salud participaron en el estudio, siendo 4 médicos, 6 técnicos y 5 profesionales
de enfermería, que actuaban en el Programa Nacional de Control de la Tuberculosis, con más de 1 año de experiencia. Resultado:
Los siguientes bloques discursivos emergieron: Detección de los casos de la tuberculosis en laboratorios; Subnotifi cación de
los casos de la tuberculosis; Barreras para la detección de los casos de la tuberculosis: largas distancias y falta de transporte; http://dx.doi.org/10.1590/0034-7167-2016-0673 http://dx.doi.org/10.1590/0034-7167-2016-0673 Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. 523 Mitano F, Sicsú AN, Sousa LO, Peruhype RC, Ballestero JGA, Palha PF. Obstacles in the detection and reporting of tuberculosis cases: a discursive analysis Mitano F, Sicsú AN, Sousa LO, Peruhype RC, Ballestero JGA, Palha PF. Y notificación de los casos para la toma de decisión. Consideraciones finales: Los discursos analizados apuntan a la filiación
ideológica que comprende la falta de políticas de inversión en el sector de la salud y del compromiso político como barreras
básicas en la detección y notificación de los casos de la tuberculosis. i
Descriptores: Tuberculosis; Política Social; Política de la Salud; Servicio de Salud; Enfermería en Salud Pública. Fernando Mitano E-mail: piqinamita@gmail.com
CORRESPONDING AUTHOR Ethical aspects The survey was approved by the National Ethics Commit-
tee of Mozambique in April 2014. Also, the same survey was
approved and authorized by the Minister of Health of Mo-
zambique. Participants were informed about the research ob-
jectives and about confidentiality/anonymity, confirming their
participation through the signing of the Free and Clarified
Consent Term. Prior to the start of the survey, all participants
were informed of the right to withdraw from the interview at
any time if they so wished. In order to preserve the anonymity
of the persons who participated in the study, the letter “S” =
subject was used in the presentation of the results, followed by
the initial letter referring to the professional category to which
the interviewee corresponds (M - Manager, P - Physician, NP
- Nursing Professional; MT - Medicine Technician) and an Ara-
bic numeral indicating the sequence of the interviews. The country is one of the 30 countries that contribute the larg-
est number of TB cases in the world and is included in the se-
lect group of countries with incidence greater than 500/100,000
inhabitants(1). The country reported 58,270 new TB cases, cor-
responding to a reporting rate of 233/100,000 inhabitants in an
estimated population of 27,216,000 in 2015(5). However, the
number of cases estimated per year by the WHO is 160,000(3),
a situation that shows the country’s failure to comply with WHO
recommendations, which set the case detection rate at 70% and
cure at 85(1) if directly observed treatment of short duration was
applied, a strategy that Mozambique began to implement before
being recommended by WHO in 1993. Despite using this strat-
egy and demonstrating efforts by the health services in an attempt
to control the disease, the country is still unable to detect and
report the majority of existing cases, and the 10 countries account
for 77% of the difference (4.3 million) between new case reports
and the estimated incidence(1). Type of study This is a qualitative exploratory study that uses the theoret-
ical-methodological reference of Discourse Analysis (DA) of
French matrix, which epistemologically is based on linguis-
tics, psychoanalysis and historical materialism(11). In a bibliographic survey in the Latin American and Carib-
bean Health Sciences (LILACS), Scientific Electronic Library
Online, (Scielo), PubMed, Scopus and Africa Index Medicus
databases, there was a shortage of studies aimed at detection
and reporting of TB cases both in the country and internation-
ally, reinforcing the importance of this study, given that it is a
topic of great relevance. INTRODUCTION In this context, the understanding of the circulating senses
produced among healthcare professionals with experience in
the treatment of TB can provide relevant information on the
obstacles to the detection and reporting of cases in a high-
burden country. Thus, the study can contribute to the formu-
lation of policies that improve the detection and reporting of
cases. In view of this, the study aims to analyze the discourses
of health professionals about the obstacles to the process of
detection and reporting of tuberculosis cases in Mozambique. Tuberculosis (TB) is one of the major causes of morbidity and
mortality in developing countries such as Mozambique and, for
that country, is one of the diseases that kill the most(1). Diagnosis
consists of one of the priority actions for TB control, since undiag-
nosed patients, thus not treated in a timely manner, are the main
source of TB transmission(2). Case detection is part of the diagnos-
tic process from the identification of the respiratory symptom to
laboratory confirmation. The reporting is an action subsequent
to the diagnostic confirmation that allows an accompaniment of
the disease, aiming at the dissemination of the information to the
management organs in order to assist in the decision making for
the control of the disease(1,3). Although several researchers empha-
size the importance of detecting and notifying TB cases for dis-
ease control(1,4-6), in Mozambique these actions fall far short of the
numbers estimated by the World Health Organization (WHO)(4). Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. RESULTS Four discursive blocks emerged: Detection of TB cases in
laboratories; Underreporting of TB cases; Obstacles for detect-
ing TB cases: long distances and lack of transportation; and
Reporting of cases for decision-making. Collection and data presentation The adversative conjunction “but” present in the discourse
(good and not that good) in the discursive sequence “but it looks
good, but it is not that good” leaves the track of double meaning
circulating, one that focuses on the positive aspect of the exis-
tence of a laboratory in each district, another that emphasizes
the need for more laboratories within the districts because the
existing ones (one for each district) are not enough. This situa-
tion leads to the need to increase laboratories at the district level. From the discursive sequence “Come on, there’s 100,000
km2 of area with a laboratory, it’s not possible!”, one can iden-
tify a discourse of physical geography, indicating the inability of
a laboratory to cover a large area occupying about 100 thousand
km2 It is emphasized that, although this number (100,000) was
randomly stated at the time of the discourse, it originated from
the representation of the unconscious, but suggests the greatness
of the territory occupied by the districts of Mozambique. The adversative conjunction “but” present in the discourse
(good and not that good) in the discursive sequence “but it looks
good, but it is not that good” leaves the track of double meaning
circulating, one that focuses on the positive aspect of the exis-
tence of a laboratory in each district, another that emphasizes
the need for more laboratories within the districts because the
existing ones (one for each district) are not enough. This situa-
tion leads to the need to increase laboratories at the district level. For data collection, an interview script was used with the
following questions guiding the interview: What are the dif-
ficulties faced in the process of detecting and reporting cases? How is the reporting of cases made? How is the flow of infor-
mation? The questions were previously tested, and the inter-
views were conducted at locations defined by participants. They were audio-recorded by means of a voice recorder and
had an average duration of 30 minutes. Data source Fifteen health professional subjects were intentionally in-
terviewed by the main researcher, 4 physicians, 6 technicians
and 5 nursing professionals, ranging in age from 25 to 55
years, who occupied the positions of managers at different
levels (central, provincial and district) . Inclusion criteria were:
to be working for more than a year in its role in the National
Tuberculosis Control Program (NTCP); and be in the exercise
of their activities at the time of data collection. It is observed that the health professional, taking the posi-
tion of manager when enunciating the signifiers “at least we
have a laboratory”, produces senses that impel the cogitation
that a laboratory, although insufficient to cover an extensive
population area, does not stop being important for case de-
tection and reporting. However, the same signifiers indirectly
leave traces of several cases that are not being detected and
reported to the national health system in Mozambique. Methodological procedures The existence of at least one laboratory in each district of
the province is indicated, on the one hand, as important in the
TB detection process, but on the other, as insufficient to cover
the population demand as it is stated by the subject in the fol-
lowing discursive sequence: Collection and data presentation From the discursive sequence “Come on, there’s 100,000
km2 of area with a laboratory, it’s not possible!”, one can iden-
tify a discourse of physical geography, indicating the inability of
a laboratory to cover a large area occupying about 100 thousand
km2 It is emphasized that, although this number (100,000) was
randomly stated at the time of the discourse, it originated from
the representation of the unconscious, but suggests the greatness
of the territory occupied by the districts of Mozambique. Theoretical and methodological reference This theoretical reference, whose pioneer is Michel
Pêcheux, appeared in the 1960s in France as a philosophical
current unsatisfied with the positivism installed at the time,
which denied the inclusion of conditions of production in the
analysis or interpretation of the phenomena under study. The
conditions of production can be understood as the histori-
cal, social and ideological contexts that permeate a discourse
when it is produced(11-12). In this sense, the DA “is intended to
highlight the senses of discourse in view of its socio-historical
and ideological conditions of production”(12). In Mozambique, a study on detection emphasized the in-
troduction of the rapid TB test to detect cases mainly in pa-
tients affected by HIV/AIDS(7). Studies in several countries,
such as Nigeria, Brazil, Afghanistan and Kenya, emphasize
case detection and reporting in specific groups such as chil-
dren, prisoners and people living in rural areas(6,8-10). But none
explores the discourses of health professionals regarding the
detection and reporting of TB cases. Anchored to the aforementioned statement, it can be deduced
that the discursive studies aim to think the sense dimensioned
in time and in the space of man’s practices, decentralizing the Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. 524 Obstacles in the detection and reporting of tuberculosis cases: a discursive analysis Mitano F, Sicsú AN, Sousa LO, Peruhype RC, Ballestero JGA, Palha PF. different discursive formations. In the third step, the discursive
process itself (ideological formation) returned to the discourse
sequences highlighted for analysis, framing itself in a theo-
retical framework on the subject under study, in a continuous
process of discretion and interpretation. notion of subject and relativizing the autonomy of the object of
linguistics(11). In DA, we understand as subject the positions oc-
cupied discursively by the individual enunciator of discourse(11). It is important to point out that individuals when they speak are
affiliated to a certain discursive formation, understood as that
which in certain ideological conditions is said in one way and
not in another. Ideology, on the other hand, is an effect of the
necessary relationship of the subject with language and history
in the production of meanings(11). In this context, meaning is
understood as a determinate relation of the subject affected by
language with history(11). Thus, with this approach, one can un-
derstand how symbolic objects (signifiers, discursive sequences,
cutouts) produce meanings and intervene in the real of meaning. Study scenario
h
d The study was conducted in Mozambique, from May to Au-
gust 2014, at three levels: central, at the Ministry of Health; pro-
vincial, in the province of Nampula; district, in eight districts
of the province of Nampula (Mecubúri, Murrupula, Muecate,
Ribaué, Nampula-Rapale, Meconta, Monapo and Mogovolas). In all districts at least we have a lab, but it looks good, but
it’s not that good. Let’s find a district that says, [...], come
on, it has 100,000 square kilometers of area with a labora-
tory, it’s not possible! (MS1) In all districts at least we have a lab, but it looks good, but
it’s not that good. Let’s find a district that says, [...], come
on, it has 100,000 square kilometers of area with a labora-
tory, it’s not possible! (MS1) Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. Data analysis The interviews were transcribed and the step of returning
the transcripts to the participants was waived. For the analysis
of the data, the steps proposed by Orlandi(11) were followed:
the first one consisted in the passage from the linguistic sur-
face to the discursive object, in which the transcription of the
interviews and successive readings were made to identify
the ideas of references that belonged to the identified discur-
sive blocks. The second step was to move from the object of
discourse to the discursive process, in which the discursive,
significant or enunciated sequences that played an important
role in the creation of meanings were discriminated against,
taking into account the signification process. It is signaled
that, at this stage, the discursive sequences were related to the On the laboratories, a speech emerged that focused on the
difficulty of acquiring a microscope, an indispensable instru-
ment for the detection of TB in patients: The main difficulty [...] of these laboratories for TB is related
to the acquisition of the instrument that is the microscope,
in this case let’s talk about the microscopy network that is
less available, not to mention the new technologies that are Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. 525 Obstacles in the detection and reporting of tuberculosis cases: a discursive analysis Mitano F, Sicsú AN, Sousa LO, Peruhype RC, Ballestero JGA, Palha PF. even more incipient. [...] the difficulty is to maintain these
instruments. The maintenance is generally not local, when
failure is necessary to take the microscope to the province
and if necessary to request another one [...]. (MS3) formations that focus on underreporting of cases as a barrier
to both the reporting system and the treatment. The difficulty is research, first diagnosis, second follow-up,
especially in the south zone of the district; we have three
sanitary units with laboratory nearby. The patients we treat
there even come from other districts. (MS11) From the discursive sequence “the main difficulty [...] of
these laboratories [...] is related to the acquisition of the instru-
ment that is the microscope”, the subject-position is observed
inscribing itself in a lamenting discursive formation that fo-
cuses denouncing the difficulty of the laboratories to func-
tion normally. Equally, the same words instigate to think that,
although there are buildings established as laboratories, they
work with difficulties, because the acquisition of the “micro-
scope” has been a problem. Data analysis Another saying that indicates the
lack of laboratory coverage and the microscope is present in
this sequence: “microscopy network that is less available, not
to mention new technologies that are even more incipient.”
This discursive sequence prompts us to consider that the in-
struments for diagnostic evaluation used are scarce. The signi-
fiers “the difficulty is to maintain these instruments,” suggests
the lack of trained professionals to maintain the microscope
at the district level, which obliges those responsible to take to
the provincial level: “it is necessary to take the microscope to
the province.” The lack of inputs has also contributed to low screening be-
cause it is not possible to screen for TB without these issues
[...] we also feel it is still low tracking in most districts, low
BK + case tracking and I do not know [...] would under-
report pediatric TB, are still having problems in many dis-
tricts, low TB reporting in children, we know that diagnosis
in children is difficult. (PS2) The signifiers “the difficulty is research”, taking into ac-
count the conditions of production, produce various effects
of meanings that work and mean. Among them, the lack of
adequate professionals and means for the detection of TB
and the lack of evidence-based studies recommending health
practices that compete for the control of TB. The complaint made by the professional health subject-po-
sition is notorious: “the lack of inputs has also contributed to
the low level of attention.” This discursive sequence suggests
the existence of diagnostic difficulties; of TB control; the lack
of implementation of a policy capable of controlling TB, and
a system of detection and reporting that mirrors the reality
of existing cases. Under these production conditions, the se-
quence suggests that these difficulties have become cyclical,
interfering negatively in TB control in the country. Also in the list of difficulties, subjects state/announce the
laboratories as not being suitable for the detection of TB: [...] infection control because in many laboratories a poorly
ventilated small compartment works, it is not possible to pro-
cess the TB samples while doing another type of activity: sam-
pling other patients while the sample is being processed TB,
it is not possible. So we also have problem with the physical
location itself, where it works the laboratory does not allow
sample processing in some places all day long. Data analysis (PS4) It is noteworthy that the words “low TB reporting in chil-
dren; we know that diagnosis in children is difficult “and”
children’s TB means the investigation is not enough “leave
within their lines the possibility of considering that subjects
state/announce not only the low detection of cases in children
but also in adults. From the discursive sequence “[...] in many laboratories
small compartments, small windows and few aired windows”,
it is noticed that the subject-position is inscribed in discursive
formations which point the question of the infrastructure as an
obstacle to the reporting and treatment of TB. In this context, it
is indicated, in these words, the denunciation of infrastructures
unfit for the detection and processing of biological material for
TB analysis. The same words state/announce the inefficiency
of case detection and biosafety, exposing health professionals
and users of the health service to an environment conducive
to contagion. The sequence also produces meanings that im-
ply that the government is finding it difficult to comply with
the guidelines of the NTCP and WHO recommendations on
the sites for the collection of the sputum sample, which should
be performed in an airy outdoors, in addition to violating Bio-
safety standards for the processing of biological samples. These
denunciations are characterized by the adjectives “small”, “re-
duced” and adverb of quantity “few”. Obstacles to tuberculosis cases: long distances and lack
of transport Health professionals state/announce the lack of transporta-
tion and long distances traveled by patients seeking health
care as one of the situations that negatively influences the de-
tection and reporting of TB cases as can be deduced from the
following discursive cutbacks: The major difficulties are many [...] mainly of transport. (MS13) The main problem is transport as we get to the units when
our access roads are damaged? (NPS5) We know that in the Mozambican reality most of the inhab-
itants live, we speak of approximately 40 to 45% of inhabit-
ants who live more than 8 km from the nearest health unit. (NPS6) Underreporting tuberculosis cases From the discursive sequence below, it is observed that the
reporting system of case reports seems to have the purpose of
controlling the performance of health professionals and not to
give the real situation of TB in the districts and in the province
as can be deduced: But quarterly the peripheral health unit is [...], compiles the
data [...], sends it to the district level and sends it to the
province. This receives analyzes and gives feedback from
what has to be agreed and then sends it to the central level
in DOTS and other TB areas. After that, within 45 days the
central level has to tell the provinces what the feedback is
in terms of performance, what has happened well or not,
and how it should improve. (NPS14) The “many” signifier, which is inserted in the semantic
field of “quantity”, produces a silencing sense of the quantity
and depth of difficulties faced by professional health subjects. The discursive sequence “how do we get to the units when
our access roads are spoiled?” Calls for both individual and
collective introspection on the transport phenomenon and
leaves evidence that its problem is not only related to its lack,
but also to the poor conditions of roads, which aggravate the
difficulty of accessibility to health units. From the discursive sequence “but quarterly the peripheral
health unit is ... compiles the data [...], sends it to the district lev-
el and [...] sends it to the province”, it can be deduced that the
subject-position, summoned by ideology and interdiscourse, as
the already-mentioned, is inscribed in a discursive formation
that focuses on the upward and downward communication as
aspects inscribed institutionally in the process of reporting of TB
cases. The action of sending information to the district and to
the province produces meanings that mean upward communi-
cation, which starts from the basis for those responsible. The discursive sequence highlighted “and some patients
live very far away” produces indirect senses of the difficulty
of the subjects affected by TB in accessing the health units; of
late diagnosis; and the difficulty of TB control. The denunciation of the geodemographic reality is repre-
sented in the following discursive sequence: “we know that in
the Mozambican reality”. The signifier “we” makes the sense
that all Mozambicans know that a good part of them live far
from the health unit. Underreporting tuberculosis cases The low detection and reporting of TB cases, especially
in children, emerge as the main guideline in the following
discursive cuts, where the subjects are enrolled in discursive [...] one of them is the distances, and some patients live very
far away. (MTS15) 526 Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. Mitano F, Sicsú AN, Sousa LO, Peruhype RC, Ballestero JGA, Palha PF. Obstacles in the detection and reporting of tuberculosis cases: a discursive analysis data in a summary form, both in manual and electronic
compilation and we take the province for planning. (PS10) Added to the problem of distances is the lack of laborato-
ries, which requires the patient to look for the health unit with
conditions to perform the TB test: It is pointed out in the above clipping that the actions of
compiling the information in a manual or electronic file sug-
gest both the double registration that can guarantee the data
conservation and the incipient process of the use of electronic
means that are being introduced in those districts belonging
to the rural area of Mozambique. Significant “we take the
province for planning” lead to understand that the information
on the reporting of the cases assists in the decision making for
the planning of activities on TB reporting. People go to the hospital, they do not have a laboratory,
they are indicated to go to other health units, but the pa-
tients do not have money to transport them. (MTS8) It is observed that the professional health subjects are in-
scribed in discursive formations of the lamenting speech that
focuses the lack of transportation as one of the obstacles in the
control of TB and the detection of the cases. It is also noted
that this lack of transportation is associated with other faults:
“the major difficulties are many”. This discursive sequence,
considering the socio-historical, economic and ideological
conditions of Mozambique, instigates to think that the lack
of means of transport is not simply a matter of cars, buses,
but other means known to the population as are the cases of
bicycles and motorcycles, which are the main means of loco-
motion of the population. Underreporting tuberculosis cases In the linguistic marks “they have lived
more than 8 km”, observes the subjected subject by its ideol-
ogy and that went through a socio-historical experience. In
this case, the sequence produces the sense that people living
for more than 8 km present difficulties from the identification
of the symptoms to the treatment of the disease. This requires
a policy design that can help reduce the distances that prevent
the detection and consequent reporting of cases. On the other hand, it is observed a downward communica-
tion symbolizing norms, orders and at the same time produc-
ing an evaluative and evaluative sense, that is, the information
that constitutes the feedback at the same time serve to evalu-
ate, qualify and value the performance of the employees: “The
central level has to tells the provinces what the feedback is
in terms of performance, what has happened well or not and
how it should improve.” The discursive sequence “gives feed-
back on what has to be right” states/announces the controlling
discourse invested with a domineering, evaluative paradigm. The “voice” of “long distances” reappears in the discursive
sequence “it has distant health units, without laboratories”. The “far-off” laboratory discourse labels leave evidence and a
sense production of the lack of detection and care for the sick
and suspected of TB. It can be understood from the following statement that the flow
of information regarding reporting has had some difficulties. However, this flow has had irregularities, unfortunately is
not processed within these periods, because there is a high
turnover of human resources in TB. It is a difficult program
to manage and often people who are placed as district man-
agers in less than 2 years have to change. So there has to be
new HR training and adaptation. So the time the informa-
tion takes from the district to the province and the province
to rectify, above all there are new district managers, and to
get here takes time. And arriving here, at the central level, Reporting of cases for decision-making From the following cuts, we observe the denunciation of
how the information is compiled. There are record books in the sanitary units and then each
health unit compiles the information according to the mod-
el of the Ministry, so we have the meeting and compile the Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. 527 Obstacles in the detection and reporting of tuberculosis cases: a discursive analysis Mitano F, Sicsú AN, Sousa LO, Peruhype RC, Ballestero JGA, Palha PF. also has its mistakes. We have to do the feedback to ask you
to look at certain data from districts A or Z are not correct
and this ends up taking more than 45 days. (MS12) qualification of the technicians, aiming in this way to increase
the reporting of the cases. The literature emphasizes that man-
agers need to make efforts to capture more resources for ac-
tion and to obtain new knowledge(1), which have been of great
importance in the detection and reporting of cases(13). A study
carried out in Mozambique revealed that health professionals
still have difficulties in knowing about patient characteristics,
diagnosis and treatment of TB(14), a situation that suggests the
need for constant training of professionals in this area. The discursive sequences “this flow has had irregularities;
is not processed within these periods “, under these condi-
tions of production, denounces the failure to send information
to higher levels. These same statements bring an indicative
paradigm that suggests the lack of use of modern technology
for the fluid transmission of information related to the report-
ing, indicating that the information only reaches the province
with the displacement of the heads of the health units. The
discursive sequence “there is a high HR turnover in TB” indi-
cates a lagged coordination between the NTCP managers in
the province and the HR managers responsible for the move-
ment of health professionals, as evidenced in the following
words: “it is a difficult program to manage and often people
who are placed as district managers in less than 2 years have
to change.” In this sense, these statements make it neces-
sary to train professionals to act in TB control, but who can
act specifically in this area and for long periods and, if they
are transferred, they are given the possibility of continuing
to work in the area and contributing to the control of TB in
other places. DISCUSSION In this study, a discursive analysis was carried out on the
obstacles in the process of detection and reporting of tubercu-
losis cases in Mozambique. In this context, from the fragments
presented, they are findings of the study: the low number of
cases of TB is related to lack of material, human and transpor-
tation resources; with inadequate reporting and information
system; and the lack of policies that focus on resolving the TB
problem and adequate infrastructures. From the speech of the MS13, it is noted that the subject
could not speak of all the difficulties faced and, “taking advan-
tage” of the bore of language, enunciated the adverb (many)
that, besides silencing, can also produce the sense of repres-
sion. It is worth mobilizing Foucault’s argument: “It is well
known that one does not have the right to say everything, that
one cannot speak at all in any circumstance, which anyone,
in short, cannot speak of anything”(17). In these conditions of
production, difficulties “passed” into the ambit of habits and
customs, which, because they had never been solved, ceased
to constitute novelty, so they were not part of worries or emer-
gencies much less urgent to the point that the subject did not
need to mention. In this context, the difficulties of access to
health services contribute to the late diagnosis and treatment
of the disease and delay the reporting of cases(18). In the first discursive block, it is observed that the health
professional (MS1), taking the position of manager, is ques-
tioned by the circulating senses anchored in the speech that
Mozambique is one of the countries with critical problems of
laboratories, infrastructures and modern technologies(1,13). The
lack of sufficient laboratories in Sub-Saharan Africa, of which
Mozambique is a part, seriously compromises the detection
and reporting of TB cases(1,8,13). It should be noted that this
reality is also present in other countries with high TB burden,
such as Nigeria, South Africa and Zimbabwe(1). The reporting rate in Mozambique remains only 34%, mak-
ing it worrying since it makes it impossible to know the true
magnitude of the disease(13). The worldwide TB case detection
rate has stagnated by about 60%, failing to reach the target of
70% originally planned for 2000(1). Reporting of cases for decision-making Also, the “difficult to manage” signifiers suggest
a health system with difficulties in articulating the various ex-
isting programs. Infrastructure is another important factor in detecting and re-
porting cases. In places where it is deficient and scarce, the de-
tection and reporting of cases have also proved to be inefficient,
which requires the design of policies that focus on the construc-
tion of adequate infrastructures for the treatment of TB(1,7-8,10). The inadequate biosafety conditions stated/announced by
subject PS4 were also reported in another study in Mozam-
bique, which revealed that guidelines for diagnosis and treat-
ment of TB were not present in all facilities, and few health
facilities had ventilation(15). Still in the list of difficulties in detecting and reporting cas-
es, MS11’s speech signals the need for operational research,
as it will provide better estimates of disease incidence and
identify program deficiencies and priority areas for action(16). The low reporting in children signaled in the speech of the
subject PS2 reflects the insufficiency of means not only for the
control of TB, but also of other diseases, which has been recur-
rent in Mozambique(1). In the ideological production conditions
that permeate TB, the reporting of children’s cases is related to
the fact that the natural difficulties inherent to the diagnosis in
children require even more attention from the professionals(1). Furthermore, TB in childhood continues to be neglected, and
the lack of estimates for childhood perpetuates and amplifies the
problem(13). These diagnostic difficulties in children also relate to
the increased prevalence of HIV in children(16). Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. DISCUSSION The words of MS13, MTS15 and MTS8 leave traces that
the lack of transport and the long distances associated with
the absence of laboratories lead to an increase in the spread
of the disease and higher costs for patients undergoing treat-
ment(19-20). Likewise, the lack of transportation is pointed out
by some authors as an obstacle in adherence to treatment(21);
and in a context such as Mozambique, where transport is a se-
rious problem and where reported cases annually are less than The senses produced by MS3 subject discourse concern-
ing the difficulty of functioning and coverage of the labora-
tories and the shortage of evaluation instrument and trained
professionals point to the need to elaborate policies that focus
the sufficient financing for this activity and the training and Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. 528 Obstacles in the detection and reporting of tuberculosis cases: a discursive analysis Mitano F, Sicsú AN, Sousa LO, Peruhype RC, Ballestero JGA, Palha PF. Contributions to the area of nursing, health or public
policy estimated(14), the mobilization of various sectors such as edu-
cation, health and transport to control TB can be important in
increasing detection and reporting of cases(21). A review of the
detection and reporting system should be done by focusing
on the increase of laboratories and qualified professionals as
well as a double registration of cases that guarantees the valid-
ity of information beyond the modernization of the means of
records. This will allow the provincial level to have immediate
access to information, which requires the use of internet and
other modern technological means in all health units(22). From the words of the subjects involved in the study it can
be concluded that the research brings important discussions: dif-
ficulties of case detection, lack of transportation, long distances,
underreporting - situations that can be verified not only in Mo-
zambique but also in other countries with high TB load. It is im-
portant to note that the words, which constitute findings, have a
great contribution both to the nursing area and to public health
in general, because, besides adding knowledge, they contribute
to the elaboration of policies that can focus on the detection of
cases, treatment and improvement of information systems, which
are elements for decision-making for TB control. FINAL CONSIDERATIONS From the symbolic material analyzed, it is pointed out that
the discourses point to the ideological affiliation that includes
the lack of investment policies in the health sector and the
political commitment, taking these absences as basic obsta-
cles in the detection and reporting of cases of tuberculosis,
which is represented by insufficient microscopes and human
resources, long distances, communication difficulties, among
other factors. This requires policy design and implementation
in order to reverse the country’s current TB situation. FUNDING This research was funded by the Coordination for the Im-
provement of Higher Education Personnel (CAPES). DISCUSSION From the speeches of MS12 and NPS14 on the way informa-
tion circulates, it signals a controlling discourse invested with
a dominant and evaluative paradigm, thus suggesting that the
information provided is linked to the obligation and not neces-
sarily to compliance with the norms(17). This situation requires
a new strategy of information dissemination and the discussion
of information for common use; standardized data collection,
compilation and analysis can be used in health facilities to moni-
tor treatment outcomes at the district level, with a view to iden-
tifying local problems that may arise at the provincial or national
level and order to consistently ensure the control of TB(1,13). Study limitations The study with only a few health professionals (managers,
physicians, nursing professionals and medical technicians),
leaving aside the laboratory workers, Community Health
Agents, patients and the community, can be pointed out as
possible limitation of the study, because it brings only the ex-
periences of some and not everyone involved in the TB case
detection and reporting process. REFERENCES Campinas: Pontes Editores; 2013. 12. Fernandes CA. Análise do Discurso: Reflexões introdutórias. 2ª ed. São Carlos: Claraluz, 2008 13. Ibanês AS, Carneiro Jr N. Panorama internacional da estratégia do tratamento diretamente supervisionado (DOTS) nas políticas
de controle de TB. ABCS Health Sci [Internet]. 2013[cited 2016 Dec 10];38(1):25-32. Available from: http://files.bvs.br/
upload/S/1983-2451/2013/v38n1/a3661.pdf 14. Noé A, Ribeiro RM, Anselmo R, Maixenchs M, Sitole L, Munguambe K, et al. Knowledge, attitudes and practices regarding
tuberculosis care among health workers in Southern Mozambique. BMC Pulmonary Med[Internet]. 2017 [cited 2017 Jun 23];17:2. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217625/pdf/12890_2016_Article_344.p 15. Brouwer M, Coelho E, Mosse CD, Brondi L, Winterton L. Leth FV. Health Care Workers Challenges in the implementation
prevention and control Measures in Mozambique. Plos One [Internet] 2014[cited 2016 Dec 3];9(12):e114364. Available from:
http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0114364&type=printable 16. García-Basteiro AL, Lópes-Varela E, Manhiça I, Macete E, Alonso PL. Mozambique faces challenges in the fight against tuberculosis. Lancet[Internet]. 2014 [cited 2016 Oct 30];383:214-6. Available from: http://www.thelancet.com/pdfs/journals/lancet/PIIS0140-
6736(14)60069-6.pdf 17. Foucault M. Vigiar e punir: nascimento da prisão. 42ª ed. Petropolis, Rio de Janeiro: Vozes, 2014. 18. Trébucq A, Schwoebel V. Numbers of tuberculosis cases dreams and reality. Int J Tuberc Lung Dis [Internet]. 2016[cited 2016 Oct
30];20(10):1288-92. Available from: http://www.ingentaconnect.com/content/iuatld/ijtld/2016/00000020/00000010/art00007 19. Hiatt T, Nishikiori N. Epidemiology and control of tuberculosis in the Western Pacific Region: update with 2013 case notification
rate. WPSR [Internet]. 2016[cited 2016 Oct 27];7(2):41–50. Available from: http://ojs.wpro.who.int/ojs/index.php/wpsar/article/
view/416/668 20. Boss JC, Smalbraak L, Macome AC, Gomes E, Leth FV, Prins JM. TB diagnostic process management of patients in a referral
hospital in Mozambique in comparison with the 2007 WHO recommendations for the diagnosis os smear-negative pulmonary
TB and extrapulmonary TB. Int Health[Internet]. 2013[cited 2016 Oct 11];5(4):302-8. Available from: https://www.ncbi.nlm.nih. gov/pubmed/24130098 21. Cardoso GCP, Cruz MM, Abreu DMF, Decotelli PV, Chrispim PPM, Borenstein JS, et al. A conformidade das ações do tratamento
diretamente observado para tuberculose na perspectiva dos profissionais de duas unidades de saúde da cidade do Rio de Janeiro. Cad. Saúde Colet[Internet]. 2012 [cited 2017 Jun 20];20(2):203-10. Available from: http://www.cadernos.iesc.ufrj.br/cadernos/
images/csc/2012_2/artigos/csc_v20n2_203-210.pdf 22. Laokris S, Drabo MK, Weil O, Kafando B, Dembélé SM, Dujardin B. Patients are paying too much for tuberculosis: a direct cost
burden evaluation in Burkina Faso. Plos One [Internet]. 2013[cited 2016 Nov 10];8(2):e56752. Available from: https://www.ncbi. nlm.nih.gov/pmc/articles/PMC3581516/pdf/pone.0056752.pdf 22. Laokris S, Drabo MK, Weil O, Kafando B, Dembélé SM, Dujardin B. Patients are paying too much for tuberculosis: a direct cost
burden evaluation in Burkina Faso. REFERENCES 1. Word Health Organization. WHO. Global Tuberculosis control: 2016[Internet]. Geneva: World Health Organization; 2016[cited
2016 Dec 13]. Available from: http://apps.who.int/iris/bitstream/10665/250441/1/9789241565394-eng.pdf?ua=1 2. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Programa Nacional de Controle da Tuberculose. Manual de
recomendações para o controle da tuberculose no Brasil. Brasília: Ministério da Saúde; 2011 3. Fátima R, Harris RJ, Enarson DA, Hinderaker SG, Qadeer E, Ali K, et al. Estimating tuberculosis burden and case detection
in Pakistan. Int J Tuberc Lung Dis [Internet]. 2014[cited 2016 Sep 15];18(1):55–60. Available from: http://ntp.gov.pk/uploads/
TB_prevalence_Pakistan_IJTLD-2014.pdf 4. Oshi DC, Chukwu JN, Nwafor CC, Meka AO, Madichie ON, Ogbudebe CL, et al. Does intensified case finding increase
tuberculosis case notification among children in resource-poor settings? a report from Nigeria. Int J Mycobacteriol [Internet]. 2016[cited 2016 Oct 10];5(1):44-50. Available from: http://www.sciencedirect.com/science/article/pii/S2212553115001612 5. Rocha MS, Aguiar FD, Oliveira GP, Saraceni V, Coeli CM, Pinheiro RS. Confiabilidade do desfecho do tratamento, usando linkage
de bases de dados para a tuberculose. Cad Saúde Colet [Internet]. 2015[cited 2016 Sep 1];23(2):150-6. Available from: http://
www.scielo.br/pdf/cadsc/v23n2/1414-462X-cadsc-23-2-150.pdf 6. Avilov KK, Romanyukha AA, Borisov SE, Belilovsky E M, Nechaeva O, Karkach AS. An approach to estimating tuberculosis
incidence and case detection rate from routine notification data. Int J Tuberc Lung Dis [Internet]. 2015 [cited 2016 Out
17];19(3):288–94. Available from: https://www.ncbi.nlm.nih.gov/pubmed/25686136 7. Cown J, Michel C, Manhiça I, Monivo C, Saize D, Creswell J, et al. Implementing rapid testing for tuberculosis in Mozambique. Bull Word Health Organ [Internet]. 2015[cited 2016 Sep 25];93(2):125-30. Available from: http://www.who.int/bulletin/
volumes/93/2/14-138560.pdf 8. Hoog AHV, Laserson KF, Githui WA, Meme HK, Agaya JA, Odeny LO, et al. High prevalence of pulmonary tuberculosis and
inadequate case finding in Rural Western Kenya. Am J Resp Crit Care Med[Internet]. 2011[cited 2016 Nov 18];181(9):45-1253. Available from: http://www.atsjournals.org/doi/pdf/10.1164/rccm.201008-1269OC 529 Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. Mitano F, Sicsú AN, Sousa LO, Peruhype RC, Ballestero JGA, Palha PF. Obstacles in the detection and reporting of tuberculosis cases: a discursive analysis 9. Valencia MS, Cezar-Vaz MR, Bruns CB, Silva PEA. O processo de detecção e tratamento de casos detberculose em um presidio. Cienc Saúde Colet[Internet]. 2016[cited 2016 Sep 28];21(7):2111-22. Available from: http://www.scielo.br/pdf/csc/v21n7/1413-
8123-csc-21-07-2111.pdf 10. Sanale A, Mergentheler C, Nasrat A, Seddlq MK, Mohmodl SD, Stevens RH. An evaluation of passive and active approaches to
improve tuberculosis notifications in Afeghanistan. PLoS One [Internet]. 2016[cited 2016 Sep 23];11(10):e0163813. Available
from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5049786/pdf/pone.0163813.pdf 11. Orlandi EP. Análise de Discurso: princípio & procedimentos. 11ª ed. REFERENCES Plos One [Internet]. 2013[cited 2016 Nov 10];8(2):e56752. Available from: https://www.ncbi. nlm.nih.gov/pmc/articles/PMC3581516/pdf/pone.0056752.pdf Rev Bras Enferm [Internet]. 2018;71(supl1):523-30. 530
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Temporospatial shifts in the human gut microbiome and metabolome after gastric bypass surgery
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1Biodesign Swette Center for Environmental Biotechnology, Arizona State University, Tempe, AZ, USA. 2Biodesign Center for Fundamental and Applied Microbiomics, Arizona
State University, Tempe, AZ, USA. 3Mayo Clinic, Division of Gastroenterology, Scottsdale, AZ, USA. 4Biological Sciences Division, Pacific Northwest National Laboratory, Richland,
WA, USA. 5William R. Wiley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA. 6School of Sustainable Engineering and the
Built Environment, Arizona State University, Tempe, AZ, USA. 7Present address: Department of Civil & Environmental Engineering, The University of Toledo, Toledo, OH, USA.
✉email: zehra-esra.ilhan@inrae.fr; Dr.Rosy@asu.edu ARTICLE
OPEN
Temporospatial shifts in the human gut microbiome and
metabolome after gastric bypass surgery Zehra Esra Ilhan
1,2✉, John K. DiBaise3, Sydney E. Dautel4, Nancy G. Isern5, Young-Mo Kim
4, David W. Hoyt
5,
Athena A. Schepmoes4, Heather M. Brewer
5, Karl K. Weitz4, Thomas O. Metz
4, Michael D. Crowell3, Dae-Wook Kang
1,2,7,
Bruce E. Rittmann1,6 and Rosa Krajmalnik-Brown
1,2,6✉ Zehra Esra Ilhan
1,2✉, John K. DiBaise3, Sydney E. Dautel4, Nancy G. Isern5, Young-Mo Kim
4, David W. Hoyt
5,
Athena A. Schepmoes4, Heather M. Brewer
5, Karl K. Weitz4, Thomas O. Metz
4, Michael D. Crowell3, Dae-Wook Kang
1,2,7,
Bruce E. Rittmann1,6 and Rosa Krajmalnik-Brown
1,2,6✉ Although the etiology of obesity is not well-understood, genetic, environmental, and microbiome elements are recognized as
contributors to this rising pandemic. It is well documented that Roux-en-Y gastric bypass (RYGB) surgery drastically alters the fecal
microbiome, but data are sparse on temporal and spatial microbiome and metabolome changes, especially in human populations. We characterized the structure and function (through metabolites) of the microbial communities in the gut lumen and structure of
microbial communities on mucosal surfaces in nine morbidly obese individuals before, 6 months, and 12 months after RYGB
surgery. Moreover, using a comprehensive multi-omic approach, we compared this longitudinal cohort to a previously studied
cross-sectional cohort (n = 24). In addition to the expected weight reduction and improvement in obesity-related comorbidities
after RYGB surgery, we observed that the impact of surgery was much greater on fecal communities in comparison to mucosal
ones. The changes in the fecal microbiome were linked to increased concentrations of branched-chain fatty acids and an overall
decrease in secondary bile acid concentrations. The microbiome and metabolome data sets for this longitudinal cohort strengthen
our understanding of the persistent impact of RYGB on the gut microbiome and its metabolism. Our findings highlight the
importance of changes in mucosal and fecal microbiomes after RYGB surgery. The spatial modifications in the microbiome after
RYGB surgery corresponded to persistent changes in fecal fermentation and bile acid metabolism, both of which are associated
with improved metabolic outcomes. npj Biofilms and Microbiomes (2020) 6:12 ; https://doi.org/10.1038/s41522-020-0122-5 www.nature.com/npjbiofilms Published in partnership with Nanyang Technological University INTRODUCTION After RYGB, the metabolic products of the gut microbiota exert
beneficial effects on host metabolism22. For example, butyrate and
propionate, which are known to induce satiety in animals23 and
humans24, were in greater concentrations in post-RYGB patients
compared to nonsurgical controls6. RYGB surgery also increased
bile acid concentrations in plasma3,8,25 and this increase has been
associated with weight loss in rats following RYGB26. An increase
in propionate and bile acids after RYGB was associated with an
increase in hormone peptide tyrosine tyrosine (PYY) in humans
and, hence, resolution of diabetes27. Finally, RYGB increased the
abundance of amino acid degradation products in feces6,8. However, these molecules in connection to microbiome have
not
been
evaluated
longitudinally
in
pre-surgical
human
populations. Roux-en-Y gastric bypass (RYGB) is an effective treatment strategy
for morbid obesity and its comorbidities, such as diabetes
mellitus1. Although the precise mechanisms leading to its success
remain unclear, RYGB alters hormonal response2, energy metabo-
lism2, and bile acid circulation3 towards weight loss outcomes. Additionally, an increasing number of studies have shown that
RYGB alters gut microbiota in humans4–11. The composition of the
gut microbiota shifts promptly in humans as soon as 1–3 months
after surgery4,5,10, and those changes have been reported to
persist 12 months post-surgery5,7,10,12. Additionally, a number of
studies4,5,7,10,11,13–15 have evaluated the fecal microbiota after
RYGB in longitudinal cohorts. Due to the invasiveness of mucosal microbiome sampling16,
studies of the human gut microbiome in obesity and after RYGB
have relied on fecal samples4,5,7,8, which underrepresent the
mucosal communities that actively interact with host immune
system and epithelial cells17. In healthy humans, composition of
mucosal and fecal microbiota varies due to differences in local
environments16,18. The composition of the mucosal microbiota
can drastically change in humans during dysbiosis, such as in
ulcerative colitis19, colorectal cancer20, and diabetes21 but, to our
knowledge, the mucosal microbiome after RYGB in humans has
not been characterized longitudinally. In this study, we characterized the temporal and spatial
structures of the microbiome and metabolome in humans before
and after RYGB surgery, using 16S rRNA amplicon gene sequen-
cing,
gas
chromatography-mass
spectrometry,
liquid
chromatography-mass spectrometry, and nuclear magnetic reso-
nance
spectroscopy. This
longitudinal
multi-omic
approach
revealed
differences
between
mucosal
and
fecal
microbial
communities
and
in
fecal
metabolites
in
morbidly
obese
individuals before and after RYGB surgery. Furthermore, we
demonstrated comparable findings from this longitudinal cohort
to those of a cross-sectional one. INTRODUCTION b Body mass index (BMI) index of participants before the surgery (pre-RYGB), 6 months (RYGB-6_mo), and
12 months (RYGB-12_mo) after the surgery. c % Excess weight loss 12 months after the surgery, 6 months after the surgery, and 6–12 months
after the surgery. The box plots represent minimum, maximum, median, first quartile and third quartile values. The gray shaded box around
median of RYGB-CS represents median absolute deviation. Statistical significance between the groups was tested with Wilcoxon signed-rank
test and p values were corrected using the Bonferroni method. **p < 0.01. INTRODUCTION Published in partnership with Nanyang Technological University Z.E. Ilhan et al. 2 35
45
55
b) Body mass index
20
20
60
c) % Excess weight loss
pre-
RYGB
RYGB-
6_mo
RYGB-
12_mo
0-12 mo
0-6 mo 6-12 mo
**
**
**
0
%
100
time after surgery
median
RYGB-CS
kg /m 2
0 mo
12 mo
pre-RYGB
(n=9)
NW (n=10)
RYGB-6_mo
(n=7)
RYGB-12_mo
(n=9)
NW (n=10)
longitudinal RYGB cohort with controls
cross
-sectional RYGB
cohort
RYGB-CS
(n=24)
+
microbiome
(16S rRNA gene
sequencing )
metabolome
(metabolites
&
bile acids)
0
no change
a) Study design
6 mo
grouping based on time after surgery
stool
stool
stool
stool
tissue
tissue
+
stool
tissue
Fig. 1
Study design and weight loss after RYGB surgery. a Study design including number of participants and sample types collected
longitudinally and cross-sectionally. b Body mass index (BMI) index of participants before the surgery (pre-RYGB), 6 months (RYGB-6_mo), and
12 months (RYGB-12_mo) after the surgery. c % Excess weight loss 12 months after the surgery, 6 months after the surgery, and 6–12 months
after the surgery. The box plots represent minimum, maximum, median, first quartile and third quartile values. The gray shaded box around
median of RYGB-CS represents median absolute deviation. Statistical significance between the groups was tested with Wilcoxon signed-rank
test and p values were corrected using the Bonferroni method. **p < 0.01. 0 mo
12 mo
pre-RYGB
(n=9)
NW (n=10)
RYGB-6_mo
(n=7)
RYGB-12_mo
(n=9)
NW (n=10)
longitudinal RYGB cohort with controls
cross
-sectional RYGB
cohort
RYGB-CS
(n=24)
+
microbiome
(16S rRNA gene
sequencing )
metabolome
(metabolites
&
bile acids)
a) Study design
6 mo
stool
stool
stool
stool
tissue
tissue
+
stool
tissue a) Study design ongitudinal RYGB cohort with controls longitudinal RYGB cohort with contro c) % Excess weight loss c) % Excess weight loss b) Body mass index b) Body mass index 35
45
55
b) Body mass index
pre-
RYGB
RYGB-
6_mo
RYGB-
12_mo
**
**
0
kg /m 2
grouping based on time after surgery 20
20
60
c) % Excess weight loss
0-12 mo
0-6 mo 6-12 mo
**
%
100
time after surgery
median
RYGB-CS
0
no change
ry no change time after surgery grouping based on time after surgery Fig. 1
Study design and weight loss after RYGB surgery. a Study design including number of participants and sample types collected
longitudinally and cross-sectionally. RESULTS AND DISCUSSION Table 1. Participant characteristics of the longitudinal cohort. Pre-RYGB RYGB-6_mo
RYGB-12_mo
NW
n
10
7
9
10
Gender F/M
5/5
–
–
7/3
Median age
50 ± 9
–
–
41 ± 15
Hypertension
(+/total)
7/9
4/7
3/9
0
Diabetes (+/total)
7/9
1/7
1/9
0
Hyperlipidemia
(+/total)
8/10
2/7
1/9
0
Arthritis (+/total)
9/10
7/7
9/9
0
Sample collection
N/A
216 ± 41 days
after surgery
455 ± 124 days
after surgery
N/A
Median and median absolute deviation values were reported for sample
collection times. RYGB surgery induced significant weight loss We studied the microbiome and metabolome of two cohorts:
longitudinal and cross-sectional populations. For the longitudinal
arm of the study, we recruited nine morbidly obese pre-RYGB
participants (a tenth participant dropped out after baseline
measurements) and monitored their weight loss and health
outcomes 6 months (RYGB-6_mo) and 12 months (RYGB-12_mo)
after RYGB surgery. The study design is presented in Fig. 1a, and
participant characteristics are summarized in Table 1. We also
compared this longitudinal population to a previously studied
cross-sectional RYGB cohort (RYGB-CS) (n = 24)6. Figure 1b, c shows the short- and longer-term effects of RYGB
surgery on weight loss. Percent excess weight loss (%EWL)
calculations, also shown in Fig. 1c, confirm that the participants
achieved the greatest weight loss during the initial 6 months and
maintained the weight loss a year after the surgery. Median %EWL
after 12 months (65 ± 10) was slightly lower than the median %
EWL of the cross-sectional group (RYGB-CS) (73 ± 15)6; however,
this difference was not statistically significant (p = 0.24). Changes in participants’ diet regimens may have contributed to
weight loss after the surgery. It is important to note that dietary
intake survey was self-administered; hence, errors in completion
could have occurred. Table 2 summarizes total dietary calories and
dietary composition. Based on total
calories reported,
the
morbidly obese participants (pre-RYGB) were consuming fewer
calories than the normal weight (NW) participants. In the United
States, it is often recommended to have morbidly obese patients
lose 10% of their excess weight prior to surgery in order to
minimize
surgical
complications1,
even
though
pre-surgical weight loss has not been associated with a reduction in post-
operative
complications28. Our
pre-RYGB
participants
were
enrolled in a pre-surgery diet program, and according to the
self-reported surveys, they appear to have restricted their calorie
intake to achieve pre-surgical weight loss. npj Biofilms and Microbiomes (2020) 12 Published in partnership with Nanyang Technological University RESULTS AND DISCUSSION p
p
y
g
When the RYGB-CS samples were incorporated into the
weighted and unweighted Unifrac analysis, both RYGB-6_mo
and RYGB-12_mo samples clustered together with RYGB-CS
samples for weighted and unweighted Unifrac, although more
strongly with the unweighted Unifrac (Supplementary Fig. 1,
ADONIS R2 = 0.2401, p = 0.003). Additionally, when fecal and
mucosal samples were analyzed together (Supplementary Fig. 1),
clustering based primarily on sample type followed by the
participant groups was observed, especially based on unweighted
Unifrac distances. Interestingly, some of the RYGB-12_mo mucosal
samples clustered with the fecal samples, indicating that after
RYGB the mucosal community structure was more similar to the
fecal community structure; however, the small sample size did not
allow us to assess the significance of this observation. In summary,
the results for the fecal microbiome are consistent with previous
reports4,7,10,12 showing that fecal microbiome structure changed
after RYGB, with changes sustained at least 1 year after surgery. 0.683), although, compared to NW individuals, carbohydrates
formed a smaller fraction of the diets of post-RYGB participants
(Table 2). Our study results are consistent with prior reports that
RYGB results in significant weight loss, especially during the first
six months after the surgery29, and remain stable or continue to
improve until up to one year after the surgery30. p
p
y
g
y
Besides weight loss, RYGB is known to lead to resolution of
many metabolic disorders, including Type II diabetes. At their
baseline measurements, seven participants had high blood
pressure and diabetes, eight of them had hyperlipidemia, and
nine of them had degenerative osteoarthritis. After RYGB, a
majority of the study participants had resolution of diabetes,
hyperlipidemia, and hypertension (Table 1), but not arthritis. The
metabolic improvements after RYGB are well known and our
observations are in agreement with previous reports31,32. after RYGB, with changes sustained at least 1 year after surgery. It is imperative to characterize changes in the microbiome of
the mucosal space and the feces due to their differences and
physiological relevance. In the lumen, substrates are usually
dietary molecules, whereas in mucosal surfaces, they are host-
derived glycans35. Another difference is the electron acceptor at
the mucosal surfaces versus the lumen36. Oxygen derived from
the eukaryotic tissues is gradually depleted in the mucosal layer
by facultative anaerobes, and, therefore, the lumen becomes
anaerobic18. RESULTS AND DISCUSSION Although the caloric
intake increased by 22% at 12 months compared to 6 months
after RYGB, the weight loss benefits were sustained. The dietary
composition of the morbidly obese participants did not signifi-
cantly change after the surgery (Wilcoxon signed-rank test, p = Published in partnership with Nanyang Technological University Z.E. Ilhan et al. 3 obscured by the great abundances of Firmicutes and Bacter-
oidetes phylotypes. Table 2. Dietary composition of the samples of normal weight (NW),
pre-surgical morbidly obese baseline (pre-RYGB), 6 months after
surgery (RYGB-6_mo), and 12 months after surgery (RYGB-12_mo). NW
(n = 10)
Pre-RYGB
(n = 9)
RYGB-
6_mo
(n = 7)
RYGB-
12_mo
(n = 9)
Calorie intake (cal)
2160 ± 680 1820 ± 710 1310 ± 510 1420 ± 465
Carbohydrate %
50 ± 7
42 ± 8
40 ± 6
37 ± 6
Fat%
33 ± 6
36 ± 6
36 ± 5
38 ± 6
Protein %
14 ± 3
19 ± 4
20 ± 8
21 ± 8
Fiber intake (g)
21 ± 9
14 ± 12
18 ± 6
14 ± 5
Stool consistency/
Bristol Stool Scale
NA
4 ± 1.2
4 ± 1.0
4 ± 1.1
Median values were presented with median absolute deviation values. Table 2. Dietary composition of the samples of normal weight (NW),
pre-surgical morbidly obese baseline (pre-RYGB), 6 months after
surgery (RYGB-6_mo), and 12 months after surgery (RYGB-12_mo). p y
yp
Based on unweighted and weighted Unifrac distances, changes
in the fecal microbiome appeared as soon as 6 months after the
surgery, and the difference on PCo2 between pre-RYGB and RYGB-
12_mo was significant (p = 0.04) (Fig. 2b, d). The ADONIS test was
used
on
Unifrac
distance
matrices
to
differentiate
overall
differences in microbiome structure based on defined groups. The ADONIS R2 values ranged from 0.086 to 0.133 (p < 0.05) based
on participant groups (NW, Pre-RYGB, RYGB-6_mo, and RYGB-
12_mo) (Fig. 2). Even though these values are relatively small in
terms of explaining the variation in the data set, ADONIS R2 values
were smaller than 0.03 when grouping was based on gender, diet,
BMI, stool consistency, or age. The ADONIS R2 results illustrate that
our data set had high heterogeneity and variability; nevertheless,
bariatric surgery had significantly greater impact on the overall
microbiome structure than any of the other factors that commonly
explain interpersonal variability, including diet and BMI. Published in partnership with Nanyang Technological University RESULTS AND DISCUSSION Microorganisms that live in the lumen are also
affected by other host-associated factors such as transit time,
frequency and composition of dietary intake, and bile acids37. RYGB altered fecal and mucosal microbiome structures To detect changes in the gut microbiome after RYGB surgery, we
analyzed the structure of fecal and mucosa-associated (mucosal)
microbiomes of morbidly obese individuals (n = 9) before and
after surgery. Rectal mucosal samples were collected at baseline
and 12 months after RYGB via unsedated flexible sigmoidoscopy. Microbial DNA was extracted from fecal and mucosal samples. We
performed weighted and unweighted Unifrac33 analyses on 16S
rRNA gene sequences using the QIIME 1.9 suite34, and the
principal component analyses (PCoA) are shown in Fig. 2. The
effects of RYGB on the microbiome were pronounced for mucosal
and fecal communities (Fig. 2a, b) for PCoA analysis based on
unweighted Unifrac distances. As demonstrated by Fig. 2a,
mucosal communities differed significantly on the PCo1 axis
when comparing pre-RYGB group to RYGB-12_mo group (p =
0.02). Additionally, the pre-RYGB group was significantly different
than the NW group, particularly on the PCo1 axis, indicating that
microbiomes of normal weight and morbidly obese individuals
differed in structure. Although PCoA based on unweighted Unifrac
distances demonstrated the impact of RYGB on mucosal and
luminal communities, PCo1 and PCo2 explained only a fraction (up
to 13%) of the variability in the data set. Even though we
controlled for factors such as, age of the participants and use of
pharmaceuticals, heterogeneity in the human population, and
other factors that influence gut microbiota composition led to a
small fraction of variability in the data set being explained by the
PCo1 and PCo2. npj Biofilms and Microbiomes (2020) 12 a) Mucosal communities - unweighted Unifrac ADONIS R2 = 0.086, p = 0.148 -0.5
0
0.5
NW
Pre-RYGB
RYGB-12_mo
PCo1
PCo2
-0.5
0
0.5
*
* NW
Pre-RYGB
RYGB-12_mo
RYGB-6_mo
-0.5
0
0.5
PCo1
-0.5
0
0.5
PCo2
* d) Fecal communities - weighted Unifrac
PCo2 (12.06%) d) Fecal communities - weighted Unifrac
PCo2 (12.06%) d) Fecal communities - weighted Unifrac
PCo1 (28.93%)
PCo2 (12.06%)
ADONIS R2 = 0.121, p = 0.053 c) Mucosal communities - weighted Unifrac
PCo2 (18.07%) PCo1 (20.1%)
PCo2 (18.07%)
ADONIS R2 = 0.133 , p = 0.009 RYGB
RYGB 12
-0.5
0
0.5
PCo1
NW
Pre-RYGB
RYGB-12_mo
-0.5
0
0.5
PCo2
**
NW RYGB-12_mo
RYGB-6_mo
pre-RYGB
NW pre-RYGB
RYGB-12_mo
NW Fig. 2
Unifrac analysis of mucosal and fecal microbiome after RYGB surgery. Microbiome communities (a mucosal) and (b fecal) before and
after RYGB surgery in comparison to NW controls based on unweighted Unifrac distances. Microbiome communities (c mucosal) and (d fecal)
before and after RYGB surgery in comparison to NW controls based on weighted Unifrac distances. Box plots represent the median distances
among the communities on PCo1 and PCo2. * indicates Mann–Whitney U-test p < 0.05 and ** indicates Mann–Whitney U-test p < 0.01. Fig. 2
Unifrac analysis of mucosal and fecal microbiome after RYGB surgery. Microbiome communities (a mucosal) and (b fecal) before and
after RYGB surgery in comparison to NW controls based on unweighted Unifrac distances. Microbiome communities (c mucosal) and (d fecal)
before and after RYGB surgery in comparison to NW controls based on weighted Unifrac distances. Box plots represent the median distances
among the communities on PCo1 and PCo2. * indicates Mann–Whitney U-test p < 0.05 and ** indicates Mann–Whitney U-test p < 0.01. Fig. 2
Unifrac analysis of mucosal and fecal microbiome after RYGB surgery. Microbiome communities (a mucosal) and (b fecal) before and
after RYGB surgery in comparison to NW controls based on unweighted Unifrac distances. Microbiome communities (c mucosal) and (d fecal)
before and after RYGB surgery in comparison to NW controls based on weighted Unifrac distances. Box plots represent the median distances
among the communities on PCo1 and PCo2. * indicates Mann–Whitney U-test p < 0.05 and ** indicates Mann–Whitney U-test p < 0.01. Figure 3a also shows the relative abundance of the aforemen-
tioned phylotypes compared to a RYGB-CS group. a) Mucosal communities - unweighted Unifrac PCo1 (6.61 %)
PCo2 (6.02 %)
ADONIS R2 = 0.096, p = 0.009 PCo2 (5.93%)
PCo1 (6.06 %)
ADONIS R2 = 0.086, p = 0.148 PCo2 (5.93%) PCo1 (6.06 %)
PCo1 (6.61 %)
c) Mucosal communities - weighted Unifrac
PCo1 (20.1%)
d) Fecal communities - weighted Unifrac
PCo1 (28.93%)
PCo2 (12.06%)
PCo2 (18.07%)
pre-RYGB
RYGB-12_mo
RYGB-12_mo
ADONIS R2 = 0.086, p = 0.148
ADONIS R2 = 0.096, p = 0.009
ADONIS R2 = 0.121, p = 0.053
ADONIS R2 = 0.133 , p = 0.009
-0.5
0
0.5
NW
Pre-RYGB
RYGB-12_mo
PCo1
PCo2
-0.5
0
0.5
-0.5
0
0.5
PCo1
NW
Pre-RYGB
RYGB-12_mo
-0.5
0
0.5
PCo2
NW
Pre-RYGB
RYGB-12_mo
RYGB-6_mo
-0.5
0
0.5
PCo1
-0.5
0
0.5
PCo2
-0.5
0
0.5
PCo2
-0.5
0
0.5
PCo1
NW
Pre-RYGB
RYGB-12_mo
RYGB-6_mo
RYGB-6_mo
pre-RYGB
NW
*
*
*
**
*
*
NW
Fig. 2
Unifrac analysis of mucosal and fecal microbiome after RYGB surgery. Microbiome communities (a mucosal) and (b fecal) before
after RYGB surgery in comparison to NW controls based on unweighted Unifrac distances. Microbiome communities (c mucosal) and (d fe
before and after RYGB surgery in comparison to NW controls based on weighted Unifrac distances. Box plots represent the median distan
among the communities on PCo1 and PCo2. * indicates Mann–Whitney U-test p < 0.05 and ** indicates Mann–Whitney U-test p < 0.01. PCo1 (6.61 %)
d) Fecal communities - weighted Unifrac
PCo1 (28.93%)
PCo2 (12.06%)
RYGB-12_mo
ADONIS R2 = 0.096, p = 0.009
ADONIS R2 = 0.121, p = 0.053
NW
Pre-RYGB
RYGB-12_mo
RYGB-6_mo
-0.5
0
0.5
PCo1
-0.5
0
0.5
PCo2
-0.5
0
0.5
PCo2
-0.5
0
0.5
PCo1
NW
Pre-RYGB
RYGB-12_mo
RYGB-6_mo
RYGB-6_mo
pre-RYGB
NW
*
*
*
rgery. Microbiome communities (a mucosal) and (b fecal) before and
Unifrac distances. Microbiome communities (c mucosal) and (d fecal)
eighted Unifrac distances. Box plots represent the median distances
U-test p < 0.05 and ** indicates Mann–Whitney U-test p < 0.01. Published in partnership with Nanyang Technological University a) Mucosal communities - unweighted Unifrac a) Mucosal communities - unweighted Unifrac b) Fecal communities - unweighted Unifrac b) Fecal communities - unweighted Unifrac obacterium, and Klebsiella. Firmicutes had many phylotypes
relative abundances were affected by the surgery. While
phylotypes—such as Streptococcus, Enterococcus, Lactococ-
illonella, and Granulicatella—were enriched, Ruminococcus,
and Roseburia were depleted after the surgery. Akkerman-
m Verrucomicrobia and Adlercruetzia and Rothia from
bacteria also were in greater abundance after RYGB. Figure 3a also shows the relative abundance of the afo
tioned phylotypes compared to a RYGB-CS group. The R
group consisted of participants who had previously und
RYGB, had lost at least 50% of their excess weight, and w
provided a stool sample 13–60 months after surgery; ther
was a more heterogeneous group by time after surger
results seen in the RYGB-CS group paralleled those seen
PCo2 (5.93%)
PCo1 (6.06 %)
a) Mucosal communities - unweighted Unifrac
PCo1 (6.61 %)
PCo2 (6.02 %)
b) Fecal communities - unweighted Unifrac
c) Mucosal communities - weighted Unifrac
PCo1 (20.1%)
d) Fecal communities - weighted Unifrac
PCo1 (28.93%)
PCo2 (12.06%)
PCo2 (18.07%)
pre-RYGB
RYGB-12_mo
RYGB-12_mo
ADONIS R2 = 0.086, p = 0.148
ADONIS R2 = 0.096, p = 0.009
ADONIS R2 = 0.121, p = 0.053
ADONIS R2 = 0.133 , p = 0.009
-0.5
0
0.5
NW
Pre-RYGB
RYGB-12_mo
PCo1
PCo2
-0.5
0
0.5
-0.5
0
0.5
PCo1
NW
Pre-RYGB
RYGB-12_mo
-0.5
0
0.5
PCo2
NW
Pre-RYGB
RYGB-12_mo
RYGB-6_mo
-0.5
0
0.5
PCo1
-0.5
0
0.5
PCo2
-0.5
0
0.5
PCo2
-0.5
0
0.5
PCo1
NW
Pre-RYGB
RYGB-12_mo
RYGB-6_mo
RYGB-6_mo
pre-RYGB
NW
*
*
*
**
*
*
NW
Unifrac analysis of mucosal and fecal microbiome after RYGB surgery. Microbiome communities (a mucosal) and (b fecal) bef
YGB surgery in comparison to NW controls based on unweighted Unifrac distances. Microbiome communities (c mucosal) and
and after RYGB surgery in comparison to NW controls based on weighted Unifrac distances. Box plots represent the median d
the communities on PCo1 and PCo2. * indicates Mann–Whitney U-test p < 0.05 and ** indicates Mann–Whitney U-test p < 0. Fecal microbiome after RYGB was similar to microbiome from a
RYGB-CS cohort Fecal microbiome after RYGB was similar to microbiome from a
RYGB-CS cohort Figure 3a shows the relative abundances of significantly enriched
or depleted genus-level phylotypes in the lumen 6 months and 1
year after RYGB surgery and in comparison to a cross-sectional
cohort
(RYGB-CS). The
surgery
significantly
altered
relative
abundances of 24 genus-level phylotypes (Wilcoxon signed-rank
test p < 0.05). The majority of enrichments or depletions of genus-
level phylotypes occurred within the first 6 months after surgery
and were sustained 1 year after surgery (Fig. 3a and Supplemen-
tary Fig. 2). The abundances of these phylotypes were significantly
different in RYGB-6_mo and RYGB-12_mo groups, compared to
the NW group (Supplementary Fig. 2) (Mann–Whitney U-test p <
0.05). One of the microbial staples of RYGB surgery is enrichment
of phylotypes from Gammaproteobacteria4,7, and our analysis
showed that RYGB also altered the abundance of genus-level
phylotypes from other phyla, including Firmicutes, Actinobacteria,
Fusobacteria, and Bacteroidetes. Differences in mucosal communities before and after RYGB
were less apparent when weighted Unifrac (Fig. 2c) was used to
calculate
the
dissimilarities
among
the
communities. With
weighted Unifrac analysis, the differences for minor taxa were We observed an increase in the abundance of Proteobacteria
phylotypes Rothia, Aggregatibacter, Granulicatella, Citrobacter, npj Biofilms and Microbiomes (2020) 12 Z.E. Ilhan et al. 4 a) Mucosal communities - unweighted Unifrac The RYGB-CS
group consisted of participants who had previously undergone
RYGB, had lost at least 50% of their excess weight, and who had
provided a stool sample 13–60 months after surgery; therefore, it
was a more heterogeneous group by time after surgery6. The
results seen in the RYGB-CS group paralleled those seen in the Janthinobacterium, and Klebsiella. Firmicutes had many phylotypes
whose relative abundances were affected by the surgery. While
many phylotypes—such as Streptococcus, Enterococcus, Lactococ-
cus, Veillonella, and Granulicatella—were enriched, Ruminococcus,
Blautia, and Roseburia were depleted after the surgery. Akkerman-
sia from Verrucomicrobia and Adlercruetzia and Rothia from
Actinobacteria also were in greater abundance after RYGB. Published in partnership with Nanyang Technological University npj Biofilms and Microbiomes (2020) 12 Z.E. Ilhan et al. 5 Akkermansia
Prevotella
Streptococcus
Fusobacterium
Megasphaera
Dorea
Veillonella
Bilophila
Haemophilus
Roseburia
Enterococcus
Lactococcus
Adlercreutzia
Oxalobacter
Rothia
Aggregatibacter
Granulicatella
Citrobacter
Janthinobacterium
Klebsiella
Blautia
Ruminococcus
NW
pre-RYGB
RYGB-6_mo
RYGB-12_mo
RYGB-CS
Parabacteroides
0 0.005
0.05
5
50
% relative abundance
A
B
F
F*
P
V
A = Actinobacteria
B = Bacteroidetes
F = Firmicutes
F*= Fusobacteria
P = Proteobacteria
V = Verrucomicrobia
a) Fecal phylotypes
Ruminococcaceae*
b) Mucosal phylotypes
Rothia
Granulicatella
Lactococcus
Streptococcus
Blautia
Dorea
Akkermansia
-0.5
0
0.5 1 1.5 2
% log10 relative
abundance
NW
pre-RYGB
RYGB-12_mo
Fig. 3
Relative abundance of genus-level phylotypes after RYGB surgery. Heat map showing which a fecal and b mucosal microbial
phylotypes were enriched or depleted after RYGB surgery. The samples were chronologically ordered based on time after surgery. The
statistical significance between pre-RYGB and RYGB-12_mo groups were based on Wilcoxon signed-rank test and p values were corrected
using Bonferroni method *p < 0.05. Akkermansia
Prevotella
Streptococcus
Fusobacterium
Megasphaera
Dorea
Veillonella
Bilophila
Haemophilus
Roseburia
Enterococcus
Lactococcus
Adlercreutzia
Oxalobacter
Rothia
Aggregatibacter
Granulicatella
Citrobacter
Janthinobacterium
Klebsiella
Blautia
Ruminococcus
NW
pre-RYGB
RYGB-6_mo
RYGB-12_mo
RYGB-CS
Parabacteroides
0 0.005
0.05
5
50
% relative abundance
A
B
F
F*
P
V
A = Actinobacteria
B = Bacteroidetes
F = Firmicutes
F*= Fusobacteria
P = Proteobacteria
V = Verrucomicrobia
a) Fecal phylotypes
Ruminococcaceae* a) Fecal phylotypes b) Mucosal phylotypes
Rothia
Granulicatella
Lactococcus
Streptococcus
Blautia
Dorea
Akkermansia
-0.5
0
0.5 1 1.5 2
% log10 relative
abundance
NW
pre-RYGB
RYGB-12_mo % log10 relative
abundance Fig. 3
Relative abundance of genus-level phylotypes after RYGB surgery. Heat map showing which a fecal and b mucosal microbial
phylotypes were enriched or depleted after RYGB surgery. a) Mucosal communities - unweighted Unifrac The samples were chronologically ordered based on time after surgery. The
statistical significance between pre-RYGB and RYGB-12_mo groups were based on Wilcoxon signed-rank test and p values were corrected
using Bonferroni method *p < 0.05. whereas Dorea and Blautia are lactate oxidizers39. Lactate-
producing Streptococcus and Lactococcus species have been used
as probiotics to enhance gut epithelial barrier and integrity40,
since lactate availability is crucial for butyrate producers and,
therefore, colon epithelium health41. RYGB-6_mo and RYGB-12_mo groups (Supplementary Fig. 2). The
results of the RYGB-CS group were sorted based on the time after
their surgery, and we found no clustering based on time after
surgery. These results support that changes in the microbiome
occurred quickly after the surgery (within 6 months) and persisted
in the long term (>60 months). In addition to lactate producers and oxidizers, we observed an
increase in the relative abundance of Akkermansia in the mucosa
after RYGB surgery. Previously, a similar trend was observed in
mice after RYGB22, and our findings confirmed this observation in
humans. Animal models have demonstrated that a weak gut
barrier contributes to the development of endotoxemia and
inflammation, which subsequently leads to insulin resistance and
an increase in adiposity42–44. Akkermansia is a known mucin
degrader, and its presence has been shown to improve the gut
epithelial barrier, reduce adiposity of the organs, and protect
against insulin resistance and obesity in humans45. However, a
recent study that investigated the link between Akkermansia
abundance in the feces of severely obese individuals after RYGB
and diabetes did not report any association between Akkermansia
abundance and glucose homeostasis after RYGB46. Overall, our
results indicate that alterations in the gastrointestinal mucosa
after RYGB may contribute to an increase in mucin-degrading,
lactate-producing, and lactate-oxidizing microorganisms. To
confirm
observations
on
genus-level
phylotypes,
we
performed unsupervised clustering and generated hierarchical
clustering heat map based on Euclidean distances among the
samples (Supplementary Fig 3). Samples formed five distinct
clusters driven by Fusobacterium, Prevotella, Ruminococcus, Para-
bacteroides, Blautia, and Akkermansia. Three of the clusters were
composed of only post-RYGB samples (RYGB-6_mo, RYGB_12-mo,
and RYGB-CS), indicating the impact of the RYGB alone on the
relative abundance of genus-level phylotypes. The sustained
changes observed in the microbiome after RYGB indicate that the
surgery-imposed changes to the gut environment/ecosystem
were persistent and permanently affected gut microbiota in a
stronger way than interpersonal variations. Published in partnership with Nanyang Technological University npj Biofilms and Microbiomes (2020) 12 RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers f Predicted relative abundance of the genes that are involved in branched chain amino acid (leucine,
valine, and isoleucine) degradation. KEGG ko00280 valine, leucine, isoleucine degradation pathway was used in the analysis. * indicates
statistical significance between pre-RYGB and RYGB-12_mo groups based on Wilcoxon signed-rank test and p values were corrected using
Bonferroni method *p < 0.05. ADONIS R2 = 0.326, p = 0.002 pre-RYGB
RYGB-6_mo
RYGB-12_mo
RYGB-CS c) PCA Plot based on NMR analysis c) PCA Plot based on NMR analysis 5.0
2.5
0.0
2.5
5.0
0
10
PC1 (22.3%)
PC2 (11.9%)
c) PCA Plot based on NMR analysis
7.5
2
pre-RYGB
RYGB-6_mo
RYGB-12_mo
ADONIS R2 = 0.134, p = 0.001 0
10
20
isobutyrate
isovalerate
umoles/g dry feces
* *
*
* *
*
pre-RYGB
RYGB-6_mo
RYGB-12_mo
RYGB-CS
f) BCAA degradation umoles/g dry feces 0
10
isoleucine
leucine
valine
*
*
*
20
30
umoles/g dry feces
RYGB-6_mo
pre-RYGB
RYGB-12_mo
RYGB-CS
e) Branched chain amino acids (BCAA)
RYGB-12_mo
RYGB-CS
f) BCAA degradation
0.3
0.1
0.2
0
**
**
**
% predicted abundance
, p e) Branched chain amino acids (BCAA) e) Branched chain amino acids (BCAA) valine leucine Fig. 4
Fecal metabolites detected with H-NMR and GC-MS. Principal component analysis based on metabolites detected by a GC-MS based
metabolomes before and after the surgery. b Principal component analysis based on GC-MS metabolome that includes RYGB-CS group
samples. c 1H-NMR based metabolomes before and after the surgery. d Branched-chain fatty acids—isobutyrate and isovalerate—measured
with NMR after RYGB surgery prospectively and retrospectively. e Concentrations of isoleucine, leucine, and valine, branched-chain amino
acids (BCAA) measured with NMR after RYGB surgery prospectively and retrospectively. The box plots represent minimum, maximum, median,
first quartile and third quartile values. f Predicted relative abundance of the genes that are involved in branched chain amino acid (leucine,
valine, and isoleucine) degradation. KEGG ko00280 valine, leucine, isoleucine degradation pathway was used in the analysis. * indicates
statistical significance between pre-RYGB and RYGB-12_mo groups based on Wilcoxon signed-rank test and p values were corrected using
Bonferroni method *p < 0.05. the impact of RYGB surgery resulted in a unique metabolic
fingerprint that was preserved in the long term. Moreover, the
similar clustering patterns with the metabolome (Fig. 4a, b) and
the microbiome (Fig. 2a) strengthen the conclusion that the
changes in the microbiome and metabolome after RYGB surgery
were linked, persistent, and stronger than interpersonal variations. RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers Our analysis of enriched or depleted phylotypes also demon-
strates that RYGB surgery led to a wide spectrum of changes in the
mucosal
space
(Fig. 3b). Six
genus-level
phylotypes
were
significantly
enriched
in
the
mucosa
after
RYGB
surgery:
Granulicatella, Lactococcus, Streptococcus, Blautia, Dorea, and
Akkermansia (Supplementary Fig. 4) (Wilcoxon signed-rank test
p < 0.05). Relative abundances of these phylotypes also were
greater in the NW mucosa, compared to the pre-RYGB mucosa. Except for Akkermansia, the microorganisms enriched post-surgery
are from the Firmicutes phylum and are known to form biofilms
and contribute to lactate metabolism38. Lactococcus, Streptococ-
cus, and Granulicatella are lactate-producing microorganisms, Post-RYGB microbiota alters the fecal metabolome
Changes in the structure of the gut microbiome after RYGB
surgery were reflected in the gut metabolome. Figure 4 shows
PCA
results
for
the
fecal
metabolomes
detected
by
gas
chromatography-mass spectrometry (GC-MS) and 1H-NMR-based
methods. Fecal water-soluble extracts were analyzed with 1H-
NMR, while lyophilized fecal matter was analyzed with GC-MS. 1H-
NMR provided mainly volatile and water-soluble compounds,
whereas GC-MS identified many metabolites of the undigested
nutrients and components of microbial cells. Published in partnership with Nanyang Technological University npj Biofilms and Microbiomes (2020) 12 Z.E. Ilhan et al. GC-MS data (Fig. 4a), RYGB-6_mo and RYGB-12_mo
olomes clustered away from pre-RYGB metabolomes
0 326
0 002) Fi
4b
l
h
GC MS
the impact of RYGB surgery resulted in a un
fingerprint that was preserved in the long term
i
il
l
i
i h h
b l
5.0
2.5
0.0
2.5
5.0
0
10
PC1 (22.3%)
PC2 (11.9%)
0
10
20
isobutyrate
isovalerate
umoles/g dry feces
* *
*
* *
*
0
10
isoleucine
leucine
valine
*
*
*
20
30
umoles/g dry feces
d) Branched chain fatty acids (BCFA)
c) PCA Plot based on NMR analysis
RYGB-6_mo
pre-RYGB
RYGB-12_mo
RYGB-CS
7.5
e) Branched chain amino acids (BCAA)
pre-RYGB
RYGB-6_mo
RYGB-12_mo
RYGB-CS
-10
-5
0
5
10
-10
-5
0
5
PC1 (16.7%)
PC2 (9.61%)
-1
-5
0
5
1
PC1 (16.7%)
PC2 (9.61%)
a) PCA Plot based on GC-MS analysis
b) PCA Plot based on GC-MS analysis
including RYGB-CS
pre-RYGB
RYGB-6_mo
RYGB-12_mo
RYGB-CS
2
pre-RYGB
RYGB-6_mo
RYGB-12_mo
f) BCAA degradation
0.3
0.1
0.2
0
**
**
**
% predicted abundance
ADONIS R2 = 0.326, p = 0.002
ADONIS R2 = 0.256, p = 0.01
ADONIS R2 = 0.134, p = 0.001
metabolites detected with H-NMR and GC-MS. RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers Principal component analysis based on metabolites detected b
s before and after the surgery. b Principal component analysis based on GC-MS metabolome that includes
H-NMR based metabolomes before and after the surgery. d Branched-chain fatty acids—isobutyrate and isovale
ter RYGB surgery prospectively and retrospectively. e Concentrations of isoleucine, leucine, and valine, branch
measured with NMR after RYGB surgery prospectively and retrospectively. The box plots represent minimum, ma
and third quartile values. f Predicted relative abundance of the genes that are involved in branched chain am
soleucine) degradation. KEGG ko00280 valine, leucine, isoleucine degradation pathway was used in the ana
nificance between pre-RYGB and RYGB-12_mo groups based on Wilcoxon signed-rank test and p values were
method *p < 0.05. Z.E. Ilhan et al. RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers -10
-5
0
5
10
-10
-5
0
5
PC1 (16.7%)
PC2 (9.61%)
-1
-5
0
5
1
PC1 (16.7%)
PC2 (9.61%)
a) PCA Plot based on GC-MS analysis
b) PCA Plot based on GC-MS analysis
including RYGB-CS
pre-RYGB
RYGB-6_mo
RYGB-12_mo
RYGB-CS
ADONIS R2 = 0.326, p = 0.002
ADONIS R2 = 0.256, p = 0.01 6 -10
-5
0
5
10
-10
-5
0
5
PC1 (16.7%)
PC2 (9.61%)
a) PCA Plot based on GC-MS analysis
ADONIS R2 = 0 326 p = 0 002 -1
-5
0
5
1
PC1 (16.7%)
PC2 (9.61%)
b) PCA Plot based on GC-MS analysis
including RYGB-CS a) PCA Plot based on GC-MS analysis b) PCA Plot based on GC-MS analysis
including RYGB-CS b) PCA Plot based on GC-MS analys
including RYGB-CS PC2 (9.61%) B
d
GC MS d t
(Fi
4 ) RYGB 6
d RYGB 12
th
i
t
f RYGB
lt d i
i
t b li
5.0
2.5
0.0
2.5
5.0
0
10
PC1 (22.3%)
PC2 (11.9%)
0
10
20
isobutyrate
isovalerate
umoles/g dry feces
* *
*
* *
*
0
10
isoleucine
leucine
valine
*
*
*
20
30
umoles/g dry feces
d) Branched chain fatty acids (BCFA)
c) PCA Plot based on NMR analysis
RYGB-6_mo
pre-RYGB
RYGB-12_mo
RYGB-CS
7.5
e) Branched chain amino acids (BCAA)
pre-RYGB
RYGB-6_mo
RYGB-12_mo
RYGB-CS
-10
-5
0
5
10
-10
-5
0
5
PC1 (16.7%)
PC2 (9.61%)
-1
-5
0
5
1
PC1 (16.7%)
PC2 (9.61%)
pre-RYGB
RYGB-6_mo
RYGB-12_mo
RYGB-CS
2
pre-RYGB
RYGB-6_mo
RYGB-12_mo
f) BCAA degradation
0.3
0.1
0.2
0
**
**
**
% predicted abundance
ADONIS R2 = 0.326, p = 0.002
ADONIS R2 = 0.256, p = 0.01
ADONIS R2 = 0.134, p = 0.001
Fig. 4
Fecal metabolites detected with H-NMR and GC-MS. Principal component analysis based on metabolites detected by a GC-MS based
metabolomes before and after the surgery. b Principal component analysis based on GC-MS metabolome that includes RYGB-CS group
samples. c 1H-NMR based metabolomes before and after the surgery. d Branched-chain fatty acids—isobutyrate and isovalerate—measured
with NMR after RYGB surgery prospectively and retrospectively. e Concentrations of isoleucine, leucine, and valine, branched-chain amino
acids (BCAA) measured with NMR after RYGB surgery prospectively and retrospectively. The box plots represent minimum, maximum, median,
first quartile and third quartile values. Published in partnership with Nanyang Technological University Published in partnership with Nanyang Technological University RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers Similar to GC-MS metabolome, 1H-NMR quantification of water-
soluble metabolites showed a distinct RYGB metabolome (Fig. 4c). The concentrations as measured by 1H-NMR of the major SCFAs of Based on GC-MS data (Fig. 4a), RYGB-6_mo and RYGB-12_mo
fecal metabolomes clustered away from pre-RYGB metabolomes
(ADONIS R2 = 0.326, p = 0.002). Figure 4b overlays the GC-MS-
based metabolomes of the RYGB-CS participants to the metabo-
lomes of longitudinal study participants illustrated in Fig. 4a. The
metabolomes of most of the RYGB-CS participants were more
similar
to
RYGB-6_mo
and
RYGB-12_mo
participants
(Mann–Whitney U-test p < 0.05, ADONIS R2 = 0.256, p = 0.01) than
to Pre-RYGB or NW participants, supporting the observation that npj Biofilms and Microbiomes (2020) 12 Z.E. Ilhan et al. 7 Table 3. Concentrations of acetate, butyrate, and propionate normalized to dry weight g stool, along with propionate-to-acetate and butyrate-to-
acetate ratios in NW, pre-RYGB, RYGB-6_mo, and RYGB-12_mo, and RYGB-CS groups. Propionate/acetate
Butyrate/acetate
μmoles/g stool
Acetate
Butyrate
Propionate
NW
0.40
0.25
110 ± 46
31 ± 16
45 ± 17
Pre-RYGB
0.37
0.24
240 ± 109
47 ± 11
75 ± 20
RYGB-6_mo
0.38
0.27
240 ± 56
61 ± 20
100 ± 16
RYGB-12_mo
0.41
0.30
160 ± 21
42 ± 9
70 ± 9
RYGB-CS
0.41
0.34
180 ± 25
69 ± 16
81 ± 12
The measurements were taken with 1H-NMR. The numbers represent median values of the groups with median absolute deviation values. Wilcoxon signed-
rank test was used to assess statistical significance. Propionate/acetate and butyrate/acetate ratios were significantly different between the Pre-RYGB and
RYGB-12_mo groups (Wilcoxon rank-signed test p = 0.004 and p = 0.002). Table 3. Concentrations of acetate, butyrate, and propionate normalized to dry weight g stool, along with propionate-to-acetate and butyrate-to-
acetate ratios in NW, pre-RYGB, RYGB-6_mo, and RYGB-12_mo, and RYGB-CS groups. The measurements were taken with 1H-NMR. The numbers represent median values of the groups with median absolute deviation values. Wilcoxon signed-
rank test was used to assess statistical significance. Propionate/acetate and butyrate/acetate ratios were significantly different between the Pre-RYGB and
RYGB-12_mo groups (Wilcoxon rank-signed test p = 0.004 and p = 0.002). of mice47. Shifts in microbial metabolism reflect another potential
mechanism explaining how microorganisms contribute to weight
loss following RYGB. Table 4. Concentrations of fecal metabolites normalized to dry weight
that were statistically different between pre-RYGB and RYGB-12_mo
samples. RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers µmoles/g stool dry weight
p value
Metabolites NW
Pre-RYGB
RYGB-
6_mo
RYGB-
12_mo
Pre-RYGB vs
RYGB-12_mo
Alanine
10.0 ± 4.3 11.8 ± 5.0
9.7 ± 2.0
6.9 ± 1.3
0.035
Cadaverine
0.2 ± 0.0
0.4 ± 0.4
0.6 ± 0.2
0.7 ± 0.3
0.031
Glucose
5.8 ± 4.1
26.1 ± 11.1
6.1 ± 4.2
3.9 ± 1.4
0.002
Glutamine
3.5 ± 1.7
5.8 ± 2.0
2.6 ± 1.6
2.5 ± 1.0
0.051
Isopropanol 0.2 ± 0.1
0.1 ± 0.0
0.2 ± 0.0
0.2 ± 0.0
0.036
Methanol
0.9 ± 0.5
1.7 ± 0.7
1.1 ± 0.6
0.7 ± 0.1
0.006
Succinate
2.9 ± 0.5
4.6 ± 0.5
2.5 ± 0.1
2.1 ± 0.3
0.028
Taurine
0.7 ± 0.0
8.0 ± 7.9
0.0 ± 0.0
0.0 ± 0.0
0.003
Threonine
5.4 ± 2.2
9.4 ± 3.7
4.7 ± 2.6
3.9 ± 1.2
0.035
Thymidine
0.2 ± 0.0
0.8 ± 0.7
0.0 ± 0.0
0.0 ± 0.0
0.009
Tyrosine
5.0 ± 2.0
7.8 ± 2.8
4.8 ± 1.4
3.4 ± 0.7
0.042
Uracil
2.4 ± 1.2
3.4 ± 1.4
1.9 ± 0.8
2.1 ± 0.5
0.035
Uridine
0.0 ± 0.0
1.3 ± 1.3
0.0 ± 0.0
0.0 ± 0.0
0.019
Valerate
7.9 ± 1.1
11.9 ± 3.1
0.0 ± 0.0
0.0 ± 0.0
0.06
Valine
7.4 ± 3.5
11.9 ± 5.2
7.8 ± 2.1
5.6 ± 1.3
0.035
Xylose
1.2 ± 1.0
3.1 ± 1.5
0.0 ± 0.0
0.0 ± 0.0
0.000
Leucine
6.6 ± 3.1
9.7 ± 4.3
6.1 ± 1.4
5.5 ± 1.1
0.042
Lysine
4.4 ± 1.2
7.4 ± 3.6
3.8 ± 1.7
2.5 ± 1.0
0.035
Isoleucine
5.4 ± 2.4
9.0 ± 4.2
5.1 ± 1.4
4.0 ± 1.1
0.028
Isovalerate
5.0 ± 2.4
5.0 ± 2.4
7.0 ± 1.6
9.3 ± 2.3
0.023
Isobutyrate
4.8 ± 1.9
6.0 ± 2.6
6.9 ± 1.6
9.1 ± 0.8
0.018
The measurements were done with
1H-NMR. The numbers represent
median values of the groups with median absolute deviation values. Wilcoxon signed-rank test was used to assess statistical significance. g
We also evaluated the concentrations of branched chain amino
acids (BCAA) and their fermentation products—branched-chain
fatty acids (BCFA)—before and after RYGB. As seen in Fig. 4d, the
fecal concentrations of two BCFAs—isobutyrate and isovalerate—
increased after surgery and this observation is consistent with
previous observations8,13. The RYGB-CS and RYGB-12_mo groups
had similar concentrations of these BCFAs. RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers Therefore, we can
deduce that an increase in the abundance of these BCFAs was
more likely associated with RYGB. Three BCAAs—leucine, iso-
leucine, and valine—were at significantly lower abundance in
RYGB-12_mo in comparison to NW and RYGB-CS groups (Fig. 4e,
Table S1). Interestingly, the concentration of BCAAs poorly
correlated with the amount of protein consumed by the
participants (Spearman’s rank correlation coefficient < 0.335). Even
though BCAA concentrations were variable, their fermentation
products were always greater post-RYGB. This observation was
further supported by the predicted abundances of the genes that
are involved in the BCAA (valine, leucine, and isoleucine)
degradation and the synthesis of BCFA production pathways as
shown in Fig. 4f. The predicted abundances of BCAA degradation
genes were significantly greater after RYGB and in the RYGB-CS
group in comparison to the pre-RYGB group. In summary, changes
in the microbiome due to RYGB surgery seemed to enhance fecal
amino acid metabolism, which may have contributed to weight
loss by producing BCFA that are capable of signaling free fatty
acid receptors48. The role of BCFAs on FFA receptor signaling
warrants further investigation. g
In addition to SCFAs and BCFAs, we analyzed a wide spectrum
of other metabolites. Most of the fecal metabolites including
sugars and amino acids, detected with 1H-NMR and GC-MS, were
at
greater
abundance
in
the
pre-RYGB
group,
and
their
concentrations dropped 12 months after surgery (Table 4). The
fecal metabolite concentration profiles of RYGB-12_mo and RYGB-
CS groups were similar, possibly due to the altered gastrointestinal
tract environment after the surgery and similarities in participant
diets. However, as shown in Table 4, besides isovalerate and
isobutyrate, concentrations of xylose also increased after RYGB
and were even higher than for NW controls. Greater abundance of
fecal xylose after RYGB would seem to indicate that the
participants adapted to more plant-based diets or that they lost
some microbial hydrolytic capabilities to metabolize xylose. The
(self-reported) participants’ fiber intake did not change signifi-
cantly after the surgery (Table 1), although it was statistically lower
in
post-RYGB
participants
compared
to
NW
participants
(p = 0.032). The measurements were done with
1H-NMR. The numbers represent
median values of the groups with median absolute deviation values. Wilcoxon signed-rank test was used to assess statistical significance. the human gut—acetate, butyrate, and propionate (Table 3)—
were
similar
for
the
RYGB-CS
and
RYGB-12_mo
groups. npj Biofilms and Microbiomes (2020) 12 Published in partnership with Nanyang Technological University RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers Propionate-to-acetate and butyrate-to-acetate ratios increased 6
and 12 months after the surgery, and the difference between
baseline and 12-month samples was statistically significant (p =
0.03). We previously reported a similar trend with our RYGB-CS
cohort6. Higher butyrate- and propionate-to-acetate ratios after
the surgery compared to baseline indicates a shift in microbial
metabolism from acetate production to butyrate and propionate
production. Butyrate and propionate have been shown to signal
free fatty acid receptors and induce a satiety response in the brain npj Biofilms and Microbiomes (2020) 12 Z.E. Ilhan et al. 8 Fig. 5
Fecal bile acids measured before and after RYGB surgery. a Fecal primary bile acid (CA cholic acid, TCA taurodeoxycholic acids, GCA
glycocholic acid, GCDCA glycochenodeoxycholic acid, TCDCA taurochenodeoxycholic acid) and b fecal secondary bile acids (TDCA
taurodeoxycholic acid, LCA lithocholic acid, GLCA glycolithocholic acid) that were statistically different after RYGB surgery. * indicates
statistical significance between pre-RYGB and RYGB-6 and RYGB-12_mo groups based on Wilcoxon signed-rank test and p values were
corrected using Bonferroni method *p < 0.05. The box plots represent minimum, maximum, median, first quartile, and third quartile values. Fig. 5
Fecal bile acids measured before and after RYGB surgery. a Fecal primary bile acid (CA cholic acid, TCA taurodeoxycholic acids, GCA
glycocholic acid, GCDCA glycochenodeoxycholic acid, TCDCA taurochenodeoxycholic acid) and b fecal secondary bile acids (TDCA
taurodeoxycholic acid, LCA lithocholic acid, GLCA glycolithocholic acid) that were statistically different after RYGB surgery. * indicates
statistical significance between pre-RYGB and RYGB-6 and RYGB-12_mo groups based on Wilcoxon signed-rank test and p values were
corrected using Bonferroni method *p < 0.05. The box plots represent minimum, maximum, median, first quartile, and third quartile values. In order to reveal microbial connections to bile acid metabo-
lism, we used Phylogenetic Investigation of Communities by
Reconstruction of Unobserved States (PICRUSt) software53 to
predict secondary bile acid biosynthesis pathway from 16S rRNA
gene abundances. PICRUSt prediction of secondary bile acid
biosynthesis pathway was greater after RYGB; however, these are
genomic predictions and not activity measurements (Fig. 6b). Table S2 summarizes the median concentrations of primary and
secondary bile acids observed in NW and RYGB-CS groups in
comparison to RYGB-12_mo and pre-RYGB groups. Published in partnership with Nanyang Technological University npj Biofilms and Microbiomes (2020) 12 Published in partnership with Nanyang Technological University RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers c Fecal bile acid and microbiome co-occurrence network based on Spearman’s rho
correlation coefficients. Fig. 6
Bile acid–fecal microbiota interactions. a Bile acid transformation reactions. Orange color = primary bile acids, green = primary
conjugated bile acids, blue = secondary bile acids, pink = secondary conjugated bile acids. CA cholic acid, DCA deoxycholic acid, GDCA
glycodeoxycholic acid, TDCA taurodeoxycholic acid, GCA glycocholic acid, TCA taurocholic acid, CDCA chenodeoxycholic acid, LCA lithocholic
acid, HCA hyacholic acid, HDCA hyodeoxycholic acid, UDCA ursodeoxycholic acid, GUDCA glycoursodeoxycholic
acid, TUDCA
tauroursodeoxycholic acid, THDCA taurohydroxydeoxycholic acid, GLCA glycolithocholic acid, TLCA taurolithocholic acid, GCDCA
glycochenodeoxycholic acid, TCDCA taurochenodeoxycholic acid, αMCA α-muricholic acid, βMCA β-muricholic acid. b Bile acid biosynthesis
genes predicted from 16S rRNA gene abundances via PICRUSt. KO numbers that were used in the prediction: K01442, K00076, K23231,
K22604, K22605, K22606, K22607, K15868, K15871, K15869, K15870, K15873, K15874, and K07007. The box plots represent minimum,
maximum, median, first quartile, and third quartile values. c Fecal bile acid and microbiome co-occurrence network based on Spearman’s rho
correlation coefficients. concentrations
of
fecal
primary
and
secondary
bile
acids
measured in this study. 52 54 We performed the same analysis with mucosal genus-level
phylotypes and bile acids (see Supplementary Fig. 5). We observed
associations with minor taxa such as Methanobacterium and bile
acids. Interestingly, Clostridium genus phylotypes negatively
correlated with a number of bile acids. Additionally, UDCA, GDCA,
and GUDCA were the bile acids that showed the greatest number
of associations with mucosal phylotypes. Given that bile acids
have been reported to modify the gut microbiome52, lower
delivery of bile acids to the colon might have played a role on
some of the microbiome compositional changes observed. Additionally, microbial bile acid metabolism can potentially have
effects on host body weight and metabolism since it was
previously shown that bile diversion to the small intestine can
recapitulate some of metabolic benefits of the RYGB indepen-
dently from the surgery57. Considering that gut microbiota can transform bile acids52,54
and concentrations of bile acids can affect gut microbiota
composition52, we performed co-occurrence-network analysis
between fecal genus-level microbial phylotypes and bile acids. As shown in Fig. 6c, phylotypes that were enriched after RYGB,
including Fusobacterium, Veillonella, Enterococcus, Akkermansia,
and Streptococcus negatively correlated with various bile acids
such as TDCA, LCA, TCDCA, GCA, GDCA, TCA, and TLCA. RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers The concen-
trations measured in the RYGB-CS group were similar to the RYGB-
12_mo group, which indicates that the response of surgical
modification on bile acid metabolism was strong and reproduci-
ble, even if the baseline time points before the surgery are
missing. Additionally, bile acid levels after RYGB groups were
similar to NW participants (Table S2). Overall, our findings indicate
that fecal concentrations of primary and secondary bile acids
declined after RYGB surgery, and levels similar to those in NW
individuals were maintained even years after the surgery. Fat and
cholesterol intake are important factors in the production and
secretion of bile acids52. As seen in Table 1, the participants did
not reduce the fat percentage of their diets, although they
consumed fewer calories after RYGB, which leads to lower
absolute amounts of fat being consumed. Lower delivery of fat
to the gastrointestinal tract might have played a role in the lower RYGB surgery decreased fecal bile acid concentrations
As RYGB is known to alter the bile acid metabolism49,50 and
contribute to remission of type 2 diabetes and weight loss51, we
quantified seven primary and 10 secondary bile acids in fecal
samples from participants before and after RYGB surgery using
liquid chromatography-mass spectrometry (LC-MS). Figure 5a, b
and Table S2 show primary and secondary fecal bile acids and
their conjugated forms measured at baseline, 6 months, and
12 months after RYGB. Fecal concentrations of primary bile acid—
cholic acid (CA)—and its glycine- and taurine-conjugated forms
(TCA and GCA) were significantly lower 6 months after the surgery
(CA p = 0.022, TCA p = 0.001, GCA p = 0.002), and they remained
at similar concentrations 12 months after surgery. Similarly,
concentrations of glycine- and taurine-conjugated forms of
chenodeoxycholic acid (CDCA), GCDCA and TCDCA, dropped
significantly 6 months after the surgery. Concentrations of
secondary bile acids, lithocholic acid (LCA), its glycine conjugated
form, GLCA, and taurodeoxycholic acid (TDCA) significantly
dropped 6 months after surgery as well (LCA p = 0.02, GLCA
p = 0.001, and TDCA p = 0.003). Figure 6a illustrates the conjuga-
tion and transformation reactions of primary bile acids and the
resulting secondary bile acids produced by gut microbiota52. Our
findings show that primary and secondary bile acids were
significantly diminished in feces after RYGB surgery. npj Biofilms and Microbiomes (2020) 12 Z.E. Ilhan et al. Published in partnership with Nanyang Technological University RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers 9 MCA
p
CDCA
p
conjugation
conjugation
GCDCA
p
TCDCA
p
conjugation
conjugation
GUDCA
s
TUDCA
s
conjugation
conjugation
TLCA
s
s
MCA
p
conjugation
conjugation
conjugation
CA
p
GCA
p
TCA
p
DCA
s
conjugation
conjugation
TDCA
s
gut microorganisms convert
primary to secondary bile acids
b) Secondary bile acid biosynthesis
RYGB-6_mo
pre-RYGB
RYGB-12_mo
0.1
0.08
0.06
0.04
0.02
0
*
c) Associations with microbial phylotypes and bile acids
a) Bile acid metabolism
s
p
p
s
primary bile acid CDCA
secondary bile acid CDCA
primary bile acid CA
secondary bile acid CA
GDCA
s
Blautia
Dorea
Corynebacterium
Eggerthella
Ruminococcus
Holdemania
Coprobacillus
Anaerotruncus
Faecalibacterium
Megasphaera
Lachnospira
Christensenella
Akkermansia
Fusobacterium
Streptococcus
Veillonella
Bilophila
Enterococcus
Serratia
Relative abundance of the
predicted genes %
Negative correlations
Positive correlations
s
GLCA
s
LCA
HCA
s
THDCA
s
HDCA
s
UDCA
s
LCA
TDCA
s
s
HDCA
CA
p
s
GLCA
s
THDCA
s
UDCA
TDCA
s
GCDCA
p
TLCA
s
TCA
p
GDCA
s
GCA
p
TCDCA
p
s
LCA
TCDCA
p
GCA
p
GDCA
s
TCA
p
TLCA
s
GCDCA
p
TDCA
s
s
UDCA
s
THDCA
s
GLCA
CA
p
s
HDCA
TDCA
s
Bile acid–fecal microbiota interactions. a Bile acid transformation reactions. Orange color = primary bile acids, green = prim
gated bile acids, blue = secondary bile acids, pink = secondary conjugated bile acids. CA cholic acid, DCA deoxycholic acid, G
deoxycholic acid, TDCA taurodeoxycholic acid, GCA glycocholic acid, TCA taurocholic acid, CDCA chenodeoxycholic acid, LCA lithoc
HCA hyacholic acid, HDCA hyodeoxycholic acid, UDCA ursodeoxycholic acid, GUDCA glycoursodeoxycholic
acid, TU
ursodeoxycholic acid, THDCA taurohydroxydeoxycholic acid, GLCA glycolithocholic acid, TLCA taurolithocholic acid, GC
chenodeoxycholic acid, TCDCA taurochenodeoxycholic acid, αMCA α-muricholic acid, βMCA β-muricholic acid. b Bile acid biosynt
predicted from 16S rRNA gene abundances via PICRUSt. KO numbers that were used in the prediction: K01442, K00076, K23
4, K22605, K22606, K22607, K15868, K15871, K15869, K15870, K15873, K15874, and K07007. The box plots represent minim
mum, median, first quartile, and third quartile values. c Fecal bile acid and microbiome co-occurrence network based on Spearman’s
ation coefficients. RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers b) Secondary bile acid biosynthesis
RYGB-6_mo
pre-RYGB
RYGB-12_mo
0.1
0.08
0.06
0.04
0.02
0
*
Relative abundance of the
predicted genes % b) Secondary bile acid biosynthesis b) Secondary bile acid biosynthesis MCA
p
CDCA
p
conjugation
conjugation
GCDCA
p
TCDCA
p
conjugation
conjugation
GUDCA
s
TUDCA
s
conjugation
conjugation
TLCA
s
s
MCA
p
conjugation
conjugation
conjugation
CA
p
GCA
p
TCA
p
DCA
s
conjugation
conjugation
TDCA
s
gut microorganisms convert
primary to secondary bile acids
b) Seconda
0.1
0.08
0.06
0.04
0.02
0
a) Bile acid metabolism
s
p
p
s
primary bile acid CDCA
secondary bile acid CDCA
primary bile acid CA
secondary bile acid CA
GDCA
s
Relative abundance of the
predicted genes %
s
GLCA
s
LCA
HCA
s
THDCA
s
HDCA
s
UDCA a) Bile acid metabolism c) Associations with microbial phylotypes and bile acids c) Associations with microbial phylotypes and bile acids
s c) Associations with microbial phylotypes and bile acids
Blautia
Dorea
Corynebacterium
Eggerthella
Ruminococcus
Holdemania
Coprobacillus
Anaerotruncus
Faecalibacterium
Megasphaera
Lachnospira
Christensenella
Akkermansia
Fusobacterium
Streptococcus
Veillonella
Bilophila
Enterococcus
Serratia
Negative correlations
Positive correlations
s
LCA
TDCA
s
s
HDCA
CA
p
s
GLCA
s
THDCA
s
UDCA
TDCA
s
GCDCA
p
TLCA
s
TCA
p
GDCA
s
GCA
p
TCDCA
p
s
LCA
TCDCA
p
GCA
p
GDCA
s
TCA
p
TLCA
s
GCDCA
p
TDCA
s
s
UDCA
s
THDCA
s
GLCA
CA
p
s
HDCA
TDCA
s Negative correlations Negative correlations Positive correlations Fig. 6
Bile acid–fecal microbiota interactions. a Bile acid transformation reactions. Orange color = primary bile acids, green = primary
conjugated bile acids, blue = secondary bile acids, pink = secondary conjugated bile acids. CA cholic acid, DCA deoxycholic acid, GDCA
glycodeoxycholic acid, TDCA taurodeoxycholic acid, GCA glycocholic acid, TCA taurocholic acid, CDCA chenodeoxycholic acid, LCA lithocholic
acid, HCA hyacholic acid, HDCA hyodeoxycholic acid, UDCA ursodeoxycholic acid, GUDCA glycoursodeoxycholic
acid, TUDCA
tauroursodeoxycholic acid, THDCA taurohydroxydeoxycholic acid, GLCA glycolithocholic acid, TLCA taurolithocholic acid, GCDCA
glycochenodeoxycholic acid, TCDCA taurochenodeoxycholic acid, αMCA α-muricholic acid, βMCA β-muricholic acid. b Bile acid biosynthesis
genes predicted from 16S rRNA gene abundances via PICRUSt. KO numbers that were used in the prediction: K01442, K00076, K23231,
K22604, K22605, K22606, K22607, K15868, K15871, K15869, K15870, K15873, K15874, and K07007. The box plots represent minimum,
maximum, median, first quartile, and third quartile values. npj Biofilms and Microbiomes (2020) 12 RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers Christensenella, a strongly heritable phylotype that was also
associated with lean body type55, was the only genus-level
phylotype that negatively correlated with the secondary bile acids
THDCA and UDCA. Previously, UDCA treatment have been
associated with weight gain in humans56. On the other hand,
Ruminococcus, Coprobacillus, Holdemania, Eggerthella, and Dorea
positively correlated with primary and secondary bile acids. Previous studies in humans reported increased levels of
circulating bile acids, especially secondary bile acids, after RYGB npj Biofilms and Microbiomes (2020) 12 Published in partnership with Nanyang Technological University Z.E. Ilhan et al. 10 as measured in blood plasma25,51,58. A recent study characterizing
bile acids in the fecal samples in women after RYGB showed
decreased concentrations of many bile acids59; hence, our results
support findings from that study. In rats, RYGB has also been
shown to increase plasma bile acid concentrations and the
secretion of weight-loss-associated hormones Peptide YY and
Glucagon Like Peptide-1 (ref. 49). However, a recent study on rats
demonstrated that the bile acid profiles in the intestines did not
change after RYGB even though microbial profiles were signifi-
cantly altered60. One difference among the reported human
studies and ours is that our measurements were in fecal samples,
whereas the others analyzed serum samples; hence, the measure-
ments are not directly comparable. Bile acid quantification is often
done in serum samples, which might reflect more physiologically
relevant concentrations. However, our findings in fecal samples
may lead to more profound understanding of microbial metabo-
lism of bile acids in the gut. Further studies on the impact of
microbial metabolism and gut levels of bile acids on host health
are warranted. Microbiome project using V4 primers with Illumina Miseq Instrument62. PANDAseq63 paired reads were analyzed using QIIME 1.9 suite34. The
details of the analysis can be found in the Supplementary Document. Briefly, OTUs were formed at 99% sequence similarity and the OTUs that
contained less than 0.005% of the total number of sequences and chimeric
sequences were omitted from the analysis as previously recommended64. We calculated alpha and beta diversity metrics of Phylogenetic Diversity
Whole Tree65, and Unifrac33. Gene abundances for bile acid biosynthesis
were predicted with Phylogenetic Investigation of Communities by
Reconstruction of Unobserved Species (PICRUSt) software53. Genus-level
phylotypes that significantly differed after RYGB were clustered based on
Euclidean distances using ClustVis66. Microbiome project using V4 primers with Illumina Miseq Instrument62. PANDAseq63 paired reads were analyzed using QIIME 1.9 suite34. Published in partnership with Nanyang Technological University Study design For the longitudinal cohort, we recruited 10 morbidly obese participants
who were scheduled to undergo RYGB surgery (pre-RYGB) and 10 normal
weight controls. The demographics of the study participants are included
in Table 1. Considering that RYGB cohorts are often composed of female
participants61, our study presents a more balanced distribution of genders
(see Table 1). In order to confirm results of cross-sectional studies with this
longitudinal study, we included 24 participants (RYGB-CS) who had
undergone RYGB surgery 13–60 months before the sample collection and
had lost at least 50% of their excess weight. Therefore, the CS population
represents long-term outcomes of RYGB surgery on gut microbiome and
metabolome. The demographics of this cross-sectional population can be
found in a previous publication6. Fecal samples collected at the specified
time points (Fig. 1a) were stored at −80 °C within 4 h of production until
analyzed. Three participants did not provide fecal samples at 6 months and
one did not provide a sample at 12 months. Distal sigmoid colon (25 cm
from the anal verge) biopsies were collected during non-sedated flexible
sigmoidoscopy following administration of a cleansing enema from 10 NW
participants and 9 prospective RYGB participants before and 12 months
after the surgery at Mayo Clinic, Scottsdale, Arizona, USA. The samples
were instantly washed and submerged in liquid nitrogen until frozen and
were kept at −80 °C until analysis. All participants filled out 4-day food
diaries and food-frequency questionnaires (within 2 weeks prior to sample
collection) with assistance of a dietitian and DietOrganizer software
(dietorganizer.com) was used to analyze the dietary composition. LC-MS analysis of fecal bile acids Fifty microliters of internal standard mixture (1.0 µg/mL) were spiked into
5 mg of lyophilized fecal samples and processed as described in the
Supplementary document. Homogenized samples were centrifuged at
13,600 × g for 20 min and the supernatants were filtered using Acrodisc
45 µm syringe-filters. Samples were cleaned-up using a 60 mg Oasis HLB
3cc cartridge (Waters Corporation, Milford, MA), dried in vacuo, and stored
at −70 °C until analysis. The extracts were analyzed with a Waters nano-
Acquity UPLC system (Waters Corporation, Milford, MA). MS analysis was
performed using an Agilent model 6490 triple quadrupole mass spectro-
meter (Agilent Technologies, Santa Clara, CA) outfitted with an in-house
nano-electrospray ionization interface. The sample preparation and bile
acid quantification procedures were based on the method of Humbert
et al.67, with modifications described in the Supplementary Document. 1H-NMR analysis of water-soluble fecal metabolites y
For each fecal specimen, approximately 1 g of wet weight was diluted with
20 mL of milliQ water and homogenized by vortexing for 3 min. The
homogenates were centrifuged at 16,110 × g for 15 min, and the super-
natants were filtered through 0.2-μm PVDF membranes (PALL Corpora-
tion). The fecal extracts were diluted with a 10% (v/v) spike of a National
Institute of Standards and Technology calibrated reference solution. The
resulting mixture was loaded into 3-mm NMR tubes (Bruker Inc), and NMR
spectra were collected using a Varian Direct Drive 600 MHz NMR
spectrometer equipped with a 5 mm triple-resonance salt-tolerant cold
probe. The 1D 1H NMR spectra of all samples were processed, assigned,
and analyzed by using Chenomx NMR Suite 8.1 with quantification of
metabolites based on spectral intensities relative to the internal standard
and as previously described6. We demonstrated the impact of RYGB surgery on the gut
microbiome, metabolome, and bile acid metabolism of humans
studied prospectively and retrospectively. We document that
changes in the human gut microbiome after RYGB in the luminal
and mucosal space. The mucosal space is a critically important site
for host–microbe interactions. Changes in the fecal metabolome
mirrored changes in the fecal and mucosal microbiome structure,
suggesting that the profile of microbial metabolism changed as a
result of major physiological, environmental, and nutritional
alterations affecting the gut after RYGB surgery. The delivery of
bile acids to the colon diminished after surgery, potentially
contributing to the altered microbiome and metabolome profiles. As a small sample size is a limitation of our study, studies with
greater sample size are needed to validate our findings. Finally,
results from a longitudinal cohort were consistent with observa-
tions from cross-sectional studies after RYGB surgery, supporting a
dominant and persistent impact of RYGB on the intestinal
microbiome. RYGB altered mucosal microbial communities increasing
Akkermansia sp. and lactate metabolizers The
details of the analysis can be found in the Supplementary Document. Briefly, OTUs were formed at 99% sequence similarity and the OTUs that
contained less than 0.005% of the total number of sequences and chimeric
sequences were omitted from the analysis as previously recommended64. We calculated alpha and beta diversity metrics of Phylogenetic Diversity
Whole Tree65, and Unifrac33. Gene abundances for bile acid biosynthesis
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methanol with sonication. Extracted metabolites were completely dried in
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analyzed by GC-MS as reported previously68. GC-MS raw data files were
processed using the Metabolite Detector software, version 2.5 beta69. All
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adaptation, distribution and reproduction in any medium or format, as long as you give
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metabolism in normal and obese diabetic rats. PLoS ONE 10, 1–17 (2015). AUTHOR CONTRIBUTIONS R.K.-B., J.K.D., B.E.R. designed the study. Z.E.I. extracted the samples for sequencing
and for the NMR analysis. S.E.D., N.G.I., and D.W.H. collected and analyzed 1H-NMR
data. A.A.S., H.M.B., K.K.W., Y.-M.K., and T.O.M. prepared and analyzed GC-MS and LC-
MS data. Z.E.I. wrote the manuscript. All authors read, edited, and approved the final
manuscript. 57. Flynn, C. R. et al. Bile diversion to the distal small intestine has comparable
metabolic benefits to bariatric surgery. Nat. Commun. 6, 7715 (2015). 58. Sayin, S. I. et al. Gut microbiota regulates bile acid metabolism by reducing the
levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist. Cell
Metab. 17, 225–235 (2013). 59. de Siqueira Cardinelli, C. et al. Fecal bile acid profile after Roux-en-Y gastric
bypass and its association with the remission of type 2 diabetes in obese women:
a preliminary study. Clin. Nutr. 38, 2906–2912 (2019). ACKNOWLEDGEMENTS Langille, M. G. I. et al. Predictive functional profiling of micr 54. Ridlon, J. M., Kang, D. J. & Hylemon, P. B. Bile salt biotransformations by human
intestinal bacteria. J. Lipid Res. 47, 241–259 (2006). 55. Goodrich, J. K. et al. Human genetics shape the gut microbiome. Cell 159,
789–799 (2014). 56. Siegel, J. L., Jorgensen, R., Angulo, P. & Lindor, K. Treatment with ursodeoxycholic
acid is associated with weight gain in patients with primary biliary cirrhosis. J. Clin. Gastroenterol. 37, 183–185 (2003). © The Author(s) 2020 ACKNOWLEDGEMENTS 50. Patti, M. E. et al. Serum bile acids are higher in humans with prior gastric bypass:
Potential contribution to improved glucose and lipid metabolism. Obesity 17,
1671–1677 (2009). We would like to thank all study participants and clinical research staff at Mayo Clinic. Research reported in this publication was supported by the National Institute of
Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under
Award Number R01DK090379. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the National Institutes of
Health. Additional support for mass spectrometry-based metabolomics analyses was
provided by the Laboratory Directed Research and Development program at Pacific
Northwest National Laboratory (PNNL) via the Signature Discovery Initiative. Metabolomics measurements were performed in the Environmental Molecular
Sciences Laboratory (grid.436923.9), a DOE Office of Science User Facility sponsored
by the Office of Biological and Environmental Research and located at PNNL in
Richland, Washington. PNNL is a multi-program national laboratory operated by
Battelle for the DOE under Contract DOE under Contract DE-AC05-76RLO 1830. We would like to thank all study participants and clinical research staff at Mayo Clinic. Research reported in this publication was supported by the National Institute of
Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under
Award Number R01DK090379. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the National Institutes of
Health. Additional support for mass spectrometry-based metabolomics analyses was
provided by the Laboratory Directed Research and Development program at Pacific
Northwest National Laboratory (PNNL) via the Signature Discovery Initiative. Metabolomics measurements were performed in the Environmental Molecular
Sciences Laboratory (grid.436923.9), a DOE Office of Science User Facility sponsored
by the Office of Biological and Environmental Research and located at PNNL in
Richland, Washington. PNNL is a multi-program national laboratory operated by
Battelle for the DOE under Contract DOE under Contract DE-AC05-76RLO 1830. 51. Pournaras, D. J. et al. The role of bile after Roux-en-Y gastric bypass in promoting
weight loss and improving glycaemic control. Endocrinology 153, 3613–3619
(2012). 52. Ridlon, J. M., Kang, D. J., Hylemon, P. B. & Bajaj, J. S. Bile acids and the gut
microbiome. Curr. Opin. Gastroenterol. 30, 332–338 (2014). 53. Langille, M. G. I. et al. Predictive functional profiling of microbial communities
using 16S rRNA marker gene sequences. Nat. Biotechnol. 31, 814–821 (2013). 53. ADDITIONAL INFORMATION Chem. 81, 3429–3439 (2009). 70. R Development Core Team, R. R: A Language and Environment for Statistical
Computing. R Foundation for Statistical Computing vol. 1 (R Foundation for Sta-
tistical Computing, 2011). 71. Y. Benjamini, Y. H. Controlling the false discovery rate: a practical and powerful
approach to multiple testing author (s): Yoav Benjamini and Yosef Hochberg
Source: Journal of the Royal Statistical Society. Series B (Methodological), Vol. 57,
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English
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A Single Lys Residue on the First Intracellular Loop Modulates the Endoplasmic Reticulum Export and Cell-Surface Expression of α2A-Adrenergic Receptor
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PloS one
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Abstract Export from the endoplasmic reticulum (ER) represents an initial step in intracellular trafficking of G protein-coupled
receptors (GPCRs). However, the underlying molecular mechanisms remain poorly understood. We have previously
demonstrated that a highly conserved Leu residue on the first intracellular loop (ICL1) is required for exit of several GPCRs
from the ER. Here we found that, in addition to Leu64 residue in the ICL1, the neighboring positively charged residue
Lys65also modulates the cell-surface transport of a2A-adrenergic receptor (a2A-AR). Mutation of Lys65 to Ala, Glu and Gln
significantly attenuated, whereas mutation of Lys65 to Arg strongly augmented a2A-AR expression at the cell surface. Consistent with the effects on the cell-surface expression of a2A-AR, mutation of Lys65 to Ala and Arg produced opposing
effects on a2A-AR-mediated ERK1/2 activation. Furthermore, confocal microscopy revealed that the a2A-AR mutant K65A
displayed a strong intracellular expression pattern and was extensively co-localized with the ER marker DsRed2-ER,
suggestive of ER accumulation. These data provide the first evidence indicating an important function for a single Lys
residue on the ICL1 in the ER export and cell-surface expression of a2A-AR. These data also suggest that the ICL1 may
possess multiple signals that control the cell-surface targeting of GPCRs via distinct mechanisms. Editor: Michael F. Olson, Beatson Institute for Cancer Research Glasgow, United Kingdom Editor: Michael F. Olson, Beatson Institute for Cancer Research Glasgow, United Kingdom Received July 19, 2012; Accepted October 19, 2012; Published December 5, 2012 Copyright: 2012 Fan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health grant R01GM076167. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by the National Institutes of Health grant R01GM076167. The funders had no role in study
analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: guwu@georgiahealth.edu exit from the ER. Incompletely or misfolded receptors are
excluded from ER-derived transport vesicles by the ER quality
control mechanism [15–17]. A Single Lys Residue on the First Intracellular Loop
Modulates the Endoplasmic Reticulum Export and Cell-
Surface Expression of a2A-Adrenergic Receptor Yi Fan1, Chunman Li1, Jianhui Guo1, Gang Hu2, Guangyu Wu1*
1 Department of Pharmacology and Toxicology, Georgia Health Sciences University, Augusta, Georgia, United States of America, 2 Department of Pharmacology, Nanjing
Medical University, Nanjing, China Yi Fan1, Chunman Li1, Jianhui Guo1, Gang Hu2, Guangyu Wu1*
1 Department of Pharmacology and Toxicology, Georgia Health Sciences University, Augusta, Georgia, United States of America, 2 Department of Pharmacology, Nanjing
Medical University, Nanjing, China Abstract It is also clear that GPCR export
from the ER is modulated by direct interactions with a multitude
of regulatory proteins such as ER chaperones and receptor activity
modifying proteins (RAMPs), which may stabilize receptor
conformation, facilitate receptor maturation and promote receptor
delivery to the plasma membrane [18–23]. More interestingly,
a number of highly conserved, specific sequences or motifs
embedded within the receptors have recently been indentified to
dictate receptor export from the ER [24–33]. Although the
molecular mechanisms underlying the function of these motifs
remain elusive, they may modulate proper receptor folding in the
ER or receptor interaction with specific components of transport
machinery [5,15,34,35]. December 2012 | Volume 7 | Issue 12 | e50416 Introduction G protein-coupled receptors (GPCRs) constitute the largest
superfamily of cell surface receptors and regulate the cellular
responses to a broad spectrum of extracellular signals, such as
hormones, neurotransmitters, chemokines, proteinases, odorants,
light and calcium ions [1–4]. All GPCRs share a common
molecular topology with a hydrophobic core of seven membrane-
spanning a-helices, three intracellular loops, three extracellular
loops, an N-terminus outside the cell, and a C-terminus inside
the cell. The proper function of GPCRs is largely determined by
the highly regulated intracellular trafficking of the receptors. GPCRs are synthesized in the ER and after proper folding and
correct assembly, they transport to the cell surface en route
through the Golgi apparatus and trans-Golgi network. As the
first step in post-translational biogenesis, the efficiency of ER
export of nascent GPCRs plays a crucial role in the regulation of
maturation, cell-surface expression, and physiological functions of
the receptors [5–8]. There are three a2-AR subtypes, designated as a2A-AR, a2B-
AR, and a2C-AR. It has been known that both a2A-AR and
a2B-AR mainly express at the cell surface, whereas a2C-AR cell-
surface expression depends on the cell types [36]. We have
identified several motifs, including the F(x)6LL motif in the C-
terminus, the RRR motif in the third intracellular loop (ICL3),
and an isolated Leu residue in the ICL1, which are essential for
export trafficking of a2B-AR [15,34,37–39]. In a continuing
effort to search for the structural determinants of a2-AR
transport, we expanded our studies to define the role of the Great progress has been made on the understanding of GPCR
export from the ER over the past decade [5,7]. However, the
underlying molecular mechanisms remain much less-well un-
derstood as compared with extensive studies on the events
involved in the endocytic and recycling pathways [9–14]. It has
been demonstrated that, similar to many other plasma membrane
proteins, GPCRs must first attain native conformation in order to December 2012 | Volume 7 | Issue 12 | e50416 1 PLOS ONE | www.plosone.org a2-AR Export and Cell-Surface Expression ICL1 in the cell-surface expression of a2A-AR. Surprisingly we
found that, in addition to Leu residue, a neighboring Lys
residue specifically modulates the ER export and cell-surface
expression of a2A-AR and this function is likely dictated by its
positively
charged
property. These
data
provide
the
first
evidence indicating that the ICL1 may possess multiple signals
that use distinct mechanisms to control the processing of a2A-
AR. Intact Cell Ligand Binding Cell-surface expression of a2A-AR and a2B-AR in HEK293
cells was measured by ligand binding of intact live cells using
the membrane impermeable ligands [3H]-RX821002 as de-
scribed previously [41,45]. Briefly, HEK293 cells cultured on 6-
well dishes were transiently transfected with 1 mg of plasmids. After 6 h the cells were split into 24-well dishes pre-coated with
poly-L-lysine. Forty-eight h post-transfection, the cells were
incubated with DMEM plus [3H]-RX821002 in a total of
300 ml
for
60 min
at
room
temperature. In
the
initial
experiments, the cells were incubated with increasing concen-
trations of [3H]-RX821002 (from 0.3125 to 20 nM) to generate
ligand dose-dependent binding curves. Because ligand binding
to the receptors is almost saturated at a concentration of
20 nM, this concentration was then used to measure the cell-
surface expression of the receptors. The binding was terminated
and excess radioligand eliminated by washing the cells twice
with ice-cold DMEM. The retained radioligand was extracted
by
digesting
the
cells
in
1 M NaOH
for
2 h
at
room
temperature. The liquid phase was collected and suspended in Introduction 4 ml of Ecoscint A scintillation fluid (National Diagnostics Inc.,
Atlanta,
GA). The
amount
of
radioactivity
retained
was
measured by liquid scintillation spectrometry. The non-specific
binding
of
a2-AR
was
determined
in
the
presence
of
rauwolscine
(10 mM). All
radioligand
binding
assays
were
performed in triplicate. Materials Antibodies against ERK1/2 and phospho-ERK1/2 were from
Cell Signaling Technology (Beverly, MA). The ER marker
pDsRed2-ER was purchased from BD Biosciences (Palo Alto,
LA). Prolong antifade reagent with DAPI was obtained from
Invitrogen Life Technologies (Carlsbad, CA). UK14,304 was from
Sigma (St. Louis, MO). [3H]-RX821002 (specific activity = 50 Ci/
mmol) was from Perkin Elmer Life Sciences. All other materials
were obtained as described previously [38,40,41]. Flow Cytometry For measurement of total receptor expression, HEK293 cells
cultured on 6-well dishes were transiently transfected with 1 mg
of GFP-tagged receptors for 24 h. The cells were collected,
washed twice with PBS and re-suspended at a density of
86106 cells/ml. Total GFP fluorescence was then measured on
a flow cytometer (BD Biosciences FASCalibur) as described
previously (38). For measurement of the cell-surface expression
of a2A-AR, HEK293 cells were cultured on 6-well dishes and
transfected with 1 mg of HA-tagged a2A-AR for 24 h. The cells
were then collected, suspended in PBS containing 1% FBS and
incubated with high affinity anti-HA-fluorescein (3F10) at a final
concentration of 2 mg/ml at 4uC for 60 min. After washing for
260.5 ml PBS containing 1% FBS, the cells were re-suspended
and the fluorescence was analyzed as described above. Measurement of ERK1/2 Activation HEK293 cells were cultured in 6-well dishes and transfected
with 1 mg of a2A-AR or its mutants as described above. After 24 h
transfection, the cells were starved for at least 3 h and then
stimulated with different concentrations of UK14,304 (0.01, 0.1
and 1 mM) for 5 min. Stimulation was terminated by addition of
150 ml of ice-cold cell lysis buffer containing 50 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium
orthovanadate and protease inhibitor cocktail (Roche). After
solubilizing the cells on ice for 20 min, 10 ml of total cell lysates
was separated by 12% SDS-PAGE and ERK1/2 activation was
determined by measuring the levels of phosphorylation of ERK1/
2 with phospho-specific ERK1/2 antibodies by immunoblotting
[47,48]. Cell Culture and Transient Transfection HEK293 and HeLa cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS),
100 units/ml penicillin, and 100 units/ml streptomycin and
transiently transfected by using Lipofectamine 2000 reagent as
described previously [44]. Transfection efficiency was estimated to
be greater than 70% based on the GFP fluorescence. Plasmid Constructions Rat a2B-AR in vector pcDNA3 was kindly provided by Dr. Stephen M. Lanier (Medical University of South Carolina,
Charleston, SC). Human a2A-AR tagged with three HA at its
N-terminus was purchased from UMR cDNA Resource Center
(Rolla, MO). a2A-AR tagged with GFP at its C-terminus was
generated as described previously [40]. The GFP and HA
epitopes have been used to label GPCRs resulting in receptors
with similar characteristics to the wild-type receptors [40,42,43]. The mutations of a2A-AR and a2B-AR were created by using
the QuikChange site-directed mutagenesis kit (Stratagene, La
Jolla, CA). The sequence of each construct used in this study
was verified by restriction mapping and nucleotide sequence
analysis. Confocal Fluorescence Microscopy For fluorescence microscopic analysis of receptor subcellular
distribution, HEK293 and HeLa cells were grown on coverslips
pre-coated with poly-L-lysine in 6-well plates and transfected with
100 ng of GFP-tagged receptors. For co-localization of GFP-
tagged receptors with the ER marker DsRed2-ER, HEK293 cells
grown on coverslips were transfected with 100 ng of GFP-tagged
receptors and 100 ng of pDsRed2-ER. The cells were fixed with
4% paraformaldehyde-4% sucrose mixture in PBS for 15 min and
the coverslips were mounted with prolong antifade reagent
containing DAPI. Images were captured using a Zessis confocal
microscope (LSM510) equipped with a 63x objective (NA = 1.3). The colocalization of the receptor with the ER marker DsRed2-
ER was determined by Pearson’s coefficient using the ImageJ
JaCoP plug-in as described [46]. Results Differential Inhibition of the Cell-surface Expression of
a2A-AR and a2B-AR by Mutation of Leu and a Positively
Charged Residue on the ICL1 g
The amino acid sequences of the ICL1 are highly conserved
in each a2-AR subtype among different species but different
between three a2-AR subtypes. The ICL1 of a2A-AR, a2B-AR
and a2C-AR have the sequence of ALK, SLR and ALR,
respectively (Fig. 1A). We have previously demonstrated that
Leu48 residue, but not Arg49 residue, in the ICL1 is essential
for the ER export and cell-surface transport of a2B-AR [38]. Here we determined the effect of mutating Leu64 and Lys65 on
the cell-surface number of a2A-AR. We first measured the
saturation binding of the radioligand [3H]-RX821002 to a2A-
AR in intact live HEK293 cells. The ligand dose-dependently
bound to a2A-AR and the binding was close to saturation at
20 nM (Fig. 1B). Wild-type a2A-AR and its mutants L64A, K65A and LK-AA
were transiently expressed in HEK293 cells and their cell-surface
expression at steady state was measured by intact cell ligand
binding using [3H]-RX821002 at 20 nM. Consistent with the
remarkable inhibitory effect of mutation of Leu48 on a2B-AR cell-
surface expression, mutation of Leu64 markedly reduced the cell-
surface number of a2A-AR by 87%. Surprisingly, in contrast to
mutation of Arg49 which did not have significant effects on a2B-
AR cell-surface expression, mutation of Lys65 to Ala significantly
attenuated a2A-AR expression at the cell surface by 52%. Double
mutation of Leu48/Arg49 in a2B-AR and Leu64/Lys65 a2A-AR
almost abolished their cell-surface transport (Fig. 1C and 1D). To
exclude the possibility that these mutations could influence a2A-
AR binding to the ligand, a2A-AR and its mutants were tagged
with HA at their N-termini and their cell-surface expression was
measured by flow cytometry following staining with anti-HA
antibodies in nonpermeabilized cells. The cell-surface expression
of the mutants L64A, K65A and LK-AA was reduced by 81, 58
and 93%, respectively, as compared with their wild-type counter-
part (Fig. 1E). To
determine
if
these
mutations
could
alter
the
total
expression of the receptors, a2A-AR and a2B-AR and their
mutants tagged with GFP at their C-termini were transiently
expressed in HEK293 cells and their overall expression was
determined by flow cytometry measuring the GFP signal. In
contrast to the cell-surface expression, these mutations did not
significantly alter the overall expression of a2A-AR (Fig. 1C) and
a2B-AR (Fig. 1D). Results These data, together our previous data [38],
demonstrate that the single Leu residue in the ICL1 plays
a general role in the cell-surface transport of GPCRs, whereas
its neighboring positively charged residue may differentially
regulate the cell-surface targeting of a2A-AR and a2B-AR. Figure 1. Effects of the mutation of Leu residues and their
neighboring positively charged residues in the ICL1 on the
cell-surface and total expression of a2A-AR and a2B-AR. (A) The
sequence of the ICL1 of a2A-AR, a2B-AR and a2C-AR. (B) Ligand dose-
dependent binding of a2A-AR in intact HEK293 cells. HEK293 cells
cultured on 6-well plates were transfected with a2A-AR and then split
onto 24-well plates. The cells were incubated with increasing
concentrations of [3H]-RX821002 (0.3125, 0.625, 1.25, 2.5, 5, 10, and
20 nM) and the ligand bound to the receptor was measured by liquid
scintillation spectrometry as described in the ‘‘Materials and Methods’’. The nonspecific binding was determined in the presence of rauwolscine
(10 mM). Similar results were obtained in at least three different
experiments. (C) Quantification of the cell-surface and total expression
of a2A-AR and its mutants in which Leu64 and Lys65 were mutated to
Ala individually or in combination. (D) Quantification of the cell-surface
and total expression of a2B-AR and its mutants in which Leu48 and
Arg49 were mutated to Ala. In (C) and (D), HEK293 cells were
transfected with a2-AR and the cell-surface expression of the receptors
was measured by intact cell binding assays using [3H]-RX821002 at
20 nM. The mean values of specific [3H]-RX821002 binding were Figure 1. Effects of the mutation of Leu residues and their
neighboring positively charged residues in the ICL1 on the
cell-surface and total expression of a2A-AR and a2B-AR. (A) The
sequence of the ICL1 of a2A-AR, a2B-AR and a2C-AR. (B) Ligand dose-
dependent binding of a2A-AR in intact HEK293 cells. HEK293 cells
cultured on 6-well plates were transfected with a2A-AR and then split
onto 24-well plates. The cells were incubated with increasing
concentrations of [3H]-RX821002 (0.3125, 0.625, 1.25, 2.5, 5, 10, and
20 nM) and the ligand bound to the receptor was measured by liquid
scintillation spectrometry as described in the ‘‘Materials and Methods’’. The nonspecific binding was determined in the presence of rauwolscine
(10 mM). Similar results were obtained in at least three different
experiments. Results (C) Quantification of the cell-surface and total expression
of a2A-AR and its mutants in which Leu64 and Lys65 were mutated to
Ala individually or in combination. (D) Quantification of the cell-surface
and total expression of a2B-AR and its mutants in which Leu48 and
Arg49 were mutated to Ala. In (C) and (D), HEK293 cells were
transfected with a2-AR and the cell-surface expression of the receptors
was measured by intact cell binding assays using [3H]-RX821002 at
20 nM. The mean values of specific [3H]-RX821002 binding were Statistical Analysis Differences were evaluated using one-way ANOVA and post-
hoc Tukey’s test, and p,0.05 was considered as statistically
significant. Data are expressed as the mean 6 S.E. December 2012 | Volume 7 | Issue 12 | e50416 PLOS ONE | www.plosone.org 2 a2-AR Export and Cell-Surface Expression Figure 1. Effects of the mutation of Leu residues and thei
neighboring positively charged residues in the ICL1 on the
cell-surface and total expression of a2A-AR and a2B-AR. (A) The
sequence of the ICL1 of a2A-AR, a2B-AR and a2C-AR. (B) Ligand dose
dependent binding of a2A-AR in intact HEK293 cells. HEK293 cell
cultured on 6-well plates were transfected with a2A-AR and then spli
onto 24-well plates. The cells were incubated with increasing
concentrations of [3H]-RX821002 (0.3125, 0.625, 1.25, 2.5, 5, 10, and
20 nM) and the ligand bound to the receptor was measured by liquid
scintillation spectrometry as described in the ‘‘Materials and Methods’
The nonspecific binding was determined in the presence of rauwolscine
(10 mM). Similar results were obtained in at least three differen
experiments. (C) Quantification of the cell-surface and total expression
of a2A-AR and its mutants in which Leu64 and Lys65 were mutated to
Ala individually or in combination. (D) Quantification of the cell-surface Functional Regulation of a2A-AR by Mutation of Lys65 Functional Regulation of a2A-AR by Mutation of Lys65
We then sought to determine if the mutation of Lys65 could
alter the function of a2A-AR by using the agonist-mediated
activation
of
ERK/12
as
readout. HEK293
cells
were
transiently transfected with a2A-AR and its mutants K65R
and K65A and their abilities to activate ERK1/2 in response to
stimulation with the a2-AR agonist UK14,304 were compared. ERK1/2 were activated by UK14,304 in a dose-dependent
fashion in cells expressing a2A-AR and the dose-dependent
activation of ERK1/2 was clearly inhibited in cells transfected
with the mutant K65A (Fig. 4A and 4B). In contrast, ERK1/2
activation
by
UK14,304
was
significantly
higher
in
cells
expressing the mutant K65R than in cells expressing wild-type
a2A-AR (Fig. 4A and 4B). These data are consistent with the
opposing effects of Lys65 mutation to Ala and Arg on the cell-
surface expression of a2A-AR. These data also suggest that
Lys65 in the ICL1 modulates not only receptor trafficking but
also receptor signaling. to the results obtained in HEK293 cells, the a2A-AR mutants
L64A, K65A and LK-AA displayed
a strong intracellular
distribution pattern in HeLa cells (Fig. 2). These data suggest that
both Leu64 and Lys65 are able to regulate a2A-AR cell-surface
expression in different cell types. to the results obtained in HEK293 cells, the a2A-AR mutants
L64A, K65A and LK-AA displayed
a strong intracellular
distribution pattern in HeLa cells (Fig. 2). These data suggest that
both Leu64 and Lys65 are able to regulate a2A-AR cell-surface
expression in different cell types. Effect of Mutation of Lys65 to Arg, Glu and Gln on the
Cell-surface Expression and Subcellular Distribution of a2A-AR Our preceding data have revealed that Lys65 plays an
important role in modulating a2A-AR cell-surface expression. To
define the possible underlying molecular mechanisms, we first
determined the role of its positively charged property. Lys65 was
mutated to the same charged Arg residue, opposite charged Glu
residue and non-charged Gln residue and the effects of these
mutations on a2A-AR expression at the cell surface were defined
by intact cell ligand binding and subcellular distribution analysis. Similar to its Ala mutation, mutation of Lys65 to Glu and Gln
inhibited a2A-AR expression at the cell surface by 67 and 36%,
respectively, as measured by intact cell ligand binding (Fig. 3A). More interestingly, mutation of Lys65 to Arg significantly
augmented the cell-surface expression of a2A-AR by 42%
(Fig. 3A). Similar results were obtained by flow cytometry to Lys65 Likely Modulates a2A-AR Transport at the ER To define the intracellular compartment where the residue
Lys65 influences a2A-AR transport, GFP-tagged a2A-AR and its
mutants K65A and K65R were co-localized with different
intracellular markers. The mutant K65A was extensively co-
localized with the ER marker DsRed2-ER (Fig. 5A), but not the
Golgi marker GM130 (data not shown). In contrast, wild-type a2A-
AR and its mutant K65R did not clearly co-localize with DeRed2- Intracellular Accumulation of a2A-AR Induced by
Mutation of Leu64 and Lys65 To further confirm the inhibitory effect of mutation of Leu64
and Lys65 on the cell-surface transport of a2A-AR, the subcellular
distribution of GFP-tagged a2A-AR and its mutants in HEK293
cells was visualized by confocal microscopy. As expected, wild-type
a2A-AR was robustly expressed at the cell surface. Mutation of
Leu64 and Lys65 to Ala individually or in combination caused
a remarkable accumulation of a2A-AR in the perinuclear region
(Fig. 2). To determine if the effect of the mutations on the subcellular
distribution of a2A-AR is cell-type specific, GFP-tagged a2A-AR
and its mutants were transiently expressed in HeLa cells. Similar December 2012 | Volume 7 | Issue 12 | e50416 PLOS ONE | www.plosone.org 3 a2-AR Export and Cell-Surface Expression measure the cell-surface expression of HA-tagged a2A-AR and its
mutants K65R, K65E and K65Q (Fig. 3B). measure the cell-surface expression of HA-tagged a2A-AR and its
mutants K65R, K65E and K65Q (Fig. 3B). 272556415 and 167856452 cpm from cells transfected with wild-type
(WT) a2A-AR and a2B-AR, respectively. Wild-type a2A-AR and a2B-AR and
their mutants were tagged with GFP and transiently expressed in
HEK293 cells. Total receptor expression was determined by flow
cytometry measuring the GFP signal as described in the ‘‘Materials
and Methods’’. (E) Quantification of the cell-surface expression of a2A-
AR and its mutants by flow cytometry. HEK293 cells were transfected
with HA-tagged a2A-AR or its individual mutant and the cell-surface
expression of the receptors was measured by flow cytometry following
staining with anti-HA antibodies in nonpermeabilized cells as described
in the ‘‘Materials and Methods’’. The data shown in (C), (D), and (E) are
percentages of the mean value obtained from cells transfected with
wild-type a2-AR and are presented as the mean 6 S.E. of at least three
different experiments. *, p,0.05 versus respective WT a2-AR. doi:10.1371/journal.pone.0050416.g001 Consistent with the measurement of the cell-surface expression
by ligand binding and flow cytometry, analysis of the subcellular
distribution of the receptors showed that wild-type a2A-AR and its
mutant K65R strongly expressed at the cell surface, whereas the
mutants K65E and K65Q were largely accumulated inside the
cell, unable to transport to the cell surface in both HEK293 and
HeLa cells (Fig. 3C). Figure 2. Effects of mutating Leu64 and Lys65 residues on the subcellular distribution of a2A-AR. GFP-tagged wild-type (WT) a2A-AR and
its mutants L64A, K65A and LK-AA were transiently expressed in HEK293 (upper panel) and HeLa cells (lower panel) and their subcellular distribution of
the receptors was revealed by detecting GFP fluorescence by confocal microscopy. The data shown are representative images of at least three
independent experiments. Green, GFP-tagged receptors; blue, DNA staining by DAPI (nuclei). Scale bar, 10 mm. doi:10.1371/journal.pone.0050416.g002 Figure 2. Effects of mutating Leu64 and Lys65 residues on the subcellular distribution of a2A-AR. GFP-tagged wild-type (WT) a2A-AR and
its mutants L64A, K65A and LK-AA were transiently expressed in HEK293 (upper panel) and HeLa cells (lower panel) and their subcellular distribution of
the receptors was revealed by detecting GFP fluorescence by confocal microscopy. The data shown are representative images of at least three
independent experiments. Green, GFP-tagged receptors; blue, DNA staining by DAPI (nuclei). Scale bar, 10 mm. doi:10.1371/journal.pone.0050416.g002 December 2012 | Volume 7 | Issue 12 | e50416 PLOS ONE | www.plosone.org 4 a2-AR Export and Cell-Surface Expression ER (Fi
5A) T
if
h
l
li
i
f h
i h
h
h
id
L 65 i
h ICL1 lik l
l
h
i
Figure 3. Effects of mutating Lys65 to Arg, Glu and Gln on the cell-surface expression and subcellular distribution of a2A-AR. (A)
Quantification of the cell surface and total expression of a2A-AR and its Lys mutants. HEK293 cells were transfected with a2A-AR and its mutants. The
cell-surface expression of the receptors was measured by intact cell binding assays using [3H]-RX821002 and total receptor expression by flow
cytometry measuring the GFP signal as described in the legends of figure 1. (B) Quantification of the cell-surface expression of a2A-AR and its mutants
by flow cytometry following staining with anti-HA antibodies in nonpermeabilized cells as described in the legends of figure 1. The data shown in (A)
and (B) are percentages of the mean value obtained from cells transfected with wild-type (WT) a2A-AR and are presented as the mean 6 S.E. of four
experiments. *, p,0.05 versus WT a2A-AR. (C) Effect of mutation of Lys65 on the subcellular distribution of a2A-AR. a2A-AR and its mutants K65R, K65E
and K65Q were tagged with GFP at their C-termini and transiently expressed in HEK293 (upper panel) and HeLa cells (lower panel). suggest that the residue Lys65 in the ICL1 likely controls the exit
of a2A-AR out of the ER. Their subcellular
distribution was revealed by detecting GFP fluorescence by confocal microscopy. The data shown are representative images of at least three
independent experiments. Green, GFP-tagged receptors; blue, DNA staining by DAPI (nuclei). Scale bar, 10 mm. doi:10.1371/journal.pone.0050416.g003 Figure 3. Effects of mutating Lys65 to Arg, Glu and Gln on the cell-surface expression and subcellular distribution of a2A-AR. (A)
Quantification of the cell surface and total expression of a2A-AR and its Lys mutants. HEK293 cells were transfected with a2A-AR and its mutants. The
cell-surface expression of the receptors was measured by intact cell binding assays using [3H]-RX821002 and total receptor expression by flow
cytometry measuring the GFP signal as described in the legends of figure 1. (B) Quantification of the cell-surface expression of a2A-AR and its mutants
by flow cytometry following staining with anti-HA antibodies in nonpermeabilized cells as described in the legends of figure 1. The data shown in (A)
and (B) are percentages of the mean value obtained from cells transfected with wild-type (WT) a2A-AR and are presented as the mean 6 S.E. of four
experiments. *, p,0.05 versus WT a2A-AR. (C) Effect of mutation of Lys65 on the subcellular distribution of a2A-AR. a2A-AR and its mutants K65R, K65E
and K65Q were tagged with GFP at their C-termini and transiently expressed in HEK293 (upper panel) and HeLa cells (lower panel). Their subcellular
distribution was revealed by detecting GFP fluorescence by confocal microscopy. The data shown are representative images of at least three
independent experiments. Green, GFP-tagged receptors; blue, DNA staining by DAPI (nuclei). Scale bar, 10 mm. doi:10.1371/journal.pone.0050416.g003 suggest that the residue Lys65 in the ICL1 likely controls the exit
of a2A-AR out of the ER. suggest that the residue Lys65 in the ICL1 likely controls the exit
of a2A-AR out of the ER. ER (Fig. 5A). To quantify the colocalization of the receptors with
the ER marker, Pearson’s coefficient was determined. Pearson’s
coefficient of the mutant K65A was significantly higher than those
of wild-type a2A-AR and the mutant K65R (Fig. 5B). These data December 2012 | Volume 7 | Issue 12 | e50416 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 5 a2-AR Export and Cell-Surface Expression Figure 4. Effects of mutating Lys65 to Ala and Arg on a2A-AR-mediated activation of ERK1/2. Discussion The molecular mechanisms underlying export from the ER and
subsequent transport to the cell surface of the GPCR superfamily
remain poorly elucidated. It has been significant efforts to define
the structural determinants for GPCR export and several highly
conserved hydrophobic and charged sequences which are required
for GPCR export from the ER to the cell surface have been
identified [5,15,18,24–34,37,38,49]. We have previously investi-
gated the role of the ICL1 in the cell-surface transport of GPCRs
and identified a Leu residue essential for ER export of a group of
GPCRs, including a2B-AR [38]. This Leu residue is extremely
conserved
amongst
the
family
A
GPCRs
and
indeed,
as
demonstrated in this manuscript, Leu64 in the ICL1 also plays
an obligatory role in a2A-AR export from the ER and transport to
the cell surface. Thus, this isolated Leu may provide a common
signal directing anterograde transport of multiple nascent GPCRs. The most important finding described in this manuscript is that
different positively charged residues on the ICL1 have differential
impacts on the cell-surface transport of distinct GPCRs. During
the studies on the function of the ICL1, we have surprisingly found
that mutation of Lys65 produced a marked inhibitory effect on the
cell-surface transport of a2A-AR. First, intact cell ligand binding
and flow cytometry to quantify the cell-surface receptors showed
that mutation of Lys65 to Ala reduced a2A-AR cell-surface
number by more than 50%. Second, the significant reduction of
receptor expression at the cell surface was supported by direct
visualization of intracellular localization of the mutated receptors. The molecular mechanisms underlying export from the ER and
subsequent transport to the cell surface of the GPCR superfamily
remain poorly elucidated. It has been significant efforts to define
the structural determinants for GPCR export and several highly
conserved hydrophobic and charged sequences which are required
for GPCR export from the ER to the cell surface have been
identified [5,15,18,24–34,37,38,49]. We have previously investi-
gated the role of the ICL1 in the cell-surface transport of GPCRs
and identified a Leu residue essential for ER export of a group of
GPCRs, including a2B-AR [38]. This Leu residue is extremely
conserved
amongst
the
family
A
GPCRs
and
indeed,
as
demonstrated in this manuscript, Leu64 in the ICL1 also plays
an obligatory role in a2A-AR export from the ER and transport to
the cell surface. (A) HEK293 cells were transfected with wild-
type (WT) a2A-AR or its mutants K65A and K65R and then stimulated with increasing concentrations of UK14,304 for 5 min. ERK1/2 activation was
determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel, a representative blot of ERK1/2 activation; Lower panel,
total ERK1/2 expression. (B) Quantitative data expressed as percentage of ERK1/2 activation obtained in cells transfected with a2A-AR and stimulated
with UK14304 at 1 mM and presented as the mean 6 S.E. of three separate experiments. *, p,0.05 versus WT a2A-AR at the same concentration of
UK14,304. doi:10.1371/journal.pone.0050416.g004 Figure 4. Effects of mutating Lys65 to Ala and Arg on a2A-AR-mediated activation of ERK1/2. (A) HEK293 cells were transfected with wild-
type (WT) a2A-AR or its mutants K65A and K65R and then stimulated with increasing concentrations of UK14,304 for 5 min. ERK1/2 activation was
determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel, a representative blot of ERK1/2 activation; Lower panel,
total ERK1/2 expression. (B) Quantitative data expressed as percentage of ERK1/2 activation obtained in cells transfected with a2A-AR and stimulated
with UK14304 at 1 mM and presented as the mean 6 S.E. of three separate experiments. *, p,0.05 versus WT a2A-AR at the same concentration of
UK14,304. doi:10.1371/journal.pone.0050416.g004 doi:10.1371/journal.pone.0050416.g004 As revealed by confocal microscopy, GFP-tagged K65A was
extensively expressed inside the cell which is in contrast to robust
cell-surface expression of its wild-type counterpart. Third, the
function of Lys65 in controlling a2A-AR transport is also
supported by receptor-mediated signal propagation as measured
by ERK1/2 activation. The reduction of ERK1/2 activation in
cells expressing K65A as compared with cells expressing wild-type
a2A-AR could be simply due to less expression of the mutated a2A-
AR at the cell surface, which are available for binding to the
ligand, indicating that mutation of Lys65 not only reduced the cell-
surface expression of a2A-AR but also attenuated receptor-
mediated signal propagation. These data strongly indicate that,
in addition to Leu64, Lys65 in the ICL1 also plays a crucial role in
regulating cell-surface transport of a2A-AR. By contrast, mutation
of the positively charged residue at the same position, Arg49, did
not influence the cell-surface transport of a2B-AR. These data
demonstrated for the first time that a single positively charged
residue on the short ICL1 is involved in the regulation of export
trafficking of GPCRs in a receptor subtype-specific fashion. December 2012 | Volume 7 | Issue 12 | e50416 Discussion Thus, this isolated Leu may provide a common
signal directing anterograde transport of multiple nascent GPCRs. The most important finding described in this manuscript is that
different positively charged residues on the ICL1 have differential
impacts on the cell-surface transport of distinct GPCRs. During
the studies on the function of the ICL1, we have surprisingly found
that mutation of Lys65 produced a marked inhibitory effect on the
cell-surface transport of a2A-AR. First, intact cell ligand binding
and flow cytometry to quantify the cell-surface receptors showed
that mutation of Lys65 to Ala reduced a2A-AR cell-surface
number by more than 50%. Second, the significant reduction of
receptor expression at the cell surface was supported by direct
visualization of intracellular localization of the mutated receptors. It is likely that Lys65 residue modulates a2A-AR transport at the
level of the ER. We found that the a2A-AR mutant K65A was
extensively
co-localized
with
the
ER
marker
DsRed2-ER,
suggesting that the mutant was unable to exit from the ER where
they are synthesized. Therefore, these studies provide another
novel regulatory mechanism for the ER export of nascent a2A-AR. As this single Lys residue also exists in several other group A
GPCRs, including angiotensin II, muscarinic and chemokine December 2012 | Volume 7 | Issue 12 | e50416 6 PLOS ONE | www.plosone.org a2-AR Export and Cell-Surface Expression Figure 5. Effect of mutation of Lys65 on the colocalization of a2A-AR with the ER marker DsRed2-ER. (A) HEK293 cells were transiently
transfected with the GFP-tagged a2A-AR or its Lys65 mutants together with pDsRed2-ER. The subcellular distribution and co-localization of the
receptors with the ER marker DsRed2-ER were revealed by confocal fluorescence microscopy as described under ‘‘Materials and Methods’’. Green, a2A
AR or its mutants tagged with GFP; red, DsRed2-ER; yellow, co-localization of a2A-AR or its mutants with the ER marker DsRed2-ER; blue, DNA staining
PLOS ONE | www.plosone.org
7
December 2012 | Volume 7 | Issue 12 | e50416 Figure 5. Effect of mutation of Lys65 on the colocalization of a2A-AR with the ER marker DsRed2-ER. (A) HEK293 cells were transiently
transfected with the GFP-tagged a2A-AR or its Lys65 mutants together with pDsRed2-ER. The subcellular distribution and co-localization of the
receptors with the ER marker DsRed2-ER were revealed by confocal fluorescence microscopy as described under ‘‘Materials and Methods’’. Discussion Green, a2A-
AR or its mutants tagged with GFP; red, DsRed2-ER; yellow, co-localization of a2A-AR or its mutants with the ER marker DsRed2-ER; blue, DNA staining December 2012 | Volume 7 | Issue 12 | e50416 PLOS ONE | www.plosone.org 7 7 a2-AR Export and Cell-Surface Expression by DAPI (nuclei). The data shown are representative images of at least three independent experiments. (B) Quantification of Pearson’s coefficient
between the receptors and the ER marker. The data are presented as the mean 6 S.E. of 20 cells from three different experiments. *, p,0.05 versus
WT a2A-AR. Scale bar, 10 mm. doi:10 1371/journal pone 0050416 g005 by DAPI (nuclei). The data shown are representative images of at least three independent experiments. (B) Quantification of Pearson’s coefficient
between the receptors and the ER marker. The data are presented as the mean 6 S.E. of 20 cells from three different experiments. *, p,0.05 versus
WT a2A-AR. Scale bar, 10 mm. doi:10.1371/journal.pone.0050416.g005 receptors [38] (data not shown), it may function as an important
code which not only directs the ER export but also controls the
cell-surface availability of these GPCRs. These data, together with
our previous studies identifying the F(x)6LL, RRR and YS motifs
[15,34,37,40], have strongly demonstrated that export trafficking
of a2-AR is coordinated by many structural determinants and
export motifs located in the intracellular domains of the receptors. receptors [38] (data not shown), it may function as an important
code which not only directs the ER export but also controls the
cell-surface availability of these GPCRs. These data, together with
our previous studies identifying the F(x)6LL, RRR and YS motifs
[15,34,37,40], have strongly demonstrated that export trafficking
of a2-AR is coordinated by many structural determinants and
export motifs located in the intracellular domains of the receptors. the side chain of Arg is larger than that of Lys, it may enhance the
possible ionic interactions between the positively charged side
chain and some negatively charged components embedded within
the plasma membrane. It is also possible that mutation of Lys to
Arg may reduce the targeting of the cell-surface a2A-AR to some
degradation pathways, such as those mediated by ubiquitination
and/or sumoylation which are carried out specifically on Lys
residues. However, whether or not Lys65 residue indeed under-
goes
ubiquitination
and/or
sumoylation
remains
unknown. References 12. Filipeanu CM, Zhou F, Lam ML, Kerut KE, Claycomb WC, et al. (2006)
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a2A-AR once transported to the cell surface. As the overall size of Second, more interestingly, mutation of Lys65 to the same
charged Arg residue enhanced the cell-surface number of a2A-AR. There are at least two possible explanations for the enhancement
of the cell-surface expression of a2A-AR induced by mutation of
Lys65 to Arg. It is possible that mutation of Lys to Arg stabilizes
a2A-AR once transported to the cell surface. As the overall size of Author Contributions Conceived and designed the experiments: GW YF. Performed the
experiments: YF CL JG. Analyzed the data: GW YF CL. Contributed
reagents/materials/analysis tools: YF GH. Wrote the paper: GW YF. Conceived and designed the experiments: GW YF. Performed the
experiments: YF CL JG. Analyzed the data: GW YF CL. Contributed
reagents/materials/analysis tools: YF GH. Wrote the paper: GW YF. Discussion Nevertheless, this study has demonstrated a crucial role for
Lys65 residue in regulating the ER export and cell-surface
expression of a2A-AR, yet the precise mechanisms of its actions
remain to be elucidated. p
p
The function of Lys65 residue in regulating the ER export and
cell-surface expression of a2A-AR is likely dictated by its specific
physiochemical and structural features. First, our data demon-
strating that mutation of Lys65 to Ala, Glu and Gln significantly
inhibited a2A-AR export from the ER and transport to the cell
surface suggest that the positively charged property is an important
factor determining the function of Lys65 in a2A-AR export. It is
possible that Lys65 plays a role in the correct folding or proper
assembly of a2A-AR in the ER and thus, its mutation results in the
misfolding and defective export of the receptor. It is also possible
that Lys65 may mediate a2A-AR interaction with other regulatory
proteins and such interactions are crucial for the receptor export
from the ER. Consistent with this possibility, a number of
accessory proteins directly interact with the intracellular loops of
GPCRs to modulate receptor export from the ER to the cell
surface [5]. This possibility is also supported by our recent studies
showing that the RRR motif in the ICL3 mediates a2B-AR
interaction with Sec24 isoforms, components of COPII transport
vesicles, to control receptor export from the ER [34]. It has become increasingly clear that the efficient trafficking and
precise positioning to specific functional destination of GPCRs are
critical aspects in controlling integrated responses of the cell to
hormones. Indeed, defective anterograde transport of GPCRs to
the cell surface en route from the ER through the Golgi is tightly
associated with the pathogenesis of a variety of human diseases,
including those induced by naturally occurring mutations or
truncations of GPCRs, leading to the accumulation of misfolded
receptors in the ER [16,50,51]. Therefore, further elucidation of
the molecular mechanisms underlying the export traffic of GPCRs
may provide a foundation for development of therapeutic
strategies by designing specific drugs to control GPCR biosynthesis
and cell-surface export trafficking. Second, more interestingly, mutation of Lys65 to the same
charged Arg residue enhanced the cell-surface number of a2A-AR. There are at least two possible explanations for the enhancement
of the cell-surface expression of a2A-AR induced by mutation of
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pharmacoperone drugs. PLoS One 6: e22784. p
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intracellular trafficking of alpha2-adrenergic receptors. Mol Pharmacol 51: 711–
720. 51. Morello JP, Bichet DG (2001) Nephrogenic diabetes insipidus. Annu Rev Physiol
63: 607–630. PLOS ONE | www.plosone.org December 2012 | Volume 7 | Issue 12 | e50416 9 9
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Cultural Study of Natural Environment Exploitation: Eating Culture of the Joseon People through the Novel “Thousand-Year Kingdom”
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1. INTRODUCTION Culture is a broad concept with numerous definitions worldwide. In seminal work, Primitive Culture, E.B. Tylor (2018) defined
that “Culture, or civilization, taken in its broad ethnographic sense, generally includes knowledge, belief, art, morals, law, custom,
and any other capabilities and habits acquired by man as a member of society” (p. 11). In this definition, Tylor equated culture with
civilization, thereby not clearly delineating the content of culture. In Vietnam, according to Tran Ngoc Them (1999), “Culture is an
organic system of material and spiritual values created and accumulated by humans through practical activities, in interaction with
the natural and social environment” (p. 10). He further posited that culture can be viewed as a system comprising four essential
elements, namely cognitive culture, community organizational culture, culture in relation to the natural environment, and culture in
relation to the social environment. Thus, the way humans interact with the natural environment is the third most important element
of every cultural system. When interacting with the natural environment, humans often take advantage of beneficial resources and
respond to harmful ones. Eating and drinking, for instance, are manifestations of utilizing the natural environment. Culturology has a relatively developed history in Europe, but in Vietnam, it is a relatively new field of research. The research
of many humanities and social sciences from the perspective of culturology is increasing in quantity and depth in quality. Particularly, cultural and literary studies have gained more attention recently. The reason for this trend, as summarized by Tran Le
Bao (2009: 68), is that literature is not just a part of a culture, subjected to direct cultural influences, but also one of the means by
which culture exists and is preserved. Literature is directly influenced by the cultural environment of its time and the unique cultural
traditions of a nation while also reflecting the unique cultural psychology of a particular era and ethnic community. On the other hand, eating is an important component when studying culture. Korean eating culture has been studied for a long
time, with massive works, especially the extensive works of Joo Young-ha, such as “Korean History on the Dining Table” and
“Culinary Studies” (2011), “Korean Cuisine: If You Don’t Eat Rice, You’ll Be Hungry” (2011), and so on. However, research on
Korean cuisine through literary works is relatively scarce. 3301 *Corresponding Author: Thuy, Thi Tran www.ijcsrr.org www.ijcsrr.org Volume 06 Issue 06 June 2023
DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023 Cultural Study of Natural Environment Exploitation: Eating Culture of the
Joseon People through the Novel “Thousand-Year Kingdom”
Tam, Minh Nguyen1, Thuy, Thi Tran2, Kiet, Tuan Tran3, Ha, Thi Thu Nguyen4
1,2,3,4 Faculty of Languages and Social Science - Ba Ria - Vung Tau University, Viet Nam ABSTRACT: The novel “Thousand-Year Kingdom” by Kim Kyung-uk is based on a true story from history when three Dutch
sailors drifted into Joseon in the 17th century. Through the narrative of Jan Jansz Weltevree - the first Westerner to set foot in
Joseon-the life, society, and people of Joseon are perceived and described in a fresh and unfamiliar way, different from the
evaluations of Koreans or Easterners. This study focuses on investigating the cultural utilization of the natural environment by the
Joseon people in this unique literary work to understand the uniqueness of their eating culture. KEYWORDS: Cultural utilization of the natural environment, Cuisine, Eating culture, Thousand-Year K 3302 *Corresponding Author: Thuy, Thi Tran International Journal of Current Science Research and Review
ISSN: 2581-8341 DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023 DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789 through literature is relatively scarce. Therefore, exploring and implementing this research topic will contribute to a
multidimensional study of Korean culture, and the author hopes that the research results will reveal many new and interesting
insights. For the above reasons, the author chose a literary work from Korea that deeply reflects the Joseon culture, especially the
culinary culture, to study the cultural utilization of the natural environment by the Korean people. This topic remains relatively
unexplored. Specifically, in this article, based on the theoretical foundation of Tran Ngoc Them’s cultural utilization of the natural
environment, the author will survey and analyze the culinary culture of the Joseon people to depict the unique cultural aspects of
Korean cuisine. 2. INTRODUCTION TO THE AUTHOR AND HIS WORK In 1627, three Dutch sailors named Weltevree, Evoken, and Denison were sailing on the ship Ouwerkerck to Nagasaki, Japan
when they were caught in a storm and shipwrecked on Jeju Island, Joseon (Korea). They were imprisoned in Jeju for about a year
before being escorted to the capital on the orders of King Joseon. According to the laws of Joseon at that time, foreigners were not
allowed to leave once they set foot on Joseon’s territory, so three of them were forced to become soldiers imperial soldiers. Evoken
was assigned to a fire-arrow unit, while Weltevree and Denison belonged to the artillery unit. Later, Weltevree was given the task
of manufacturing cannons to serve the military. In the following spring, an envoy from the Tatar Empire (the ethnic Manchu people who would later establish the Qing Dynasty)
arrived in Joseon. If the Tatar envoy discovered the presence of Western foreigners in Joseon, they would suspect that Joseon was
in contact with foreign powers and plotting betrayal. As a result, all three Dutch people were detained. On the day the Tatar envoy
departed, Denison disappeared after deceiving the guards. He intercepted the Tatar envoy and pleaded for their help to leave Joseon. The Joseon court was furious, and Denison was punished severely. Later, Weltevree and Evoken were exiled to the southern region,
where they coincidentally discovered cannons left behind by the Ming Dynasty from the war against Japan. They planned to upgrade
the cannons and make them more powerful. Weltevree focused on researching the production of cannons under the gentle
supervision of a friendly camp commander. However, his successor was different from him. Due to his passion for banquets and
his desire to assert his authority, this person prohibited Weltevree from manufacturing cannons any longer. The monotonous life
drove Weltevree to seek a way to escape Joseon. However, his escape plan was thwarted by Evoken, who wished to stay in Joseon In the following spring, an envoy from the Tatar Empire (the ethnic Manchu people who would later establish the Qing Dynasty)
arrived in Joseon. If the Tatar envoy discovered the presence of Western foreigners in Joseon, they would suspect that Joseon was
in contact with foreign powers and plotting betrayal. As a result, all three Dutch people were detained. On the day the Tatar envoy
departed, Denison disappeared after deceiving the guards. 2. INTRODUCTION TO THE AUTHOR AND HIS WORK Kim Kyung-uk, born in 1971 in Gwangju, is a distinguished South Korean author. He earned a bachelor’s degree in English
Literature from Seoul National University and subsequently obtained a Ph.D. in Korean Literature from the same prestigious
institution. Kim embarked on his literary journey in 1993 as a student with the Young Writer's Award bestowed by “The World of
Authors” for the novel The Lonely. Since then, he has garnered numerous awards, including the 37th Han-kuk Il-bo Literature
Award in 2004 for the short story “Jang Guk-yeong Has Died, So What?,” the 53rd Modern Literature Award in 2007 for “99%”
the 40th Dong-in Literature Award in 2009 for “Dangerous Reading” and the esteemed 3rd Kim Seung-ok Literature Award in
2015 for "Forever Young." In 2016, his mesmerizing piece “Heaven’s Door” earned him the distinction of the 40th Ideal Literary
Award. Other notable works by Kim include “No Coffee in Baghdad Café” (1996), “Meeting Betty” (1999), “Who Killed Kurt
Cobain?” (2003), “Like a Fairy Tale” (2010), “God Does Not Have Grandchildren” (2011), “The Golden Apple” (2013), “Time
of Dogs and Wolves” (2016), and “The Man Who Looks in the Mirror” (2018). The novel “The Thousand-Year Kingdom” is based on the true story of three Dutch sailors who drifted ashore in Joseon during
the 17th century. The novel depicts the lives of these foreign individuals in a foreign land through the narrative of Jan Jansz
Weltevree. “The Thousand-Year Kingdom” vividly portrays the inner lives of the main character as well as aspects of social and
political life in Joseon during the early 17th century. In 1627, three Dutch sailors named Weltevree, Evoken, and Denison were sailing on the ship Ouwerkerck to Nagasaki, Japan
when they were caught in a storm and shipwrecked on Jeju Island, Joseon (Korea). They were imprisoned in Jeju for about a year
before being escorted to the capital on the orders of King Joseon. According to the laws of Joseon at that time, foreigners were not
allowed to leave once they set foot on Joseon’s territory, so three of them were forced to become soldiers imperial soldiers. Evoken
was assigned to a fire-arrow unit, while Weltevree and Denison belonged to the artillery unit. Later, Weltevree was given the task
of manufacturing cannons to serve the military. 1. INTRODUCTION Among these works, Han Kyung Ran’s “Classical Literature:
Investigating Author’s Consciousness through Food in the Works of Huh Gyun and Seong Yak-yong” (2014) is one of the few
studies that explore the topic of cuisine through literary works. This study examines the author’s sense of restraint portrayed through
food and the author’s consciousness reflected in food (instincts, entertainment, solace, and perception of reality) through Huh
Gyun’s work “Tinh So Phu Bao Cao” and Jeong Yak-yong’s collection of poems “Tra Son Thi Van Tap”. In this study, Han Kyung
Ran also explored previous studies that focus on two directions. Direction of studying classical works on culinary culture Direction of studying food consumption in classical works In line with the trends and research directions of foundational studies, the author chose to study literary works on the culinary
culture of the Korean people, specifically the culinary culture of the Joseon era in the 17th century. Furthermore, contrary to the
significance of literature as a means of expressing the unique culture of a specific era and community, research on cultural aspects In line with the trends and research directions of foundational studies, the author chose to study literary works on the culinary
culture of the Korean people, specifically the culinary culture of the Joseon era in the 17th century. Furthermore, contrary to the
significance of literature as a means of expressing the unique culture of a specific era and community, research on cultural aspects Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
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Page No. 3301-3308 3. Cultural Utilization of the Natural Environment: Eating in Korean Joseon Era through the novel “A Thousand-Year
Kingdom”. According to Tran Ngoc Them (1999), the culture of interacting with the natural environment can be classified into the culture
of utilizing the natural environment, including food, the culture of adapting to the natural environment, including clothing, housing,
and transportation. Ideally, all three aspects would be presented simultaneously. However, due to the limitations of this article, the
author will only analyze the eating culture of the Korean people during the Joseon era. The study of the culture in relation to
clothing, housing, and transportation will be addressed in the upcoming study. 3.1 Meal Structure: Emphasis on Rice and Porridge 3.1 Meal Structure: Emphasis on Rice and Porridge Eating and drinking is a culture that utilizes the natural environment. Similar to Vietnam, the meal structure of the Joseon
people clearly reflects the influence of the traditional agricultural culture of rice farming. It is a plant-based meal structure in which
rice, made from grains, takes the central role and is mentioned first in the meal. In the novel “A Thousand-Year Kingdom”, rice
made from rice grains is also mentioned first when discussing the meal: “Looking at the rounded rice grains like small balls, Evoken
said it was rice balls. He said the Japanese people also eat it. They must have added salt to it, so it has a slightly salty taste.” (Kim
Kyung Uk, 2007: 46). The origin of rice balls in Korean cuisine is not clear. However, it may have appeared since ancient times
when people needed a portable meal for long journeys or on battlefields where it was not feasible to cook rice. During times of
scarcity, the Korean people would make rice balls from plain rice or rice without seasonings to eat. For this reason, rice balls are
considered a symbol of difficult times. The character Weltevree described the Joseon people’s rice balls as “like small balls.” Here,
we can see cultural differences reflected in the naming of the same concept. While the Japanese refer to this dish as “onigiri,”
derived from the verb “nigiru,” meaning “to grasp” or “to squeeze” in Japanese, the Vietnamese call it “cơm nắm,” and the Koreans
call it “jumeok-bap,” derived from the noun “jumeok,” meaning “fist” or “to clench one’s hand” in Korean. 3303 *Corresponding Author: Thuy, Thi Tran International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 06 Issue 06 June 2023
DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023
www.ijcsrr. DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023 DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789 displayed by the Joseon court towards the Tatar, the war between the two nations was inevitable. In that winter, an army of 120,000
Tatar soldiers advanced towards Joseon. When news of the impending war reached King Joseon, the royal family, the noble officials,
and a force of 12,000 troops quickly moved to Nam Han Mountain Fortress. The enemy forces surged in like an unstoppable wave. The war on Joseon soil suddenly became the war of Weltevree and Evoken. In one battle, amidst the deafening roar of the cannons,
Evoken pushed Weltevree aside and fell dead upon him. Thus, all of Weltevree’s comrades perished, and he was left alone to fight
in a foreign land among unfamiliar people and beliefs. Nevertheless, he still whispered to himself, “Even if the enemy retreats, I
will continue to fight. The battle for the soul has just begun.” When Weltevree first set foot in Joseon, he was amazed and startled by what he saw. Among the many “peculiar” aspects of
Joseon society, he was particularly interested in the cuisine and eating culture. He paid meticulous attention to the food and described
it vividly in his writings. Through Weltevree’s descriptions, the author will analyze the culture of the Korean people during the
Joseon era from the cultural perspective of Tran Ngoc Them (1999). 2. INTRODUCTION TO THE AUTHOR AND HIS WORK He intercepted the Tatar envoy and pleaded for their help to leave Joseon. in contact with foreign powers and plotting betrayal. As a result, all three Dutch people were detained. On the day the Tatar envoy
departed, Denison disappeared after deceiving the guards. He intercepted the Tatar envoy and pleaded for their help to leave Joseon. The Joseon court was furious, and Denison was punished severely. Later, Weltevree and Evoken were exiled to the southern region,
where they coincidentally discovered cannons left behind by the Ming Dynasty from the war against Japan. They planned to upgrade
the cannons and make them more powerful. Weltevree focused on researching the production of cannons under the gentle
supervision of a friendly camp commander. However, his successor was different from him. Due to his passion for banquets and
his desire to assert his authority, this person prohibited Weltevree from manufacturing cannons any longer. The monotonous life
drove Weltevree to seek a way to escape Joseon. However, his escape plan was thwarted by Evoken, who wished to stay in Joseon
instead of risking his life elsewhere. As a result, Weltevree was discovered and imprisoned for a period of time. At the same time,
news of the current commander's authoritarian and dictatorial rule reached the ears of the King of Joseon, resulting in his dismissal
and exile to Jeju Island. The newly appointed commander granted freedom and restored the opportunity for Weltevree to research
and improve the cannons. Finally, Weltevree successfully manufactured the cannons. Meanwhile, the Tatar king changed the country’s name to “Qing,” meaning “purity,” and proclaimed himself the Emperor. Thus, a nomadic tribe on the steppe became a ruling dynasty over all of China. The emerging empire demanded Joseon become its
vassal state instead of maintaining a friendly diplomatic relationship. If Joseon refused, the Tatar Empire would invade and conquer
Joseon. However, the Joseon court never agreed to the Tatar’s demands for submission. Furthermore, with the hostile attitude Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
Page No. 3301-3308 Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
Page No. 3301-3308 International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 06 Issue 06 June 2023
DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023
www.ijcsrr.org International Journal of Current Science Research and Review w
www.ijcsrr.org DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023 DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789 brought out wine and refreshments to entertain him, he declined, stating that it was already late and wanted to leave. Since he had
not eaten, the family continued serving him cakes and fruits, but he remained steadfast in his decision to leave because he had not
yet eaten rice. Upon the vizier’s return, the household members recounted their fear that Ming envoy Nguyệt Sa might be hungry,
so they served him food five times. However, he continued to assert that he had not eaten and left. Hearing this, the vizier regrettably
said, “The people of Joseon perceive that if they do not eat rice, they are hungry. It seems that you have forgotten to offer him rice.” Nguyệt Sa mentioned here is none other than Lee Jeong-gun (1564-1653), who is recognized as one of the four major mid-
Joseon writers. This period shows the mindset of the people during the Joseon era, when rice is regarded as the essential component
of a meal. They believed that regardless of the variety of other dishes, it is essential to consume rice or grains to consider the meal
complete. In addition, this belief explains why rice is considered fundamental when offering ancestral rituals in Korean culture. The
absolute faith in rice led to its use as a form of currency or taxation. Rice became a substitute for currency (Kim Young-ha, 2011:
74-77). The Kingdom of a Thousand Years also addressed this issue: “The king appointed us as his close guards and pledged to
provide us with 70 catties (a unit of weight) of rice every month” (Kim Kyung Uk, 2007: 88). The typical meal structure of the Joseon people emphasizes the importance of rice. However, what was the meal structure
during formal banquets hosted by officials or in the royal court? The main food in the meal structure of royal banquets and the
households of high-ranking Joseon officials first include various types of meat and fish. In the work, Park Yeon and his two
companions were summoned to the royal court and were entertained by King Taejo of Joseon (1595-1649). International Journal of Current Science Research and Review Weltevree described
the cuisine of the Joseon royal court as follows: “There was an abundance of food and drinks, from thinly sliced and grilled beef,
to marinated and fragrant grilled pork, pan-fried fish wrapped in egg, stir-fried mushrooms, noodles, and wine...” (Kim Kyung Uk,
2007: 86) 1. The meal structure in the homes of high-ranking Joseon officials, where Weltevree was later invited to attend, was also
diverse and rich, no less than the royal court banquets. There were “various ingredients, such as beef, pork, chicken, fish, eggs,
mushrooms, vegetables…” marinated with spices and seasonings before being grilled, simmered, or boiled. Thinly sliced and
skillfully prepared beef and pork, thinly sliced fish with a layer of white or egg yolk, and neatly fried eggs were some of the
delicacies served (Kim Kyung Uk, 2007: 102). Through Weltevree’s description, it can be seen that the meal structure of Joseon banquets was rich in various types of meat
such, as beef, pork, chicken, etc. As for seafood, except for fish, Park Yeon’s work does not mention any representative seafood. Despite Joseon being a peninsula with a coastline stretching 8,460 km from east to west, in reality, crabs and shellfish were rare
delicacies even for the royal court and the noble class of Joseon. Go Yeon Hee (2013:119) examined ancient records and found the
reason: “The book Capital of Goryeo meticulously describes Goryeo through the eyes of Chinese envoys, stating that there is an
abundance of seafood in Goryeo, and even the lowest-ranking individuals can easily buy and consume them. However, when
examining later documents, it is found that it is unknown whether it was due to the need to pay tribute with a lot of seafood, but the
low-ranking individuals of Joseon could not easily enjoy them,” and that “crabs were among the extremely rare dishes in Joseon
period.” According to Weltevree the next component in the structure of Joseon banquet meals is mushrooms and vegetables. Weltevree
mentions vegetables in general without specifying each type. It can be assumed that the variety of green leafy vegetables in the
eastern country would be much greater than in his homeland, and he could not possibly know all their names. Therefore, similar to Vietnamese culture, the culinary culture of the Korean people during the Joseon period utilized plant-
based resources such as vegetables. 3304 *Corresponding Author: Thuy, Thi Tran Volume 06 Issue 06 June 2023
A
i
ij
1 All quotations from the book Kingdom of a Thousand Years by Kim Kyung Uk (2007) in this article are translated by
Nguyen Thi Thu Ha. 3304 *Corresponding Author: Thuy, Thi Tran 3. Cultural Utilization of the Natural Environment: Eating in Korean Joseon Era through the novel “A Thousand-Year
Kingdom”. Meanwhile, Park Yeon,
a Westerner, referred to this dish as “rice ball.” It is further observed that the meal structure during this difficult period consisted
solely of rice balls mixed with a small amount of salty salt, without any other food. The meal structure with rice as the main and only ingredient is also reflected in the dish known as “súp gạo tẻ” (white rice
porridge). “The foreign soldiers brought us bowls of white rice porridge and removed the iron and rope shackles that bound us. Their sudden act of kindness made me uneasy. I did not know if this would be our last meal. I ate the food they brought with relish
and gratitude.” (Kim Kyung Uk, 2007:49). Therefore, simply consuming a bowl of white rice porridge could be considered
equivalent to having a complete meal. The meal structure that emphasizes dishes prepared from grains or rice among the Koreans during the Joseon era contributed
to the belief that one must “eat rice” to be considered truly “eating” like a Korean. Kim Young-ha (2011) also supported this idea
in his research on Korean cuisine, where he references content from a Korean-language book titled “Mậu ngọ yến hành lục” (Record
of Banquets and Travels in the Year of the Wood Horse), specifically Volume 4 written by Seo Yu-moon (1762-1822) during the
Joseon period. When Wolsa Lee Sang-gong went on an embassy to Ming, a vizier invited him to choose a date to visit his house. On the day of
the appointment, the general was busy with affairs at the palace, so the family members invited Nguyệt Sa to stay and wait for the
vizier to return. However, since Nguyệt Sa arrived before the mealtime, when the family members conveyed the vizier’s words and 3303 *Corresponding Author: Thuy, Thi Tran Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
Page No. 3301-3308 International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 06 Issue 06 June 2023
DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023
www.ijcsrr.org 3.2 Eating etiquette The scholars of the Joseon dynasty adhered to strict eating and driking etiquette. The elementary school textbooks at that time
taught children about the proper manners for eating and drinking. Scholars of Neo-Confucianism in Joseonalso believed in the
importance of moderation during meals (Joo Young-ha, 2011: 88-89). However, Weltevree made a different observation about the
eating habits of the Joseon people in the novel “A Thousand-Year Kingdom.” “All heretics eat excessively. They eat and drink
without pause. Their hurried eating and drinking aim for extreme pleasure but fall into transient excitement. They do not eat to live
but live to eat. Male heretics grab food with their mouths and swallow it whole like hungry ghosts. Their gluttony devours everything,
as if consuming raw and swallowing death itself, clinging tightly to the source of life” (Kim Kyung Uk, 2007: 105). After finishing
their meals, they “rub their bellies, swollen like sails filled with wind, and feel content and happy" (Kim Kyung Uk, 2007: 106). Thus, it can be said that the people of Joseon might not have been aware of the amount of food they consumed, but according to
Weltevree, a foreigner, he found that Koreans in Joseon were generally people who ate a lot. In fact, not only Park Yeon in the
novel "A Thousand-Year Kingdom,” but also the Japanese and French people made similar observations. In the late 19th century,
when missionaries came to Korea, a French missionary referred to the people of Joseon as “great eaters of Asia.” The book “Thuc
Thieu” by Yi-ik (1681-1763) also mention: “The efforts of our people in consuming food are unparalleled. Recently, someone visited Yuku (now Okinawa), and the people
there sarcastically said, “Your custom is to constantly stir the rice vigorously with an iron pot and eat with metal spoons, so how
could you not be poor?” Overall, they were people who had migrated to our country before, so they understood the situation well”
(Joo Young-ha, 2011: 72). In Korea, the book “Toa Vi Luc” by Oh Hee-moon (1539-1613) also mentioned that despite being in a period of war, they
cooked up to 7 hob (a measure of volume) of rice for each meal (Joo Young-ha, 2011: 72-73). It is indeed a large quantity for each
person to consume half a kilogram of rice per day. International Journal of Current Science Research and Review Volume 06 Issue 06 June 2023 DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789 Regarding the taste of the food, Weltevree says, “The cuisine of heretics is both stimulating and tender. All dishes emit the
smell of garlic” (Kim Kyung Uk, 2007:86). This is the distinctive taste of Korean cuisine that leaves a strong impression on
foreigners. Korean dishes tend to use a variety of spices and seasonings to create a unique “complex flavor” rather than preserving
the natural taste of the ingredients. Korean dishes do not just linger on the tip of the tongue; they spread throughout the mouth with
a subtle, gentle flavor that is not overly sweet, fatty, or spicy. Koreans use soy sauce, scallions, garlic, sesame salt, sesame oil, chili
powder, pepper, and more to season their dishes, Hence, a typical dish would require at least 5 to 6 types of seasonings to create the
stimulating yet tender taste of Korean cuisine. In addition, their food presentation is meticulous and vibrant in color, and Park Yeon exclaimed: “What captivated my gaze the
most was the decoration and vibrant colors of the dishes. The high-quality dishes were shaped like towers, meticulously crafted by
skilled artisans. Why would the heretics of this kingdom focus all their intellectual and cultural light solely on the dining table? The
heretics elevated their cuisine to the level of artistry” (Kim Kyung Uk, 2007:102-103). Even desserts were arranged like towers:
“As soon as the table was cleared, the servants immediately brought in a new small table. On the table, there were indeed peaches,
apples, pears, dried persimmons, and walnuts piled up high like a tower. They were dessert items" (Kim Kyung Uk, 2007:106). The
style of arranging food into tall cylindrical towers is only seen in the structure of banquet meals. The banquet dishes are stacked on
top of each other, and the height varies depending on social status. Surprised by this arrangement, "Evoken, who had traveled the
world and tasted diverse dishes, could not help but exclaim in awe. Denison couldn’t keep his mouth shut” (Kim Kyung Uk,
2007:102-103). These foreign visitors were greatly impressed by the banquet table arrangement of the Joseon Koreans to the point that they
doubted whether the Joseon people had poured all their intellect and culture of their civilization onto the dining table. 3305 *Corresponding Author: Thuy, Thi Tran International Journal of Current Science Research and Review They perceived
that the Joseon table is filled with exquisite artistic qualities, completely different from their own country's culture. International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 06 Issue 06 June 2023 International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 06 Issue 06 June 2023
DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023
www.ijcsrr.org International Journal of Current Science Research and Review However, the Joseon Peninsula, which European visitors likened to “a sea face in a hurricane”
has a large number of mountain ranges covering 70% of its total area. This is why the Korean people made extensive use of wild
vegetables in their meal structure. Although Weltevree does not mention rice or grains when describing the dishes at the banquet, he does mention wine: “The
wine is clear in color but has a deep taste” (Kim Kyung Uk, 2007:86). The clear-colored wine referred to is wine made from rice,
including well-known Korean wines such as Thakju, Cheongju, or Soju. Therefore, rice is not only used to make rice balls or
porridge but is also utilized by Koreans in the production of rice wine for drinking. 3304 *Corresponding Author: Thuy, Thi Tran Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
Page No. 3301-3308
1 All quotations from the book Kingdom of a Thousand Years by Kim Kyung Uk (2007) in this article are translated by
Nguyen Thi Thu Ha. Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
Page No. 3301-3308 International Journal of Current Science Research and Review (Kim Kyung Uk, 2007: 103) South Korea is one of the countries heavily influenced by Confucianism, and Confucianism during the Joseon period (1392-
1910) reached its peak. The perspective of Confucianism emphasizes that the relationship between men and women is the foundation
of all human relationships, but in reality, it emphasizes the differentiation between males and females. The absence of women and
girls in the banquet for guests mentioned above is an expression of gender discrimination during the Joseon era. Joo Young-ha
(2011) also states that during the Joseon period, when eating, fathers and sons or grandsons would sit at the same table, while
mothers and daughters would eat separately in the kitchen area, and they would only use one spoon to eat rice due to the scarcity of
eating utensils during that time. Being influenced by Confucianism, people in East Asia, such as Japan, Korea, and Vietnam would not be surprised by this
phenomenon. However, people from the Western culture may find it difficult to understand and express different thoughts. In “A
Thousand Years of the Kingdom,” Evoken reflects on this phenomenon as follows: “Captain, it is clear that these people are afraid
that their women will fall in love with us, so they have to hide them carefully. I heard that a Chinese man locked up his wife in a
separate room for ten years to isolate her because he could not bear to see her smiling at the neighboring men. (...) However, it turns
out that when it comes to jealousy, the Chinese cannot compare to the men of this kingdom. That is why they had to establish the
rule that men can only move around during the day, while women can only go out at night, right?” Evoken finds it difficult to understand the phenomenon of women not being allowed to travel during the day and the absence
of women sitting at the same table as men, even within the same family. It is likely that Evoken has never heard of Confucianism -
a belief system that has greatly influenced Northeast Asian countries and Vietnam - so it is understandable that he is perplexed. Being a free-spirited individual who craves love, Evoken speculates that perhaps the people of Joseon Korea were afraid that their
women would fall in love with Western foreigners. International Journal of Current Science Research and Review DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023 dining with Koreans during the Joseon era feeling perplexed, even thinking they are being mocked. This can be clearly observed in
the novel “A Thousand Years of Kingdom.” “We apologize for not preparing many dishes.” “We apologize for not preparing many dishes.” isn’t that man making fun of us now?” Evoken said in shock. “Captain, isn’t that man making fun of us now?” Evoken said in shock. “Captain, isn’t that man making fun of us now?” Evoken said in shock. “Captain, isn’t that man making fun of us now?” Evoken said in shock. Evoken asked the young manager if they were being teased. The young official responded with a smile Evoken asked the young manager if they were bein “That is the way of greeting in this country.” “That is the way of greeting in this country.” The behavior of the Joseon people bears a slight resemblance to that of the Vietnamese, as the hosts always strive to generously
and graciously treat their guests. Therefore, when they saw the three foreign guests eating voraciously, instead of feeling offended,
the high-ranking officials and nobles were delighted. However, when the guests did not eat much or declined the food, the high-
ranking officials and nobles could not hide their disappointed expressions. Another strange point that foreign visitors discovered when dining at the noble houses of Joseon was the absence of women
sitting and eating together. “Does this house not have women?” Evoken asked the young official. “Does this house not have women?” Evoken asked the young official. It was only then that I realized that all the people sitting in the room were men. It was only then that I realized that all the people sitting in the room were men. It was only then that I realized that all the people sitting in the room were men. “Men and women cannot sit and eat together,” the young official replied. (Kim Kyung Uk, 2007: 103) was only then that I realized that all the people sitting in the room were men. Men and women cannot sit and eat together,” the young official replied. (Kim Kyung Uk, 2007: 103) d women cannot sit and eat together,” the young official replied. 3306 *Corresponding Author: Thuy, Thi Tran 3.2 Eating etiquette Describing the phenomenon of overeating among the people of Joseon in a straightforward, direct, and vivid manner, Weltevree
said, “They (the people of Joseon) do not eat to live but live to eat. Men of different religions pick up food and swallow it whole
like hungry ghosts.” Surely, the people of Joseon at that time ate a lot and hastily, which surprised Weltevree. Regarding the etiquette of treating guests at home, the people of Joseon would often say, “We apologize for not preparing many
dishes,” even though a continuous array of food is presented before and during the banquet. This inevitably leaves foreign guests 3305 *Corresponding Author: Thuy, Thi Tran Volume 06 Issue 06 June 2023
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Page No. 3301-3308 International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 06 Issue 06 June 2023
DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023
www.ijcsrr.org International Journal of Current Science Research and Review However, the deep-rooted reason behind these rules of dining and social
interactions in Joseon Korea lies in the long-standing Confucian ideology that emphasizes male dominance and control in the
country. In addition, Weltevree also mentions the smoking culture of the Joseon people. “People of different religions smoked using
long tobacco pipes made from bamboo. At both ends of the bamboo, there were tobacco containers and mouthpieces made of brass. The length of the pipe was half a vadem (1.698m), so it was impossible to light it by oneself.” (Kim Kyung Uk, 2007: 120). The tobacco pipe used by the Joseon people of Korea was made of bamboo, symbolizing for the agricultural and rural countries. This feature is similar to the tobacco pipe, a cultural symbol of Vietnam, which is also a cultural symbol. However, the difference
lies in the length of the Korean pipe, which is over half a meter, making it impossible for the smokers to light it by themselves. It is
no wonder that during the late Joseon period, a profession related to tobacco emerged in Korea. We can observe the occupation of
tobacco harvesters in the painting “Removing Tobacco” by Kim Hong Do. When the act of tobacco harvesting became a profession,
it can be understood that the people of Korea during this period favored tobacco to the extent that Weltevree had to comment: “In
this kingdom, people of all ages, both men and women, enjoy smoking. Even young children casually hold tobacco pipes in their
mouths while walking on the street. When holding a tobacco pipe, the faces of the followers of different religions light up with a Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
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Available at: www.ijcsrr.org
Page No. 3301-3308 DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023 DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789 look of contentment and happiness. A child holding a pipe as tall as their body smiles brightly like an angel. The followers of
different religions indulge in pleasure. In this country, not being able to smoke tobacco is considered disgraceful.” (Kim Kyung Uk,
2007: 120). Through Weltevree’s comments, we can understand that people from all walks of life, regardless of gender or age,
including children, were swept up in the “storm” of smoking tobacco during the Joseon period in Korea. International Journal of Current Science Research and Review Tobacco brought a sense
of pleasure and ecstasy to the people of Joseon, and not being able to smoke was not just a feeling of discomfort but also a sense of
shame and embarrassment. Therefore, the widespread utilization of cigarettes is self-evident. However, when was tobacco brought into Joseon and through which route? While there are no exact written records, the legend
suggests that tobacco was introduced to Joseon from Japan during the years 1608-1616, around the time of the Japanese invasions
of Korea. According to the “Nhan To Thuc Luc” (Records of Nhan To), tobacco was imported into Joseon in 1616. However, the
smoking method was mentioned in the book Chi phong loại thuyết2, indicating that tobacco might have been introduced even earlier. Initially, when tobacco was newly introduced to Joseon, it was very expensive, with the price of one catty of tobacco reaching a
significant amount of silver in 1624, making it affordable only for the noble class. However, within a few years, it became widely
popular. Hendrik Hamel (1630-1692) also described the fondness of the Joseon people for tobacco in his book “Hamel’s Journal”
- a book he wrote after escaping from 13 years of captivity in Joseon3. In this work, the author examined the food and drinks recounted by Weltevree in the book “A Thousand-Year Kingdom” to
depict the structure of meals in Joseon-era Korea. In addition, the author relies on the evidence provided by Weltevree regarding
the etiquette rules of eating and drinking during the Joseon period to generalize the social behavior through the manner of hosting
guests at the homes of officials and the Joseon royal court. 4. CONCLUSION This article has examined the Korean cuisine presented in the work “A Thousand-Year Kingdom” by Kim Kyung Uk, while
analyzing it from a cultural perspective. The research findings indicate that during the Joseon era, the Korean people placed great
importance on rice-based dishes, specifically “cơm nắm” (rice balls) and rice porridge. These dishes were the main and first ones
mentioned in the meal structure of the Korean people. In this work, “cơm nắm” and white rice porridge are not only the main dishes
but also the only ones associated with individuals in difficult circumstances. In addition, the royal cuisine and banquet culture of
Joseon Korea is depicted in a majestic and elaborate manner through Weltevree’s accounts, showcasing a wide variety of intricately
decorated dishes resembling towers. This unique feature in the Korean table-setting culture is highlighted. Furthermore, tobacco
also holds significant meaning as it helps depict the social and cultural landscape of 17th-century Joseon. It created a widespread
“obsession” that permeated all levels of society, from commoners to the aristocracy and even the royal court of the Joseon dynasty. Through this study, we also recognize the similarities between Korean and Vietnamese cultures in various aspects, such as the
emphasis on rice-based cuisine, observance of etiquette when hosting guests, and the cultural practice of smoking with long bamboo
pipes. 3307 *Corresponding Author: Thuy, Thi Tran
p
y 2 The first Korean encyclopedia was compiled by Lee Su Gwang in 1614 (the reign of King Gwanghaegun).
3 Reference: https://terms.naver.com/entry.nhn?docId=3403538&cid=58389&categoryId=58389 3. Han Kyung Ran, “Khảo sát ý thức tác giả thông qua ẩm thực: trọng tâm là tác phẩm của Huh Gyun và Jeong Yak-yong”,
Tạp chí Ngôn ngữ và văn hoá số 16, Vol.16 (3~32) 2014. [한경란, “음식을 통해 본 작가의 고찰 – 허균과 정약용을
중심으로”, 한국어와 문화 제16 집 (3~32) 2014.] 1. E.B.Tylor (2018), Văn hoá nguyên thuỷ, Nxb. Tri Thức (Huyền Giang dịch). 7. Trần Ngọc Thêm (1999), Cơ sở văn hoá Việt nam, Nxb. Giáo dục. Cite this Article: Tam, Minh Nguyen, Thuy, Thi Tran, Kiet, Tuan Tran, Ha, Thi Thu Nguyen (2023). Cultural Study of Natural
Environment Exploitation: Eating Culture of the Joseon People through the Novel “Thousand-Year Kingdom”. International
Journal of Current Science Research and Review, 6(6), 3301-3308 6. Trần Lê Bảo (2009), “Giải mã văn hoá trong tác phẩm văn học – dẫn chứng từ nền văn học Trung Quốc”, Tạp chí Nghiên
cứu Trung Quốc số 2 (90) – 2009, trang 68-79. 3308 *Corresponding Author: Thuy, Thi Tran 7. Trần Ngọc Thêm (1999), Cơ sở văn hoá Việt nam, Nxb. Giáo dục. REFERENCES 1. E.B.Tylor (2018), Văn hoá nguyên thuỷ, Nxb. Tri Thức (Huyền Giang dịch). 1. E.B.Tylor (2018), Văn hoá nguyên thuỷ, Nxb. Tri Thức (Huyền Giang dịch). Khảo sát ý thức tác giả thông qua ẩm thực: trọng tâm là tác phẩm của Huh Gyun và Jeong Yak-yong”,
và văn hoá số 16, Vol.16 (3~32) 2014. [한경란, “음식을 통해 본 작가의 고찰 – 허균과 정약용을 3. Han Kyung Ran, “Khảo sát ý thức tác giả thông qua ẩm thực: trọng tâm là tác phẩm của Huh Gyun và Jeong Yak-yong”,
Tạp chí Ngôn ngữ và văn hoá số 16, Vol.16 (3~32) 2014. [한경란, “음식을 통해 본 작가의 고찰 – 허균과 정약용을
중심으로”, 한국어와 문화 제16 집 (3~32) 2014.] 4. Joo Young Ha (2011), Sự lý thú của Hàn Quốc học, Nbx. Hội nhà văn (Nguyễn Thị Thu Vân dịch). 3307 *Corresponding Author: Thuy, Thi Tran Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
Page No. 3301-3308
2 The first Korean encyclopedia was compiled by Lee Su Gwang in 1614 (the reign of King Gwanghaegun). 3 Reference: https://terms.naver.com/entry.nhn?docId=3403538&cid=58389&categoryId=58389 2 The first Korean encyclopedia was compiled by Lee Su Gwang in 1614 (the reign of King Gwanghaegun). 3 Reference: https://terms.naver.com/entry.nhn?docId=3403538&cid=58389&categoryId=58389 Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
Page No. 3301-3308 International Journal of Current Science Research and Review
ISSN: 2581-8341
Volume 06 Issue 06 June 2023
DOI: 10.47191/ijcsrr/V6-i6-24, Impact Factor: 6.789
IJCSRR @ 2023
www.ijcsrr.org 6. Trần Lê Bảo (2009), “Giải mã văn hoá trong tác phẩm văn học – dẫn chứng từ nền văn học Trung Quốc”, Tạp chí Nghiên
cứu Trung Quốc số 2 (90) – 2009, trang 68-79. 7. Trần Ngọc Thêm (1999), Cơ sở văn hoá Việt nam, Nxb. Giáo dục. 3308 *Corresponding Author: Thuy, Thi Tran Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
Page No. 3301-3308
Cite this Article: Tam, Minh Nguyen, Thuy, Thi Tran, Kiet, Tuan Tran, Ha, Thi Thu Nguyen (2023). Cultural Study of Natural
Environment Exploitation: Eating Culture of the Joseon People through the Novel “Thousand-Year Kingdom”. International
Journal of Current Science Research and Review, 6(6), 3301-3308 Volume 06 Issue 06 June 2023
Available at: www.ijcsrr.org
Page No. 3301-3308
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Ovarian Sex Hormones Modulate Compulsive, Affective and Cognitive Functions in A Non-Induced Mouse Model of Obsessive-Compulsive Disorder
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Frontiers in behavioral neuroscience
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cc-by
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ORIGINAL RESEARCH
published: 09 November 2016
doi: 10.3389/fnbeh.2016.00215 Ovarian Sex Hormones Modulate
Compulsive, Affective and Cognitive
Functions in A Non-Induced Mouse
Model of Obsessive-Compulsive
Disorder Swarup Mitra 1,2†, Cristiane P. Bastos 2,3†, Katherine Bates 4, Grace S. Pereira 3
and Abel Bult-Ito 2,4* Swarup Mitra 1,2†, Cristiane P. Bastos 2,3†, Katherine Bates 4, Grace S. Pereira 3
and Abel Bult-Ito 2,4* 1 Department of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, AK, USA, 2 IDeA Network
of Biomedical Research Excellence (INBRE), University of Alaska Fairbanks, Fairbanks, AK, USA, 3 Núcleo de Neurociências,
Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo
Horizonte, Brazil, 4 Department of Biology and Wildlife, University of Alaska Fairbanks, Fairbanks, AK, USA There is currently a lack of understanding of how surgical menopause can influence
obsessions, compulsions and associated affective and cognitive functions in female
obsessive-compulsive disorder (OCD) patients. Early menopause in women due to
surgical removal of ovaries not only causes dramatic hormonal changes, but also
may induce affective and cognitive disorders. Here, we tested if surgical removal
of ovaries (ovariectomy, OVX), which mimics surgical menopause in humans, would
result in exacerbation of compulsive, affective and cognitive behaviors in mice
strains that exhibit a spontaneous compulsive-like phenotype. Female mice from
compulsive-like BIG, non-compulsive SMALL and randomly-bred Control strains were
subjected to OVX or sham-surgery. After 7 days animals were tested for nest
building and marble burying to measure compulsive-like behavior. The elevated
plus maze and open field tests measured anxiety-like behaviors, while memory
was assessed by the novel object recognition. Acute OVX resulted in exacerbation
of compulsive-like and anxiety-like behaviors in compulsive-like BIG mice. No
significant effects of OVX were observed for the non-compulsive SMALL and Control
strains. Object recognition memory was impaired in compulsive-like BIG female
mice compared to the Control mice, without an effect of OVX on the BIG mice. We also tested whether 17 β-estradiol (E2) or progesterone (P4) could reverse
the effects of OVX. E2, but not P4, attenuated the compulsive-like behaviors in
compulsive-like BIG OVX female mice. The actions of the sex steroids on anxiety-like
behaviors in OVX females were strain and behavioral test dependent. Altogether,
our results indicate that already existing compulsions can be worsened during
acute ovarian deprivation concomitant with exacerbation of affective behaviors and
responses to hormonal intervention in OVX female mice can be influenced by genetic
background Received: 07 September 2016
Accepted: 25 October 2016
Published: 09 November 2016 Received: 07 September 2016
Accepted: 25 October 2016
Published: 09 November 2016 Keywords: compulsive-like behavior, ovariectomy, strain differences, surgical menopause, 17 β-estradiol,
rogesterone Edited by: Edited by:
Allan V. Kalueff,
St. Petersburg State University,
Russia Reviewed by:
Eddy A. Van Der Zee,
University of Groningen, Netherlands
Guilherme Bracarense Filgueiras,
Universidade Estadual de Londrina,
Brazil *Correspondence:
Abel Bult-Ito
abultito@alaska.edu
†These authors have contributed
equally to this work. *Correspondence:
Abel Bult-Ito
abultito@alaska.edu
†These authors have contributed
equally to this work. †These authors have contributed
equally to this work. INTRODUCTION One such study showed that, OCD
is not a rare comorbidity during post menopause (Uguz et al.,
2010), while another study demonstrated that the symptoms are
more related with menarche and decreases during menopause
(Guglielmi et al., 2014). Clinical and genetic data for OCD corroborate the hypothesis
of sexual dimorphism, which reveals differences in clinical
manifestations between males and females (Labad et al., 2008;
Torresan et al., 2009). Obsessions for cleaning and compulsive
contamination are more prevalent in females than males, while
males have higher rates of symmetrical and sexual obsessions
when compared to females (Noshirvani et al., 1991; Lensi et al.,
1996; Bogetto et al., 1999; de Mathis et al., 2008; Labad et al.,
2008). There is also a sex difference to treatment response
(Mundo et al., 1999). Women typically have a later onset when
compared to men and display a bi-modal distribution with the
first peak occurring between 13–16 years of age and the second
peak around 22–32 years. These are puberty and child bearing
stages in a women’s life, respectively (Brandes et al., 2004) when
sex hormone (estrogen and progesterone, P4) levels are known to
fluctuate. In animal studies, acute administration of estradiol (E2)
in pre-pubertal female rats exerted an anti-compulsive-like
effect (Flaisher-Grinberg et al., 2009), while male mice with
estrogen deficiency (aromatase enzyme knockout) displayed
compulsive-like behavior (Hill et al., 2007). In OVX rats,
concurrent administration of E2 and P4 was able to reduce
compulsive-like lever pressing behavior (Fernández-Guasti et al.,
2006). Most of these studies were conducted on induced
(drug or gene knockouts) models and did not investigate the
associated comorbidities like anxiety and cognitive impairments,
while only one study looked at the effect of P4 and E2 in
the ovariectomized condition (Fernández-Guasti et al., 2006). Moreover, though OCD has a compelling genetic basis (Nestadt
et al., 2010) the role of genetic background in influencing steroid
actions in OCD condition during menopause has never been
explored. It is well established that a plausible cause of OCD is
abnormal cortical-striatal-thalamic circuitry activation (Ahmari
et al., 2013) and altered serotonergic (Schilman et al., 2010),
glutamatergic (Arnold et al., 2004; Egashira et al., 2013; Porton
et al., 2013) and GABAergic (Egashira et al., 2013) systems. INTRODUCTION challenges are encountered during the menopause transition
due to the natural decline in ovarian function, the primary
source of estrogen and P4 (Luine, 2014). Natural menopause
is also associated with cognitive deficits and mood disorders
(Weber et al., 2012; Dumas et al., 2013). Such dysfunction
in mood and cognitive functions has also been reported in
women with surgical menopause (Chen et al., 2013; Faubion
et al., 2015). Physiological challenges during surgical menopause
are much more drastic due to a sudden depletion of ovarian
sex steroids as compared to progressive menopause which
follows fluctuating patterns of steroid levels (Bachmann, 2001;
Rodriguez and Shoupe, 2015; Rodríguez-Landa et al., 2015). This
results in greater predisposition to mood and anxiety disorders
when compared to natural menopause (Rodríguez-Landa et al.,
2015). Obsessive-compulsive disorder (OCD) is characterized by
intrusive thoughts (obsessions) and/or repetitive behaviors
(compulsive rituals) in response to the obsessions (American
Psychiatric Association, 2013). OCD has a lifetime prevalence of
around 2.3% in the United States (Ruscio et al., 2010) and it has
been listed as a common mental disorder in adults (Eaton et al.,
2008). The obsessive beliefs lead to compulsive symptoms among
patients. For example, contamination obsessions can result in
compulsive cleaning (Wheaton et al., 2010). Moreover, OCD can
negatively impact cognitive and affective functions in humans. Human studies involving neurocognitive tests and image analysis
showed impairments in non-verbal (Kashyap et al., 2013), spatial
working (van der Wee et al., 2007; Nakao et al., 2009) and visual
memories (Dirson et al., 1995). Associated comorbidities like
depression (Peris et al., 2010; Remijnse et al., 2013) and anxiety
disorders (Nestadt et al., 2001) are also very common in the OCD
condition. The impact of acute ovarian dysfunction during surgical
menopause on compulsive behaviors and comorbid affective
behaviors in females are currently poorly understood. In addition
to younger women, one out of eight women after the age
of 55 undergoes bilateral oophorectomy (surgical removal of
ovaries) before reaching natural menopause due to benign
diseases, prophylaxis against cancer and autoimmune disorders
(Shuster et al., 2010; Erekson et al., 2013; Cox and Liu, 2014). Existing studies have investigated obsessions and compulsions
only during and after progressive menopause with contradictory
evidence (Vulink et al., 2006). Citation: Mitra S, Bastos CP, Bates K, Pereira
GS and Bult-Ito A (2016) Ovarian Sex
Hormones Modulate Compulsive,
Affective and Cognitive Functions in a
Non-Induced Mouse Model of
Obsessive-Compulsive Disorder. Front. Behav. Neurosci. 10:215. doi: 10.3389/fnbeh.2016.00215 November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org Ovarian Steroids in Surgical Menopause Mitra et al. Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2016 | Volume 10 | Article 215 INTRODUCTION Interestingly, female hormones, such as estrogen and P4, regulate
various neurotransmitter signaling pathways in brain regions
implicated in OCD (Dreher et al., 2007; Karakaya et al., 2007;
Benmansour et al., 2009; Alonso et al., 2011; Quinlan et al., 2013;
Barth et al., 2015). During the estrous phase, circulating estrogen
levels are higher and serotonin release is lower in striatal neurons
(Yang et al., 2015), while in the frontal cortex, estrogen depletion
by ovariectomy (OVX) decreases 5-HT2A receptor density and
mRNA levels (Cyr et al., 1998). On the other hand, P4 increases
dopamine release mediated by NMDA receptor activation in
striatal neurons (Cabrera and Bregonzio, 1996) and decreases
NMDA binding density in the frontal cortex after OVX (Cyr
et al., 2000). Therefore, ovarian sex hormones may account for
the sex differences observed in OCD. How do already existing compulsions in females get affected
during acute sex hormone deprived conditions when compared
to non-compulsive females are not clearly known. The co morbid
anxiety and cognitive functions associated with OCD during
such a physiological state and the role of genetic background
in influencing steroid actions demands investigation. According
to Maio et al. (2014), our mice developed through selective
breeding for phenotypes of increased or decreased amounts of
compulsive-like behavior can be a heuristic tool for studying
OCD, especially the replicate BIG strains (BIG1 and BIG2). An unpublished study from our lab has shown that there is Women are subjected to hormonal fluctuations during their
entire life span, which may lead to significant alterations in mood
and cognition (Soares and Zitek, 2008). However, remarkable November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 2 Ovarian Steroids in Surgical Menopause Mitra et al. variation in compulsive-like and affective behaviors between
the two replicate BIG strains that mimics heterogeneity as
seen in subgroups of OCD patients. This study however did
not look into hormonal deprivation and manipulations. We
therefore investigated the hypothesis that acute deprivation of
estrogen and P4 through OVX for 7 days will increase the
compulsive-and anxiety-like behavior and impair novel object
recognition memory in compulsive-like mouse strains. We also
hypothesized that the administration of estrogen (E2) and P4 will
attenuate the exacerbation in compulsive-like, anxiety-like and
cognitive behaviors in compulsive-like strains. Though rodents
do not have menopause, surgical removal of the ovaries can
cause depletion of E4 and P4 (Kato et al., 2013). Surgical Procedures g
For the OVX study, animals were divided into two groups
for each strain. One group was sham operated while the
other group was OVX (removal of ovaries). All animals in
the hormone replacement studies were OVX. For the surgical
procedures, females were exposed to isofluroane (4% induction
and flow rate of 1.5–2 L/min) anesthesia. Abdominal incisions
were made longitudinally and bilaterally in the region below
the last lumbar vertebra. The ovary, oviduct and top of the
fallopian tubes were tied and removed in the OVX group. For the sham-operated mice, the procedure remained the same
except that the ovaries were not removed but only identified
(Fonseca et al., 2013). The abdominal wall and the skin were
sutured as described by Capettini et al. (2011). All animals were
provided ibuprofen in the drinking water 24 h prior to surgery
and maintained for 3–4 days post surgery as needed for pain
management. MATERIALS AND METHODS The University of Alaska Fairbanks Institutional Animal Care
and Use Committee approved the animal care and experimental
procedures (IACUC assurance numbers 568518 and 631126). E2 Administration For the E2 administration study, BIG1 and BIG2 females
were subdivided into two treatment groups: vehicle and E2
(n = 12 females per group). Seven days after OVX, the
vehicle group received a single subcutaneous injection of
corn oil while the E2 group received 0.1 mg/kg (acute
dosage of E2 produces comparable proestrus levels (Walf
et
al.,
2006))
of
E2
(Sigma,
St. Louis,
MO,
USA)
in
corn oil 44 h before behavioral assessments (compulsive
and
anxiety
tasks;
Walf
et
al.,
2008b). For
the
object
recognition task, the mice were injected immediately after
the training session and were tested 4 h later (Walf et al.,
2008a). A total gap of 5 days between each behavior was
employed. Experimental Subjects p
j
The mouse model of OCD used for this study was developed
from house mouse (Mus musculus) strains bidirectionally
selected for nest-building behavior (Lynch, 1980; Bult and Lynch,
2000). The stock population for the original selection experiment
(Lynch, 1980) was a cross among eight inbred strains, i.e., A,
AKR, BLB/c, C3H/2, C57BL, DBA/2, Is/Bi, and RIII, to yield
the HS/Ibg outbred strain (McClearn et al., 1970; Lynch, 1980). This resulted in two BIG strains (BIG1 and BIG2) that use
a forty-fold larger amount of cotton for their nest than the
two SMALL strains (SML1 and SML2) and two randomly-bred
control strains (C1 and C2) that show intermediate levels of
nesting (Lynch, 1980; Bult and Lynch, 2000). The BIG strains
engage in excessive and repetitive nest building (considered to
be homologous to hoarding in humans; Warneke, 1993) and
marble burying behavior which is dose-dependently attenuated
by fluoxetine and clomipramine, but not desipramine, treatment,
making the BIG mice a novel non-induced model for OCD
(Greene-Schloesser et al., 2011). INTRODUCTION We therefore
used bilateral OVX as the sex hormone deprived surgical
menopause model to achieve the experimental endpoints in this
study. (BIG1 and BIG2), randomly-bred Control strains (C1, C2) and
SMALL (SML1, SML2) strains, were used. For the hormone
replacement studies in OVX females, only compulsive-like
BIG1 and BIG2 female strains were used. All mice were
80–90 days of age during testing. All data were collected by an
individual blinded to the outcome of the study. (BIG1 and BIG2), randomly-bred Control strains (C1, C2) and
SMALL (SML1, SML2) strains, were used. For the hormone
replacement studies in OVX females, only compulsive-like
BIG1 and BIG2 female strains were used. All mice were
80–90 days of age during testing. All data were collected by an
individual blinded to the outcome of the study. P4 Administration For the P4 administration study, BIG1 and BIG2 females were
subdivided into vehicle and P4 groups (n = 9 females per group). Following 7 days of OVX, the vehicle group received corn oil
while the P4 group received 4 mg/kg of P4 1 h before behavioral
testing. For the object recognition task, the mice were injected
immediately after the training session and the test was performed
4 h later (Walf et al., 2008a). A gap of 3 days between the end
of each behavioral test and the next injection was employed. An
acute dosage of P4 used in this study approximates circulating
and central P4 levels observed during the proestrus phase (Walf
et al., 2006). Hormone Administration
E2 Administration All
mice
were
raised
in
polypropylene
cages
(27 cm × 17 cm × 12 cm) and provided with wood shavings
under a 12:12 light-dark cycle at 22 ± 1◦C. Weaning of the
pups was conducted at 19–21 days of age. All mice were housed
with same-sex and same-strain littermates until the end of all
the experiments. All mice were singly housed just before the
behavioral assessments and were returned to their home cages
with their littermates following each test. Food (Masuri Rodent
Diet #5663, Purina Mills, LLC, St. Louis, MO, USA) and water
were available ad libitum. Frontiers in Behavioral Neuroscience | www.frontiersin.org Nest-Building Nest-building behavior was used to assess the compulsive-like
phenotype of the female mice (Greene-Schloesser et al., 2011). All mice were housed individually and were allowed to access a
pre-weighed cotton roll placed in the cage top food hopper. The
amount of cotton used by the mice after 24 h was determined
by weighing the cotton roll. As all other behavioral assessments
in the P4 administration experiment were performed after 1 h
of P4 administration, nest building was measured for 1 h and
24 h of cotton availability, starting 1 h after the injection, to
be able to capture the short-term effects of P4 and also to be
able to compare this behavior to the 24-h nesting score of the
E2 administration experiment. Plasma Steroid Levels To establish that acute OVX leads to depletion of E2 and P4
plasma E2 and P4 levels were determined in plasma samples For the OVX study, female mice (Mus musculus) of six
different mouse strains i.e., two each of compulsive-like strains Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2016 | Volume 10 | Article 215 3 Ovarian Steroids in Surgical Menopause Mitra et al. mouse was placed in the central square facing an open arm and
was allowed to explore the maze for 5-min duration (Frye et al.,
2008). The time spent on the open arms was determined by the
ANYMaze video tracking program (Stoelting Co., Wood Dale,
IL, USA). The maze was cleaned before each test. (n = 5–7 per group) of OVX and sham operated compulsive-like
BIG female strains (BIG1 and BIG2). All samples were assayed
in duplicates using Cayman ELISA kits (Ann Arbor, MI, USA)
as per the manufacturer’s instructions. Data collection was
accomplished with a Biotek EL808 spectrophotometric plate
reader and analyzed by Prism software. Marble Burying The
marble-burying
test
was
also
used
to
measure
compulsive-like behavior (Takeuchi et al., 2002; Thomas
et al., 2009; Greene-Schloesser et al., 2011; Angoa-Pérez et al.,
2013). All mice were individually introduced to a polypropylene
cage (37 cm × 21 cm × 14 cm) containing 20 glass marbles
(10 mm in diameter) evenly spaced on 5 cm deep wood shavings
firmly pressed into a bedding without access to food or water
for 20 min. The total number of marbles buried at least 2/3 in
the 20-min period was quantified as compulsive-like digging
behavior (Greene-Schloesser et al., 2011). After the 20-min
test, the animals were returned to their home cages with
littermates. Statistical Analysis All data were analyzed using Statistical Analysis System (SAS)
software. A general linear model (GLM) repeated analysis
of variance (ANOVA), with strain (BIG, SMALL, Control),
replicate nested within strain (1, 2), treatment (OVX, sham), and
strain by treatment interaction effects was used to statistically
evaluate the effects of OVX on nest building behavior (grams of
cotton), marble burying behavior (number of marbles buried),
open field behavior (time in seconds in center), elevated plus
maze behavior (time in seconds on open arms), and novel object
recognition memory (RI). If the replicate nested within strain
effect was significant, the strain effect was tested over the replicate
effect. If the replicate effect was not significant, the strain effect
was tested over the error term. Open Field The open field test was performed to evaluate anxiety-like
behavior in female mice (Crawley, 1985; Meerlo et al., 1999). Female mice were singly housed outside the testing room just
prior to testing. The open field apparatus consisted of an open
field arena (40 cm × 40 cm × 30 cm). For testing, animals
were placed in the center of the field and allowed to explore
the arena for 3 min. Entries into the central square (20 cm
× 20 cm) (Greene-Schloesser et al., 2011) were recorded by
ANYMaze video tracking system (Stoelting Co., Wood Dale,
IL, USA). Total number of line crossings was also assessed for
sham and OVX strains. The apparatus was cleaned before each
test. A GLM ANOVA, with treatment (OVX, sham), strain (BIG1,
BIG2), and replicate by treatment interaction effects, was used to
statistically evaluate the effects of OVX on E2 (pg/mL) and P4
(ng/mL) plasma levels. A GLM ANOVA, with treatment (E2, vehicle or P4, vehicle),
strain (BIG1, BIG2), and strain by treatment interaction effects,
was used to statistically evaluate the effects of females, sex
hormone replacement in compulsive-like OVX females on nest
building behavior (1 and 24 h nesting scores), marble burying
behavior, open field behavior, elevated plus maze behavior and
novel object recognition memory. Frontiers in Behavioral Neuroscience | www.frontiersin.org Compulsive-Like Behaviors g
The novel object recognition test was performed to measure
object recognition memory (Antunes and Biala, 2012). Mice were
allowed to explore the open field arena (40 cm × 40 cm × 30 cm)
without any objects for 3 min during the habituation phase on
day 1. Twenty-four hours later on day 2, the training session
was performed and mice were introduced to two similar objects
(plastic toys) within a 5 cm distance in the open field arena for
3 min. Mice were then taken out of the arena and returned to
their home cages. After 4 h, one of the objects was replaced
with a novel object of different shape and size. Animals were
then reintroduced into the arena and allowed to explore the
objects for 3 min in the testing phase. Time spent exploring the
familiar and novel objects was recorded with ANYMaze video
tracking software (Stoelting Co., Wood Dale, IL, USA). The
preference of one object over another was assessed through the
Recognition Index (RI: time spent on novel object divided by the
time spent on novel and familiar object together; Fonseca et al.,
2013). Compulsive-Like Marble Burying Compulsive-Like Marble Burying
Acute OVX resulted in more marbles buried in BIG1 (post hoc
t(22) = 3.248, p < 0.004) and BIG2 (post hoc t(22) = 3.193,
p < 0.005) females when compared to the sham operated groups
(F(1,134) = 18.15, p < 0.0001). No significant differences were
observed between OVX and sham operated SMALL (SML1: post
hoc t(21) = 0.525, p > 0.60; SML2: post hoc t(22) = 1.028, p > 0.31)
and Control (C1: post hoc t(22) = 1.732, p > 0.09; C2: post hoc
t(22) = 0.650, p > 0.52) strains (Figure 1B), which explains the
significant strain by treatment interaction effect (F(2,134) = 3.49;
p < 0.034). BIG females buried more marbles than the
SMALL females, with the Control mice showing intermediate
values (F(2,3) = 24.24, p < 0.015). The replicate nested within
strain effect was also significant (F(3,134) = 5.56, p < 0.0013),
predominantly due to the SML1 females burying fewer marbles
than the SML2 females (sham: post hoc t(21) = 2.324, p < 0.05;
OVX: post hoc t(22) = 2.922, p < 0.008). FIGURE 1 | Compulsive-like behavior in BIG, SMALL and Control
strains. The data represent the mean (± SEM) for (A) nesting score in grams
between 0–24 h in nest-building test and (B) number of marbles buried in
marble burying test of the two replicates of the BIG, SMALL and Control
strains. ∗(p < 0.05), ∗∗(p < 0.001) and ∗∗∗(p < 0.0001) indicates significant
differences between sham and ovariectomy (OVX) groups. #(p < 0.05),
##(p < 0.001) and ###(p < 0.0001) indicate significant differences between
replicate strains. Elevated Plus Maze When
significance
was
found
appropriate
pairwise
comparisons were performed using the studentized range
test. The nesting scores were square root transformed to obtain
a more normal distribution (Bult and Lynch, 1996, 1997, 2000),
while the data are presented as non-transformed nesting scores. Anxiety-like
behavior
was
further
substantiated
by
the
elevated plus maze test. The plus maze apparatus consisted
of two open arms (5 cm × 40 cm) and two closed arms
(5 cm × 40 cm × 20 cm) at right angles to each other. Each Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2016 | Volume 10 | Article 215 4 Ovarian Steroids in Surgical Menopause Mitra et al. RESULTS p > 0.98) OVX strains when compared to their sham operated
controls (Figure 1A), which explains the significant strain by
treatment interaction effect (F(2,134) = 65.91, p < 0.0001). The
BIG strains built bigger nests than the SMALL and Control
mice (F(2,3) = 70.84, p < 0.0001). The replicate nested within
strain effect was also significant (F(3,134) = 10.59, p < 0.0001),
predominantly due to the BIG1 females building bigger nests
than the BIG2 females (sham: post hoc t(22) = 5.188, p < 0.0001;
OVX: post hoc t(22) = 2.666, p < 0.05). Acute OVX Increased Compulsive-Like
Behavior in Compulsive-Like BIG Strains Compulsive-Like Nesting Compulsive-Like Nesting
Acute OVX in compulsive-like BIG1 (post hoc t(22) = 8.983,
p < 0.0001) and BIG2 (post hoc t(22) = 11.51, p < 0.0001)
females resulted in significant increases of nesting behavior
when compared to the sham operated ones (F(1,134) = 77.60,
p < 0.0001). No significant increases of compulsive-like nesting
were observed in the SMALL (SML1: post hoc t(21) = 0.0045,
p > 0.99; SML2: post hoc t(22) = 0.794, p > 0.43) and Control
(C1: post hoc t(22) = 0.270, p > 0.78; C2: post hoc t(22) = 0.0150, Acute OVX Increased Anxiety-Like
Behavior in Compulsive-Like BIG Strains The data represent the mean (± SEM) for (A) time spent on center in open field and
(B) total number of line crossings and (C) time spent on open arm in elevated plus maze of the two replicates of the BIG, SMALL and Control strains. ∗∗∗(p < 0.0001)
and ∗(p < 0.05) indicates significant differences between sham and OVX groups. ###(p < 0.0001) indicate significant differences between replicate strains. FIGURE 2 | Anxiety-like behavior in BIG, SMALL and Control strains. The data represent the mean (± SEM) for (A) time spent on center in open field and
(B) t t l
b
f li
i
d (C) ti
t
i
l
t d
l
f th t
li
t
f th BIG SMALL
d C
t
l t
i
∗∗∗(
0 0001) FIGURE 2 | Anxiety-like behavior in BIG, SMALL and Control strains. The data represent the mean (± SEM) for (A) time spent on center in open field and
(B) total number of line crossings and (C) time spent on open arm in elevated plus maze of the two replicates of the BIG, SMALL and Control strains. ∗∗∗(p < 0.0001)
and ∗(p < 0.05) indicates significant differences between sham and OVX groups. ###(p < 0.0001) indicate significant differences between replicate strains. and BIG2 (t(22) = 4.934, p < 0.0001) females when compared
to the sham groups (F(1,134) = 30.14, p < 0.0001). No significant
differences were observed in Control (C1: post hoc t(22) = 0.0676,
p > 0.94 and C2: post hoc t(22) = 1.403, p > 0.17) and SMALL
(SML1: post hoc t(21) = 0.1833, p > 0.85 and SML2: post hoc
t(22) = 0.4394, p > 0.66) OVX strains when compared to the sham
operated mice (Figure 2C), which explains the significant strain
by treatment interaction effect (F(2,134) = 17.50, p < 0.0001). The
BIG females spent the most time on the open arms, followed by
the Control females, and the SMALL mice showed the highest
level of anxiety-like behavior (F(2,134) = 26.84; p < 0.0001). The replicate nested within strain effect was not significant
(F(3,134) = 0.86, p > 0.46). Acute OVX Increased Anxiety-Like
Behavior in Compulsive-Like BIG Strains Anxiety-Like Open Field Behavior
In the anxiety-like open field test, the BIG1 (post hoc t(22) = 5.697,
p < 0.0001), BIG2 (post hoc t(22) = 5.008, p < 0.0001), C1 (post hoc
t(22) = 6.272, p < 0.0001), C2 (post hoc t(22) = 5.927, p < 0.0001),
and SML1 (post hoc t(21) = 2.296, p < 0.032) OVX females
spent significantly less time in the center when compared to
the sham groups (F(1,134) = 119.24, p < 0.0001). No significant
difference was observed between OVX and sham groups in SML2
(post hoc t(22) = 1.286, p > 0.21) females for the time spent
in the center (Figure 2A), which explains the significant strain
by treatment interaction effect (F(2,134) = 10.55; p < 0.0015). No significant strain effect was found (F(2,3) = 6.51, p > 0.08),
although the Control strains tended to be the least anxious and
the SMALL strains the most, while the BIG strains tended to
be intermediate. The replicate nested within strain effect was
significant (F(3,134) = 22.87, p < 0.0001), predominantly due
to the C1 females spending less time in the center than the
C2 females (sham: post hoc t(22) = 5.582, p < 0.0001; OVX: post
hoc t(22) = 5.927, p < 0.0001). For total number of line crossings,
as a measure of locomotor activity (Figure 2B), no differences
were observed among the strains (F(2,3) = 0.10, p > 0.90) and
between sham and OVX groups (F(1,134) = 0.03, p > 0.80). FIGURE 1 | Compulsive-like behavior in BIG, SMALL and Control
strains. The data represent the mean (± SEM) for (A) nesting score in grams
between 0–24 h in nest-building test and (B) number of marbles buried in
marble burying test of the two replicates of the BIG, SMALL and Control
strains. ∗(p < 0.05), ∗∗(p < 0.001) and ∗∗∗(p < 0.0001) indicates significant
differences between sham and ovariectomy (OVX) groups. #(p < 0.05),
##(p < 0.001) and ###(p < 0.0001) indicate significant differences between
replicate strains. Anxiety-Like Elevated Plus Maze Behavior
Acute OVX resulted in less time spent on the open arms in
the elevated plus maze test for BIG1 (t(22) = 6.320, p < 0.0001) November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 5 Mitra et al. Ovarian Steroids in Surgical Menopause FIGURE 2 | Anxiety-like behavior in BIG, SMALL and Control strains. Plasma E2 and P4 Levels Declined in BIG
Strains Following Acute OVX Plasma E2 and P4 Levels Declined in BIG
Strains Following Acute OVX
Plasma
E2
levels
in
acute
OVX
BIG1
(post
hoc
t(9) = 5.501 p < 0.0001) and BIG2 (post hoc t(10) = 6.948
p < 0.0001) mice were significantly and similarly (strain:
F(1,19) = 0.00; p > 0.99; strain by treatment interaction:
F(1,19) = 2.99; p > 0.10) reduced when compared to the
sham
females
(treatment:
F(1,19)
=
63.23;
p
<
0.0001;
Figure 4). Acute OVX Increased Anxiety-Like
Behavior in Compulsive-Like BIG Strains sham operated C2 mice (post hoc t(22) = 2.569, p < 0.05), while
the other strains did not show significant differences (BIG1: post
hoc t(22) = 0.02763, p > 0.97; BIG2: post hoc t(22) = 0.2579,
p > 0.78; C1: post hoc t(22) = 0.4236, p > 0.66; SML1: post hoc
t(21) = 0.6280, p > 0.52; SML2: post hoc t(22) = 0.9393, p > 0.34). The Control females had significantly higher RIs than the BIG
and SMALL mice (F(2,134) = 37.70; p < 0.0001). The replicate
nested within strain (F(3,134) = 0.53, p > 0.66) and the strain by
treatment interaction (F(2,134) = 0.92, p > 0.40) effects were not
significant. Acute OVX Did Not Affect Recognition
Index (RI) for Compulsive-Like BIG Strains
in Novel Object Recognition (F(1,43) = 0.79, p > 0.37) and strain by treatment interaction
(F(1,43) = 1.24, p > 0.27) effects were not significant, indicating
that the BIG1 and BIG2 OVX females had similar marble burying
scores and responses to E2. P4 administration did not cause significant changes in the FIGURE 4 | Ovarian E2 and P4 plasma levels in BIG strains. The data
represent the mean (± SEM) for plasma (A) 17β-estradiol (E2) levels and
(B) progesterone (P4) levels of the BIG1 and BIG2 strains. ∗∗(p < 0.001) and
∗∗∗(p < 0.0001) indicates significant differences between sham and OVX
groups. ###(p < 0.0001) indicate significant differences between replicate
strains. (F
0 79 p > 0 37) and strain b
treatment interaction FIGURE 3 | Novel object recognition in BIG, SMALL and Control
strains. The data represent the mean (± SEM) for the recognition index (RI) in
the novel object recognition test between sham and OVX groups of the two
replicates of the BIG, SMALL and Control strains. ∗(p < 0.05) indicates
significant differences between sham and OVX groups. FIGURE 3 | Novel object recognition in BIG, SMALL and Control
strains. The data represent the mean (± SEM) for the recognition index (RI) in
the novel object recognition test between sham and OVX groups of the two
replicates of the BIG, SMALL and Control strains. ∗(p < 0.05) indicates
significant differences between sham and OVX groups. P4 levels were significantly reduced in the BIG2 (post hoc
t(12) = 8.665 p < 0.0001) but not in BIG1 (post hoc t(10) = 1.993
p > 0.058) OVX females when compared to their sham
counterparts (F(1,19) = 61.17; p < 0.0001; Figure 4), which
explains the significant strain (F(1,19) = 17.30; p < 0.0005) and
strain by treatment interaction (F(1,19) = 18.93; p < 0.0004)
effects. Compulsive-Like Nesting Was Attenuated by E2 But
Not P4 Administration Acute E2 administration resulted in a significant and similar
(strain by treatment interaction: F(1,43) = 0.00, p > 0.94) decline
of nesting scores in the BIG1 (post hoc t(22) = 3.000, p < 0.007)
and BIG2 (post hoc t(22) = 3.814, p < 0.001) OVX females when
compared to the vehicle controls (F(1,43) = 19.51; p < 0.0001;
Figure 5A). The BIG1 females used more cotton for their nest
compared to the BIG2 mice (F(1,43) = 5.69, p < 0.022), which
replicated the results in Figure 1A. FIGURE 4 | Ovarian E2 and P4 plasma levels in BIG strains. The data
represent the mean (± SEM) for plasma (A) 17β-estradiol (E2) levels and
(B) progesterone (P4) levels of the BIG1 and BIG2 strains. ∗∗(p < 0.001) and
∗∗∗(p < 0.0001) indicates significant differences between sham and OVX
groups. ###(p < 0.0001) indicate significant differences between replicate
strains. (F(1,43) = 0.79, p > 0.37) and strain by treatment interaction
(F(1,43) = 1.24, p > 0.27) effects were not significant, indicating
that the BIG1 and BIG2 OVX females had similar marble burying
scores and responses to E2. No significant differences were observed in the 1 h
(F(1,32) = 0.96; p > 0.33) and 24 h (F(1,32) = 3.47; p > 0.05)
nesting scores of acute P4 treated BIG1 and BIG2 OVX
females
when
compared
to
the
vehicle
treated
controls
(Figures 6A,B). For 1 h and 24 h nesting scores, the
strain (F(1,32) = 0.37; p > 0.54; F(1,32) = 1.05; p > 0.31,
respectively) and strain by treatment interaction (F(1,32) = 2.06;
p > 0.16; F(1,32) = 0.00; p > 0.95, respectively) effects were not
significant. P4 administration did not cause significant changes in the
number of marbles buried by BIG1 and BIG2 OVX females when
compared to the vehicle control mice (F(1,32) = 2.64; p > 0.11;
Figure 6C). The strain (F(1,32) = 3.22, p > 0.08) and strain by
treatment interaction (F(1,32) = 1.64, p > 0.20) effects were also
not significant. Acute OVX Did Not Affect Recognition
Index (RI) for Compulsive-Like BIG Strains
in Novel Object Recognition The RI was significantly reduced in OVX females compared to
sham operated mice (F(1,134) = 3.94; p < 0.05; Figure 3) with
the C2 OVX females having a significantly lower RI than the November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 6 Mitra et al. Ovarian Steroids in Surgical Menopause FIGURE 3 | Novel object recognition in BIG, SMALL and Control
strains. The data represent the mean (± SEM) for the recognition index (RI) in
the novel object recognition test between sham and OVX groups of the two
replicates of the BIG, SMALL and Control strains. ∗(p < 0.05) indicates
significant differences between sham and OVX groups. P4 levels were significantly reduced in the BIG2 (post hoc
t(12) = 8.665 p < 0.0001) but not in BIG1 (post hoc t(10) = 1.993
p > 0.058) OVX females when compared to their sham
counterparts (F(1,19) = 61.17; p < 0.0001; Figure 4), which
explains the significant strain (F(1,19) = 17.30; p < 0.0005) and
strain by treatment interaction (F(1,19) = 18.93; p < 0.0004)
effects. Acute E2 and P4 Administration in OVX
Female Mice
Compulsive-Like Nesting Was Attenuated by E2 But
Not P4 Administration
Acute E2 administration resulted in a significant and similar
(strain by treatment interaction: F(1,43) = 0.00, p > 0.94) decline
of nesting scores in the BIG1 (post hoc t(22) = 3.000, p < 0.007)
and BIG2 (post hoc t(22) = 3.814, p < 0.001) OVX females when
compared to the vehicle controls (F(1,43) = 19.51; p < 0.0001;
Figure 5A). The BIG1 females used more cotton for their nest
compared to the BIG2 mice (F(1,43) = 5.69, p < 0.022), which
replicated the results in Figure 1A. No significant differences were observed in the 1 h
(F(1,32) = 0.96; p > 0.33) and 24 h (F(1,32) = 3.47; p > 0.05)
nesting scores of acute P4 treated BIG1 and BIG2 OVX
females
when
compared
to
the
vehicle
treated
controls
(Figures 6A,B). For 1 h and 24 h nesting scores, the
FIGURE 4 | Ovarian E2 and P4 plasma levels in BIG strains. The data
represent the mean (± SEM) for plasma (A) 17β-estradiol (E2) levels and
(B) progesterone (P4) levels of the BIG1 and BIG2 strains. ∗∗(p < 0.001) and
∗∗∗(p < 0.0001) indicates significant differences between sham and OVX
groups. ###(p < 0.0001) indicate significant differences between replicate
strains. Compulsive-Like Marble Burying was Attenuated by
E2 But Not P4 Administration E2 and P4 Treatment Showed Strain Dependent
Decreases in Anxiety-Like Behavior in the Open Field
In the acute E2 administration group, the BIG1 OVX females
(post hoc t(22) = 4.245, p < 0.0005) spent more time in the
center when compared to the vehicle group (F(1,43) = 16.70, BIG1 (post hoc t(22) = 6.447, p < 0.0001) and BIG2 (post hoc
t(22) = 7.606, p < 0.0001) OVX females buried significantly less
marbles in the acute E2 treatment group when compared to the
vehicle groups (F(1,43) = 85.67, p < 0.0001; Figure 5B). The strain November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 7 Mitra et al. Ovarian Steroids in Surgical Menopause FIGURE 5 | E2 administration in OVX mice. The data represent the mean (± SEM) for (A) nesting score in grams, (B) number of marbles buried, (C) time in center
in open field, (D) time spent on open arms in elevated plus maze and (E) RI in novel object recognition of the BIG1 and BIG2 strains. ∗∗(p < 0.001) and
∗∗∗(p < 0.0001) indicates significant differences between vehicle and E2 treatment groups. #(p < 0.05) and ###(p < 0.0001) indicate significant differences between
replicate strains. FIGURE 5 | E2 administration in OVX mice. The data represent the mean (± SEM) for (A) nesting score in grams, (B) number of marbles buried, (C) time in center
in open field, (D) time spent on open arms in elevated plus maze and (E) RI in novel object recognition of the BIG1 and BIG2 strains. ∗∗(p < 0.001) and
∗∗∗(p < 0.0001) indicates significant differences between vehicle and E2 treatment groups. #(p < 0.05) and ###(p < 0.0001) indicate significant differences between
replicate strains. BIG2 females responded to E2 in a similar direction as the
BIG1 females. p < 0.0002; Figure 5C). No significant difference in the time
spent in the center was observed between E2 and vehicle
groups in BIG2 OVX females (post hoc t(22) = 1.564 p > 0.13). The significant strain effect (F(1,43) = 17.05; p < 0.0003) was
due to BIG1 E2 administered females performing better than
the BIG2 E2 administered females (post hoc t(22) = 4.178,
p < 0.0005). Compulsive-Like Marble Burying was Attenuated by
E2 But Not P4 Administration The strain by treatment interaction effect was not
significant (F(1,43) = 3.77, P > 0.058), which showed that the P4 administered groups in both BIG1 (post hoc t(16) = 4.311,
p < 0.0005) and BIG2 (post hoc t(16) = 3.904, p < 0.001)
OVX females spent significantly more time in the center
when compared to their respective vehicle control groups
(F(1,32) = 33.75; p < 0.0001; Figure 6D). The strain (F(1,32) = 2.17,
p > 0.15) and strain by treatment interaction (F(1,32) = 0.08, Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 8 Mitra et al. Ovarian Steroids in Surgical Menopause FIGURE 6 | P4 administration in OVX mice. The data represent the mean (± SEM) for (A) nesting score in grams between 0–1 h, (B) nesting score in grams
between 0–24 h, (C) number of marbles buried, (D) time in center in open field, (E) time spent on open arms in elevated plus maze and (F) RI in novel object
recognition of the BIG1 and BIG2 strains. ∗(p < 0.05) and ∗∗∗(p < 0.0001) indicates significant differences between vehicle and P4 treatment groups. #(p < 0.05)
indicate significant differences between replicate strains. FIGURE 6 | P4 administration in OVX mice. The data represent the mean (± SEM) for (A) nesting score in grams between 0–1 h, (B) nesting score in grams
between 0–24 h, (C) number of marbles buried, (D) time in center in open field, (E) time spent on open arms in elevated plus maze and (F) RI in novel object
recognition of the BIG1 and BIG2 strains. ∗(p < 0.05) and ∗∗∗(p < 0.0001) indicates significant differences between vehicle and P4 treatment groups. #(p < 0.05)
indicate significant differences between replicate strains. strain by treatment interaction (F(1,43) = 0.39, p > 0.53) effects
were also not significant. p > 0.77) effects were not significant, indicating that the
BIG1 and BIG2 OVX females spent similar times in the center
and responded similarly to P4. Overall, P4 administration significantly increased the time
spent on the open arms compared to the vehicle groups
(F(1,32) = 8.20, p < 0.0073; Figure 6E), which was significant
when comparing the BIG2 P4 treated to the BIG2 vehicle OVX
females (post hoc t(16) = 2.692, p < 0.014). Compulsive-Like Marble Burying was Attenuated by
E2 But Not P4 Administration Although the trend was
in the same direction as the BIG2 OVX females, no significant
difference was observed between P4 and vehicle groups of
BIG1 OVX females (post hoc t(16) = 1.358, p > 0.18). The strain E2 and P4 Improved Recognition Index (RI) in Object
Recognition Memory With a Replicate Effect Seen in
E2 Treatment Overall, E2 administration significantly increased performance
in the novel object recognition test compared to the vehicle
treated OVX females (F(1,43) = 26.95, p < 0.0001; Figure 5E),
which was significant for the BIG2 OVX females compared
to their vehicle treated counterparts (post hoc t(22) = 5.358,
p < 0.0001). Although the trend was in the same direction as the
BIG2 OVX females, no significant difference was observed in the
RI of E2 treated BIG1 OVX females compared to vehicle treated
females (post hoc t(22) = 1.946, p > 0.06), which explains the
significant strain by treatment interaction effect (F(1,43) = 5.55,
p < 0.024). The strain effect was not significant (F(1,43) = 1.93,
p > 0.17), which indicates that the BIG1 and BIG2 females had
overall similar memory scores. A prior study has shown that acute E2 administration
along with P4 to OVX rats reduced compulsive perseverance
in the T-maze (Fernández-Guasti et al., 2006). This is similar
to our findings in the compulsive-like mice though, E2 alone
had an attenuating effect in the OVX state. Interestingly,
previous findings show that acute P4 administration reduced
compulsive-like marble burying behavior in male rats (Umathe
et al., 2009). However, in the current study we did not see an
anti-compulsive effect of P4 treatment in BIG mice. This could be
due to various factors, including using mouse strains and females
in our study compared to male rats in the (Umathe et al., 2009)
study. P4 administration enhanced the performance in the novel
object recognition test for both the BIG1 (post hoc t(16) = 3.855,
p < 0.001) and the BIG2 (post hoc t(16) = 4.726, p < 0.0001) OVX
females compared to their vehicle control groups (F(1,32) = 114.3;
p < 0.0001; Figure 6F). The BIG1 OVX females had higher
RIs compared to the BIG2 OVX females (strain: F(1,32) = 7.32;
p < 0.011, irrespective of treatment group (strain by treatment
interaction: F(1,32) = 0.36; p > 0.55)). In the anxiety-like measures, the OVX BIG strains spent less
time in the center of the open field and also explored the open
arm less in elevated plus maze when compared to the sham
groups. The SMALL and the Control OVX strains showed no
significant changes in open field and elevated plus maze when
compared to their sham counterparts. Therefore, OVX worsened
anxiety-like behaviors in compulsive-like condition specifically. P4, But Not E2, Treatment Had an Effect on
Anxiety-Like Elevated Plus Maze Behavior No significant differences in the time spent on the open arm in
the elevated plus maze test was observed between E2 and vehicle
groups of BIG1 and BIG2 OVX females (F(1,43) = 1.81; p > 0.18;
Figure 5D). In addition, the strain (F(1,43) = 0.22, p > 0.63) and November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 9 Ovarian Steroids in Surgical Menopause Mitra et al. (F(1,32) = 0.63, p > 0.43) and strain by treatment interaction
(F(1,32) = 0.89, p > 0.35) effects were not significant, indicating
that the BIG1 and BIG2 OVX females spent similar times on the
open arms, and the BIG1 females responded to P4 in a similar
direction as the BIG2 females. higher nesting scores when compared to BIG2 sham females. This variation in compulsive-like nesting behavior was also
seen post acute OVX but was abolished in the E2 treatment
regimen. What is more intriguing is the fact that BIG1 and
BIG2 females did not exhibit variation in compulsive-like marble
burying. This is an interesting finding indicating heterogeneity
in the BIG strains based on compulsive-like traits and genetic
background as often seen in subgroups of OCD patients
(Fontenelle et al., 2005; Grados and Riddle, 2008; Leckman
et al., 2009). Whether E2 might be more effective compared
to P4 in reducing OCD symptoms in postmenopausal females,
as seen in our OVX compulsive-like mice, remains to be
elucidated. E2 and P4 Improved Recognition Index (RI) in Object
Recognition Memory With a Replicate Effect Seen in
E2 Treatment Acute administration of E2 resulted in increased time spent in
the central square of the open field in BIG1, but not BIG2 strains,
indicating a strain dependent effect in the E2 treatment response. The acute dosage and the time frame of administration of E2 in
our study previously also showed anxiolytic effects in the open
field in acutely OVX mice (Walf et al., 2008b) and rats (Walf
and Frye, 2009). In the plus maze no significant effect of E2 was
observed in the compulsive-like mice, which could be due to the
fact that the open field and the elevated plus maze tests measure
different aspects of emotionality associated with anxiety (Ramos,
2008; Anchan et al., 2014). Frontiers in Behavioral Neuroscience | www.frontiersin.org DISCUSSION In the current study we showed that acute OVX for 7 days
resulted in a significant increase in the compulsive-like nesting
and marble burying behaviors of BIG1 and BIG2 female mice. No increase in nesting and marble burying was observed for
the Control and SMALL strains, which shows the specificity
of the OVX effects for the compulsive-like condition. The
exacerbations in compulsive-like behaviors in BIG mice were
attenuated by acute subcutaneous administration of E2, but not
P4. Human studies have shown that gonadal steroids trigger
or precipitate mood disorders in women with a history of an
already existing disease condition when compared to women
without it (Hay et al., 1994; Schmidt et al., 1998; Clayton and
Ninan, 2010). Onset and exacerbation of OCD associated with
pregnancy and postpartum has been shown in human studies
(Neziroglu et al., 1992; Williams and Koran, 1997; Labad et al.,
2005; Uguz et al., 2007; Forray et al., 2010) establishing a strong
link between reproductive events and OCD. However, there
is lack of literature on how obsessions and compulsions and
associated affective and cognitive behaviors vary during induced
menopause. According to a review (Forray et al., 2010) a large
variation exists in human studies on onset and exacerbation of
OCD during reproductive events and one of the contributing
factors could be innate differences in patient populations. In
congruence with this we found that BIG1 sham females had P4 treatment, on the other hand, decreased anxiety-like
behavior in open field for both BIG1 and BIG2 strains. For
the elevated plus maze, however, P4 was effective only in
the BIG2 and not the BIG1 strain. These results indicate
a strain specific response to anxiety-like behavior due to
P4 administration. Strain specific effects of E2 and P4 on
behavioral responses have been sparsely explored in rodent
studies. Only one study has shown significant strain specific
effects of E2 on depressive-like forced swim behavior (Koss
et al., 2012). Behavioral responses to alterations in gonadal
steroids have been found to vary in women with and without
premenstrual syndrome (Schmidt et al., 1998), which might be
similar to the differences in behavioral responses to E2 and P4 in
the BIG1 and BIG2 strains. November 2016 | Volume 10 | Article 215 10 Ovarian Steroids in Surgical Menopause Mitra et al. The association of memory impairment with OCD is not
clear. American Psychiatric Association (ed.). (2013). Diagnostic and Statistical
Manual of Mental Disorders 5 Edn., Washington, DC: American Psychiatric
Press. DISCUSSION Many clinical studies have failed to find any evidence
that OCD is associated with memory deficits (McDonald, 1991;
Dirson et al., 1995; Radomsky and Rachman, 1999). In addition,
no impairment in declarative and short-term memory has been
found in OCD patients compared to normal controls (Roth
et al., 2004; Demeter et al., 2013). However, many others have
reported working memory impairments in patients (Martin et al.,
1995; Nakao et al., 2009). Our BIG mice showed a larger object
recognition memory deficit than the Control mice, both in the
sham and OVX groups. However, the SMALL mice also showed
a similar memory deficit and, therefore, whether this memory
deficit in the BIG mice was due to a genetic correlation between
compulsive-like behaviors and object recognition memory, or
was due to genetic differences between the two strains caused
by founder effects or random drift (Bult and Lynch, 1996, 2000),
remains to be elucidated. Also as the BIG and SMALL strains had
a RI below 0.5 they appeared to avoid the new object, while the
control mice had an index above 0.5 and appeared to favor the
new object. compulsive-like mouse strains only, which was restored by
E2 and not P4. This effect was similar for the spontaneously
compulsive-like BIG1 and BIG2 strains, unlike the associated
anxiety-like and cognitive-like behaviors, which displayed
differences between the BIG1 and BIG2 strains for both E2 and
P4 effects on these behaviors. We therefore hypothesize that
the associated comorbidities in the surgical menopause state in
the OCD condition might vary among individuals because of
genetic differences. Future studies will focus on investigating
effects of E2 and P4 on the chronic OVX state and also the
potential signaling pathways in the brain of compulsive-like
mice. Overall, the results presented here strengthen the face,
predictive and construct validities of the mouse model for
investigating heterogeneity associated with OCD during ovarian
failures in females. Anchan, D., Clark, S., Pollard, K., and Vasudevan, N. (2014). GPR30 activation
decreases anxiety in the open field test but not in the elevated plus maze test in
female mice. Brain Behav. 4, 51–59. doi: 10.1002/brb3.197 ACKNOWLEDGMENTS We thank the Biological Research and Diagnostics (BiRD)
Facility animal quarters staff for excellent routine animal care
and assistance with surgeries. We thank Malabika Maulik and
Shailesh Narayan Khatri for their valuable comments on the
manuscript. The current study supports a complex interplay of genetic
background and sex steroids during acute ovarian dysfunction
in the compulsive-like condition. We report exacerbation
of compulsive-like behaviors with trait specific intra-strain
variation during acute ovarian failure in the spontaneously FUNDING Research reported in this publication was supported by an
Institutional Development Award (IDeA) from the National
Institute of General Medical Sciences of the National Institutes
of Health under grant number P20GM103395 to SM. The
content is solely the responsibility of the authors and does
not necessarily reflect the official views of the NIH. The
work was also supported by College of Natural Sciences and
Mathematics (CNSM) and the Office of the Vice-Chancellor
for Research to SM and AB-I. CPB received a fellowship
from CAPES/Brazil for this project. These funding sources
did not have a role in the study design, data collection,
analysis, interpretation and submission of this article for
publication. AUTHOR CONTRIBUTIONS SM, CPB and KB conducted all experiments and performed
data analysis. SM and CPB lead manuscript writing efforts. AB-I
and GSP made significant contributions to research design, data
interpretations and manuscript preparation. Acute OVX did not result in significant object recognition
memory
impairment
in
the
compulsive-like
condition. Contradictory evidence exists as to whether acute OVX
leads to object recognition memory impairment in normal
mice (c57 strain). While few studies have shown memory
loss during acute OVX (Gresack and Frick, 2006; Rhodes and
Frye, 2006), others show the opposite (Willard et al., 2011;
Fonseca et al., 2013; Bastos et al., 2015). However, chronic OVX
has consistently caused poor performance in the novel object
recognition test (Fonseca et al., 2013; Bastos et al., 2015). Though
there was no overall effect in novel object recognition memory
among sham and OVX strains, E2 treatment improved the RI
in only the BIG2 but not the BIG1 strain. P4 treatment however
improved the RI in both the strains. In the current context of
investigation object recognition was performed to evaluate short
term or working memory impairments associated with OCD in
the mouse strains. However, object recognition encompasses
just one aspect of assessing otherwise very complex memory
consolidation and cognition process in rodents. In future studies
we aim to include a more robust assessment of both short-term
and spatial memory components. Angoa-Pérez, M., Kane, M. J., Briggs, D. I., Francescutti, D. M., and
Kuhn, D. M. (2013). Marble burying and nestlet shredding as tests of repetitive,
compulsive-like behaviors in mice. J. Vis. Exp. 2013:50978. doi: 10.3791/
50978 American Psychiatric Association (ed.). (2013). Diagnostic and Statistical
Manual of Mental Disorders 5 Edn., Washington, DC: American Psychiatric
Press.
Anchan, D., Clark, S., Pollard, K., and Vasudevan, N. (2014). GPR30 activation
decreases anxiety in the open field test but not in the elevated plus maze test in
female mice. Brain Behav. 4, 51–59. doi: 10.1002/brb3.197
Angoa-Pérez, M., Kane, M. J., Briggs, D. I., Francescutti, D. M., and
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compulsive-like behaviors in mice. J. Vis. Exp. 2013:50978. doi: 10.3791/
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conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Walf, A. A., and Frye, C. A. (2009). Effects of two estradiol regimens on
anxiety and depressive behaviors and trophic effects in peripheral tissues
in a rodent model. Gend. Med. 6, 300–311. doi: 10.1016/j.genm.2009. 04.004 Copyright © 2016 Mitra, Bastos, Bates, Pereira and Bult-Ito. This is an open-access
article distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution and 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. Walf, A. A., Koonce, C. Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2016 | Volume 10 | Article 215 REFERENCES J., and Frye, C. A. (2008a). Estradiol or diarylpropionitrile
administration to wild type, but not estrogen receptor beta knockout, mice
enhances performance in the object recognition and object placement tasks. Neurobiol. Learn. Mem. 89, 513–521. doi: 10.1016/j.nlm.2008.01.008 November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 14
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Kinetics and Mechanism of Oxidation of Diphenyl Sulphoxide by Chloramine-B in Aqueous Acid Medium
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Refereoces Polarographic measurements were performed
with Sx IQ-• mol dm-• CJ11 and 0.001% Triton
X·lOO at 1=0.5 mol dm- 8 (KN0 8 ) and at 35±0.1". Purified nitrogen gas was passed through the solu·
tions to remove dissolved oxygen. 1. J, BJJCRRUM, "Metal Ammlne Formation In Aqueous
Solution .. , Haase, Copenhagen, 19U: M. CAI.VIN and
K. W. Wn,SoN, J. Am. Ohsm. 8oc.,1945, 67, 200!1. J 2. H. M, IRVING and H. B. BOSSOTTI, .J, Ohsm. Soc., 1954,
2904; 195!1, 889'1. J !1. G. P. BJtNGUPTA, N. N. Gaosu and P. BANYAI., J. Indian Okem. Soc., communicated. Polarograms were recorded on a Toshniwal
CL02B digital polarograph. The DME had t=4 s
(open-circuit) at herr=49 em (35.25 em in case of
Cdli-BP system). All potentials refer to SCE. pH
measurements were made on a ELICO LI-120
digital pH-meter equipped with glass and calomel
electrodes. 4. G. BCHWARZltNBACH,
"Oomplexometric Titrations",
Methuen, London,1956, p. 60. J p
6. G. P. SENGUPTA and 0. B. BKRA, J, lnorg, NuoZ. Ohem.,
1981. 43, 21!19. J
11
11
h 6. G. P. SJtNGOPTA and 0. B. BaRA, J, I11aia11 Ohem. Soc.,
1984, 61, 806. h '[, T. B. HARltiNS and H. FR!tiSJCR, J, Am. Ohem. Soc.,
1956,78, 1148; H. T. B. BRITTON, Endeavour, 1943, 2,
160. The protonation constants of amino acids were
determined by Irving and Rossotti method 4 • 8. K. E. JA.BAI.PURWAI.A, K. A. VANKATACHAI.A:U: and
M. B. K.ABADI,J. Inorg. Nucl, Ohsm., 1964, 26, 1011,
102'1. 9. T. Moar.r.JtR, D. F. MAR'l'IN, L. c. TnoMPSON, B. F~tRRU!l, G. R. F~tiSTiti, and W. J. RAND.\!. I., Ohem. !Uv., 1966, 65, 1. 10
A
E
M ,, INDIAN CHBM. SOC., VOL, 67, APRIL 1990 ,, INDIAN CHBM. SOC., VOL, 67, APRIL 1990 unfavourable negative contribution of entropy may
arise due to the restriction of degrees of freedom
of solvent molecules in the vicinity of the metal
ion and this effect is very much pronounced with
metal ions of high charge10 and nature of the
ligand. The log K values reported here are
accurate to±0.3 log units and the values of l:l.G. l:l.H and l:l.S are reliable to±0.04 kcal mol-1 , ±0.5
kcal moi- 1 and 2 cal K- 1 mol-1 respectively. amino acids as secondary ligands have also been
studied by pH-titration method•. As no polarogra-
phic studies on ternary complexes of Cd11 with
glycine (GL), DL·c(.·alanine (AL)
and DL-valine
(VL) as primary ligands and BP as secondary ligand
have so far been made, we report the results herein. Experimental The order of stability of trivalent metal com-
plexes follows the sequence, Prlli > Ndiii < Smiii >
Gdiii. Such difference in the trend of stability
constants is not uncommon in the case of lantha-
nides11 and this may be traced to be due to varying
degree of stabilisations arising out of interaction
of 4/metal orbitals with ligand field 111• Stock solutions of the ligands glycine (B.D.H.),
oL-c(.·alanine (B.D.H.), DL·valine (SO's) and 2,2'·
bipyridyl (AnalaR) were prepared in double-distilled
water. Stock solution of CdCI 9.6H90 {SISCO)
was prepared in nitric acid to prevent hydrolysis
and its metal content was determined by titration
with
EDTA 8 •
'Mercury for polarography' (E. Merck) was used for the DME. '*Present address : Department of Chemistry, Faculty of
Science, P. 0. llox 9480, Garyounis University, Benghazi,
Libya. Results and Discussion The com-
plexes with BP were studied pH range 3-6 and the
pH was adjusted with HN0 8 •
The concentration
of free ligand was calculated from the pH and the
total amount present using appropriate pK values. The stability of mixed-ligand complexes can be
compared with that of simple complexes. The
equilibrium constant for the reaction, [Cd(L)+]+[Cd(BP)H] ~
[Cd(L)(BP)+]+[Cd~~+]
is given by the relation, {3) 6. log K=log /311 - (log {J01 +log /J10)
{3) 6. log K=log /311 - (log {J01 +log /J10)
{3 6. log K=log /311 - (log {J01 +log /J10) Plots of 6.£1111 vs log X for Cd11-amino acid and
-BP systems were not linear, thereby indicating the
stepwise complexation. The experimental data were
processed by the method of DeFord and Hume 8 • The statistical value of 6. log K for coordina-
tion of two different bidentate ligands for an
octahedral coordination sphere is -0.4 log units10
(for other situations,
the statistical values of
D. log K.'s may be less but are always negative). The
respective calculated values of 6, log K for ternary
systems, for example for I : I : 1 Cdli·L-BP com-
plexes, are - 0.90, -0.97 and -1.27. These higher
negative values suggest that the mixed species are
less stable than the simple 1 : 1 complexes, or in
other words, BP binds better to Cd11 than to the
binary Cd(L)+ complexes. This is due to the fact
that more coordination positions are available on
Cdli than on ML complexes. All the three amino acids and BP formed three
complexes i.e. Cd(L)+,
CD(L) 11,
Cd(L)jj and
Cd(BP)u. Cd(BP):+,
Cd(BP):+. The
overall
stability constants are given in Table 2. TABI.B 2-0VltRAT.I, S'l'ABII.I'l'Y 00NS'l'AN'l'S FOR BINARY
OOlloiPI,ltXJtS 01' 0ADMIUM(II) TABI.B 2-0VltRAT.I, S'l'ABII.I'l'Y 00NS'l'AN'l'S FOR BINARY
OOlloiPI,ltXJtS 01' 0ADMIUM(II)
I-0.5 mol dm-• (KNO,), Temp.=95"
System
log ~ 1
log {J •
Odii-GL
!.!5
6.96
Odii-AL
4.18
6.711
Cdii-VL
9.65
6.01
Odii-BP
4,60
7.80
log~. 9.99
9.06
8.06
9.89 Relative stability of a mixed complex with
respect to the parent binary complexes can also be
evaluated from the expression, p
i[Cd(L)p]+i [Cd(BP)p]~[Cd(L)m(BP)D]
where, m+n=p. Ternary systems : The mixed-ligand systems
were studied by keeping the concentration of amino
acid constant
while varying that of BP (pH
8.5- 9.5). The concentration of BP was varied in
the range (0.5- 6.0) x IO-• mol dm-s. Results and Discussion Dissociation constants (Table 1) of ligands were
calculated from the pH-titration data using Rossotti
and Rossotti's method•. For BP. the values given
are taken from literature8 • 10. A. E. MARTBI.I. and M. C.AI.VIN, ''Chemistry of Metal
Chelate Compounds", Prentice Hall, New York, 1961!, p. 59. J 11. J. L. BJSAR, G. B. CaoPPIN and J. V. QUAGI.I.AWO, J. Inorg. NucZ. Ohem., 196!1,14, 1601 ; 196!1, 25, 573. L K
d
Di
F " d 12. L. K. A. STAVgLY and T. R.'I.NDAI.I., Discuss. Fat"a.da.y
Soc., 1958, 16, 157. TABI.lt 1-DISSOCIA'l'ION CONSTANTS OF LIGANDS
I-0.5 mol dm-• (KN01 ), Temp .... 85°
Ligand
pK,
pK. Glyolne8
9.55
DJ:.·<~.-Alanine•
9.70
DL•Valine"
9.51
2,111-Bipyridylb
:1.40
11.80
•Present work. bRei. 6. Polarographic Studies on Mixed Ligand Com-
plexes of Cadmlum(n) with some Amino
Acids as Primary Ligands and 2,2'-Bipyri-
dyl as Secondary Ligand
ZUBAIDA KHATOON and KABIR·UD•DIN*
Department of Chemistry, Allgarh Muslim University,
Aligarh-20.\ 00.\
Mtat~usct"tpt ncesvea 17 .B'tbruarfll989, nvisea 11 September 1989,
accepted 10 No"ember 1989 Polarographic Studies on Mixed Ligand Com-
plexes of Cadmlum(n) with some Amino
Acids as Primary Ligands and 2,2'-Bipyri-
dyl as Secondary Ligand
ZUBAIDA KHATOON and KABIR·UD•DIN*
Department of Chemistry, Allgarh Muslim University,
Aligarh-20.\ 00.\
Mtat~usct"tpt ncesvea 17 .B'tbruarfll989, nvisea 11 September 1989,
accepted 10 No"ember 1989 Binary systems : The studies of binary and
ternary systems were made under identical condi·
tions 7 • The reduction of Cdu gave a single, reversible
and diffusion-controlled wave in absence and
presence of ligands. An increase in ligand concen-
tration or pH shifted the half-wave potential of Cdii
to more negative values indicating the complex
formation between the metal ion and the ligand. IN lERACIION of cadmium til) with amino acids
and 2,2'-bipyridyl (BP) has been investigated
polarographically by many workers1 • The ternary
systems of Cd11 with BP as primary ligand and '*Present address : Department of Chemistry, Faculty of
Science, P. 0. llox 9480, Garyounis University, Benghazi,
Libya. In case of amino acids as ligand, pH was adjus·
ted with KOH. The total concentration of amino 344 NOTBS acids was varied in the range (0.83-4.00) x to-s
mol dm-•. The complexes of Cd11 with amino acids
were studied in the pH range 7.0- 10.5. where, A= 1 +Po1[L-]+.Bos[L -]• +Po alL -]•
B=P1o+.Bu[L -]+Pu[L -]•
C==l'to+Pu[L -] Results and Discussion On increa-
sing the concentration of BP, the half-wave poten-
tials shifted towards more negative values. The
shifts were always greater than those observed for
single ligand systems. Schaap and McMasters
method 11 was used to determine the overall stability
constants of the mixed complexes. The mixing constant (Km) is given by 11 g
g
y
Km = [Cd(L)m(BP)Dl/[Cd(L)p]miP[Cd(BP)p]ulp
_
11
/ Rmlp pnlp
-l'mn l'op
po or log Km =log fJmD -lfp(m log /lop+n log fJpo) or log Km =log fJmD -lfp(m log /lop+n log fJpo) The stabilisation constant (K.) for ligands of
equal denticity is given by The equation used at constant ionic-strength is log K. =-log Km -log
~ ! 1
m. n. The equation used at constant ionic strength is
Foo(L, BP)==antilog (0 ·4!~nF b,.E118 +log ~)
=A+B(BP]+C[BP]11
{1)
where,
A= 1 +Po1[L-]+.Bos[L -]• +Po alL -]•
B=P1o+.Bu[L -]+Pu[L -]•
C==l'to+Pu[L -]
(2)
d
d
h
bi
bili
f q
g
Foo(L, BP)==antilog (0 ·4!~nF b,.E118 +log ~)
=A+B(BP]+C[BP]11
{1)
where,
A= 1 +Po1[L-]+.Bos[L -]• +Po alL -]•
B=P1o+.Bu[L -]+Pu[L -]•
C==l'to+Pu[L -]
(2)
d P D
d P
h
bi
bili
f Foo(L, BP)==antilog (0 ·4!~nF b,.E118 +log ~)
=A+B(BP]+C[BP]11
{1) Calculated values of log Km and log K, for 1 : 1 : 1,
1 : 2 : 1 and 1 : 1 : 2 complexes are listed in Table 4
for the ternary systems. The positive values of
mixing constants and stabilisation constants show
that the mixed complexes are relatively more
stable, than the simple binary complexes. The
stability order of 1 1 1 : I ternary complexes with
respect to the primary ligand (amino acid), was
found to be glycine ) alanine ) valine. {1) where, Results and Discussion MYRZABA:BV.A, lillectrokhim., 197ll, 14,1734.,
G
A
B
S
G
d
A
O 2. G. B. MAI.IK, B. P. SINGH and J.P. TANDON, Indian J. Ohem., Beet. A, 1977, 15, 838; Monatsh. Ohem., 1977,
108, 168 ; 1. D. JoSHI, Vidfla, 1976, 193, 199; J. Indian
Ohem. Boc., 1984, 61. 901. J The order with respect to oxidant (1.7) was
found out by half-life method 8 •
For all the runs,
rates were determined when 40% of the reaction was
over and from that rate constant was calculated. The fairly constant k values confirm the fractional
order with respect to oxidant (Table 1). The rate
of the reaction was independent of [sulphoxide]. The k values are fairly constant within the limits
of experimental error (Table 1). The rate increased
with increase in [Acid] and the plot of log k vs
log [H+] was linear with a gradient 1.82 (r, 0.997). 3. 0. N. Rsu,r.v, R. W. ScHMID and F, S. BADJUt, J, Chem. Educt., 1959, 36, 555, 619. J 4. H. M. IRVING and R. B. RossoTTI, J, Ohem. Boc., 195!1,
9904. 5. F. J. C. RossoTTI and H. B. Rossot'l'r, Acta Chem. Bcand., 1965, 9, 11'17. J 6. W. A. E. MCBRYD!t, Can. J, Cllem., 1965, 43, 3472, W. A. E. MCBRYD!t, Can. J, Cllem., 1965, 43, 347
R P MARTIN M M PB'l'I'l' BA114 BI
d J P
B 7. R. P. MARTIN, M. M. PB'l'I'l'·BA114:BI. and J.P. BCHARP'Y,
"Metal Ions in Biological Systems", Marcell Dekker,
New York, 1978, Vol. 2, Oh.l. d
J A
Ch
B
5 8. D. D. DEFORD and D. N. HOM:B, J, Am. Chem. Boc., 1951,
73,5821. Ch
B TABI.:B 1-EP'P'KCT OY RBACTANTII CONCIUfTRA'l'ION ON
RATIC Oil OXIDATION Olil' DIPB:BNYI. B"OX.PHOXIDB BY
0HI.ORAMINK•B (OAB)
Medium: Aqueous acetio a.oid (l: 1, v/v), [H+j=0.95
mol dm-•, Temp. =308 K
(Sulphoxlde J o
X 10"
moldm-•
3.U
3.44
8.44
s.u
8,44
8.44
1.98
11.'15
4.13
5.50
6.88
[OAB10
k
AoOH*
k
X 101
X 102 8
%
X 101 II
mol dm-•
0.93
1.68
1.65
2.32
~.1.78
3.70
1.63
1.68
1.63
1.63
1.68
6.8
6.9
6.11
6.6
6.11
6.7
6.4
6.3
6.5
7.11
6.8
50
55
60
65
101'•
(mol dm-•)
1.11
11.8
3.5
4.7
6.9
7.6
9.7
13.5
8.5
6.3
6.9
5.1
•[OAB)0 -1,68Xlll1 mol dm-•; (Snlphoxide]0 •3.44Xlll2
mol am-•. ,
9. W. B. SCHAAP and D. L. ll'lcJIUSTaRs,J • .A.m. Chem. Boc.,
1961, 83, 4699. J. INDIAN CBBM. Soc., VOL. 67, APRIL 1990 J. INDIAN CBBM. Soc., VOL. 67, APRIL 1990 Acknowledgement sulphate was
employed to maintain the ionic
strength. The authors thank the Chairman, Department
of Chemistry, for facilities. One of the authors
(Z.K.) thanks C.S.I.R., New Delhi, for the award
of a Senior Research Fellowship. The kinetic studies were carried out in SO% (v/v)
aqueous acetic acid at an ionic strength of 0.23 mol
dm- 11 under pseudo-first order conditions
(at
35±0.5"). The reaction was followed by estimating
the unreacted CAB iodometrically. Refereaces The stoichiometry (l : 1) of the oxidation was
determined with varying amounts of oxidant in
excess over the substrate. The products were
detected by tic. 1. B. A. Asn-Er.-NABKY and M. 8. Er.·EZABY, J, Inorg. Nucl. Ohem., 1978, 40, 789 ; B. L. JAIN, J. KISHAN and
R. 0. KAPOOR, Indian J, Ohsm.. Bee,, A, 1979. 18,
138; A. R. AGGARWAl., K. B. PANDlCYA and R. P. Bums, Indian J, Ohem., Beet. A, 1981, 20, 752 ; Ann. Okim. (Rome), 1981, 71, 387; R. KUMARJ, 0. P. S. CHAND:BI. and 0. M. Gu:r~A. J. Indian Ohem. Boc.,
1985,62, 740; E. A. MBMBKTKAZI:BV, M. U. ABII.OVA,
A. M. BuAI.DYB.AKVA, S. I. ZaDANOV and G. 1\il,
MYRZABA:BV.A, lillectrokhim., 197ll, 14,1734., -
+
C 11H 11SO.NC1Na +(C11H 11)sSO+Hs0-+
C 11H 11SO.NHs +(C.,H11 ) 11SO. + NaCl -
+
C 11H 11SO.NC1Na +(C11H 11)sSO+Hs0-+
C 11H 11SO.NHs +(C.,H11 ) 11SO. + NaCl Results and Discussion . J
i! Oh
S TABI.:B 1-EP'P'KCT OY RBACTANTII CONCIUfTRA'l'ION ON
RATIC Oil OXIDATION Olil' DIPB:BNYI. B"OX.PHOXIDB BY
0HI.ORAMINK•B (OAB) 10. B. P. MITCH:Bt.I.and H. BIGKI., J, i!m. Ohem. Soc., 1978,
160,1564. ~IA.RCUS 11. Y. ~IA.RCUS and I. E.r.IltZKK, Oo()Td. Ohem. Rev., 1969, 4,
273. Kinetics and Mechanism of Oxidation of
Ciphenyl Sulphoxide by Chloramine-&
in Aqueous Acid Medium
G. MANOALAM* and SUBBIAH MEENAKSHJSUNDARAM
Department of Chemistry, Faculty of Enalneering and
Technology, Aunamalai University,
.~
AnnamalaJnagar-608 002
Manuscrip~ received 18 April 1989, rtWUed 18 Beptembsr 1989,
accepted 10 Navember 1989 THE oxidation kinetics of diphenyl sulphoxide with
chloramine-B have been studied in aqueous
acetic acid medium. The reaction involves an
initial slow step followed by a rapid one, indicating
that the reaction follows complicated kinetics. TH The rate increased with the decrease in dielectric
constant of the medium (Table 1). The plot of
log k vs 1/D was linear with a positive slope, indi-
cating positive ion-dipole interactions. Addition
of one of the reaction products benzenesulphon•
amide (BSA) had no effect on the rate (Table 1). The rates decreased with the increase in ionic
strength of the medium (Table 1). Addition of
Cl- increased the rate very sharply. The reaction
was too fast and it could not be followed. (2) (2) and PoD and Pmo are the binary stability constants of
the simple metal complexes with amino acids and
bipyridyl, respectively ; /311, P11 and /311 are the
overall stability constants for the formation of the
ternary complexes, Cd(L)(BP)+, Cd(L) 1 (BP) and
Cd(L)(BP):, respectively. The values of overall
stability constants for the ternary systems are
given in Table 3. respectively ; /311, P11 and /311 are the
bility constants for the formation of the
omplexes, Cd(L)(BP)+, Cd(L) 1 (BP) and
:, respectively. The values of overall
onstants for the ternary systems are
able 3. VBRAI,I, Sl'ABII,I'l'Y CO!!iS'l'ANl'S :1'011. TJtRNARY
00MPI,JtXltS OF 0ADlloiiUM(II)
l dm-• (KNO,), Temp.=91i"
log {J11
log /111
BP
8.15
10.119
BP
7.18
10.05
BP
6.98
9,21
log fJu
10.17
10.14
9.89
TABI.Jt 4-VALUltS O]il Alog K, log Km AND log Ka Jil'OR
Cdii-L-BP SYS'l'ltMS
1-=0.Ii mol dm-• (KN01 ), Temp.=95°
Primary
llga.nd (L)
m
n
'P
Alog K
logKm
log Ka
GL
1
1
2
-0.90
1.011
D.'lll
II
1
8
-1.03
0.99
0.51
1
II
9
-1.58
0.49
0.01
AL
1
1
2
-0.97
0.80
0.50
II
1
8
-1.117
0.79
0.116
1
II
3
-1.34
0.57
0.09
VL
1
1
2
-1.117
0.32
0.011
II
1
8
-1.40
0.56
0.08
1
II
9
-1.62
0.09
-0.99
345 TABI.Jt 4-VALUltS O]il Alog K, log Km AND log Ka Jil'OR
Cdii-L-BP SYS'l'ltMS
1-=0.Ii mol dm-• (KN01 ), Temp.=95°
Primary
llga.nd (L)
m
n
'P
Alog K
logKm
log Ka
GL
1
1
2
-0.90
1.011
D.'lll
II
1
8
-1.03
0.99
0.51
1
II
9
-1.58
0.49
0.01
AL
1
1
2
-0.97
0.80
0.50
II
1
8
-1.117
0.79
0.116
1
II
3
-1.34
0.57
0.09
VL
1
1
2
-1.117
0.32
0.011
II
1
8
-1.40
0.56
0.08
1
II
9
-1.62
0.09
-0.99
345 TABI.Jt 4-VALUltS O]il Alog K, log Km AND log Ka Jil'OR
Cdii-L-BP SYS'l'ltMS TABI.Jt 9-0VBRAI,I, Sl'ABII,I'l'Y CO!!iS'l'ANl'S :1'011. TJtRNARY
00MPI,JtXltS OF 0ADlloiiUM(II)
1=0.5 mol dm-• (KNO,), Temp.=91i"
System
log {J11
log /111
OdlLGL-BP
8.15
10.119
Odll-AL-BP
7.18
10.05
Cdli-VL-BP
6.98
9,21
log fJu
10.17
10.14
9.89 345 Experimental Diphenyl sulphoxide was prepared by the
Friedel-Crafts reaction of benzene with thionyl
chloride1 • Chloramine-B (CAB) {sodium N-chloro-
benzenesulphonamidc) was prepared by partial
chlorination of benzenesulphonamide 11 •
Sodium The reaction was studied
at four different
temperatures 35, 40, 42.5 and 45". The values of 346
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Endoscopic removal of a gastric pharmacobezoar induced by clomipramine, lorazepam, and domperidone overdose: a case report
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von Düring et al. Journal of Medical Case Reports
https://doi.org/10.1186/s13256-019-1984-0
(2019) 13:45
CASE REPORT
Open Access
Endoscopic removal of a gastric
pharmacobezoar induced by clomipramine,
lorazepam, and domperidone overdose: a
case report
Stephan von Düring1*, Corinne Challet2 and Laurent Christin1
Abstract
Introduction: Gastric pharmacobezoars are a rare entity that can induce mechanical gastric outlet obstructions and
sometimes prolong toxic pharmacological effects. Certain medications, such as sustained-release forms, contain
cellulose derivatives that may contribute to the adhesion between pills and lead to the creation of an aggregate
resulting in a pharmacobezoar. Case reports are rare, and official guidelines are needed to help medical teams
choose proper treatment options.
Case presentation: Our patient was a 40-year-old Caucasian woman with borderline personality disorder and
active suicidal thoughts who was found unconscious after a massive drug consumption of slow-release
clomipramine, lorazepam, and domperidone. On her arrival in the emergency room, endotracheal intubation was
preformed to protect her airway, and a chest x-ray revealed multiple coffee grain-sized opaque masses in the
stomach. She was treated with activated charcoal followed by two endoscopic gastric decontaminations 12 h apart
in order to extract a massive gastric pharmacobezoar by manual removal of the tablets.
Conclusion: This case demonstrates that in the case of a massive drug consumption, a pharmacobezoar should be
suspected, particularly when cellulose-coated pills are ingested. Severe poisoning due to delayed drug release from
the gastric aggregate is a potential complication. Detection by x-ray is crucial, and treatment is centered on
removal of the aggregate. The technique of decontamination varies among experts, and no formal
recommendations exist to date. It seems reasonable that endoscopic evaluation should be performed in order to
determine the appropriate technique of decontamination. Care should be patient-oriented and take into account
the clinical presentation and any organ failure, and it should not be determined solely by the suspected medication
ingested. Thus, serum levels are not sufficient to guide management of tricyclic antidepressant intoxication.
Keywords: Pharmacobezoar, Clomipramine, Acute tricyclic antidepressant poisoning, Drug overdose, Sustainedrelease formulation
Background
A gastric bezoar results from an aggregation of foreign
bodies in the stomach. The major types are phytobezoars
(vegetable matter), trichobezoars (hair), and pharmacobezoars (ingested drugs) [1]. Pharmacobezoars are a rare
entity and result from an aggregation of pills ingested at
* Correspondence: stephan@vonduring.ch
1
Department of Internal Medicine, Emergency Medicine and Critical Care
Medicine, Groupement Hospitalier de l’Ouest Lémanique (GHOL), Nyon
Hospital, Chemin Monastier 10, 1260 Nyon, Switzerland
Full list of author information is available at the end of the article
the same time. Certain substances are more likely to
bond than others [2]. All types of gastric bezoars can induce mechanical gastric outlet obstruction, but
pharmacobezoars also present the risk of a prolonged
drug release with multiple peak plasma concentrations
and an increased risk of toxicity [3].
It is difficult to know when to suspect a pharmacobezoar,
but they should always be considered in cases of massive
drug consumption. Clinical presentations are not always
© The Author(s). 2019 Open Access 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.
von Düring et al. Journal of Medical Case Reports
(2019) 13:45
helpful. Abdominal x-ray may be helpful, but only when
the tablets are radio-opaque.
In this case report, we discuss the proper management
of multidrug gastric pharmacobezoars, because they are
a rare entity and no formal guidelines exist on how to
deal with them. Our patient’s case demonstrates that abdominal x-rays can be helpful to confirm initial
suspicions, but an early gastric endoscopy is necessary
for the diagnosis and management and furthermore
must be repeated until full extraction is achieved.
Case presentation
A 40-year-old Caucasian woman with training in human
resources but unemployed since 2014 due to borderline
personality disorder and active suicidal thoughts, was
found unconscious at home by her husband. She was a
nonsmoker and a social drinker. Her medical treatment
consisted of clomipramine 150 mg once daily and lorazepam 2.5 mg twice daily. She was rapidly transported to
the emergency room (ER) by ambulance with an oxygen
mask. On arrival, her vital signs were as follows: blood
pressure of 119/62 mmHg, heart rate of 62 beats/min,
and temperature of 35.0 °C. She was unalert with a
Glasgow Coma Scale score of 5/15 (E1 V1 M3) and
presented no protective airway reflexes. The result of
her cardiopulmonary examination was normal, and we
found no abdominal distention or guarding and no
masses on palpation. Neurological examination revealed
an unconscious patient with a slight reactive bilateral
miosis and no focal neurological deficits on cranial nerve
or peripheral neurological examination. Laboratory
Page 2 of 5
findings were within normal range, including a complete
blood count (hemoglobin of 133 g/L, white cell count of
6.2 × 109/L, platelet count of 153 × 109/L), coagulation
test, full electrolytes, kidney and liver function tests. Arterial blood gas showed a nonhypoxemic respiratory
acidosis (pH 7.34, partial pressure of oxygen 56.9 kPa,
partial pressure of carbon dioxide 6.2 kPa, bicarbonate
24.8 mmol/L). We proceeded to perform an endotracheal intubation (propofol 50 mg, fentanyl 50 μg,
suxamethonium chloride 70 mg, rocuronium 50 mg, propofol 100 mg/h, and a slow drip of 250 ml of Ringer’s
lactate solution) followed by a chest x-ray that revealed
multiple coffee grain-sized opaque masses in the
stomach. Empty blister packs found around her by
paramedics suggested an ingestion of up to 8,625 g of
slow-release clomipramine (Anafranil SR® 75 mg; Novartis Pharma Schweiz AG, Switzerland), 125 mg of lorazepam (Temesta® 2.5 mg; Pfizer PFE Switzerland GmbH),
and 160 mg of domperidone (Motilium® 10 mg;
Janssen-Cilag AG). In accordance with the national poisons information center (Tox Info Suisse, Zürich,
Switzerland), we started a multidose activated charcoal
(AC) regimen (60 g loading dose in the ER, completed
by 30 g every 6 h for 24 h), followed by a gastric endoscopy that found an important pharmacobezoar extending from the fundus to the great curvature of the
stomach (Fig. 1). Three liters of normal saline were used
in the stomach to fragment the aggregate, and a manual
extraction of the tablets was performed with a wire basket with partial success. The patient was admitted to the
intensive care unit (ICU) for mechanical ventilation and
Fig. 1 Gastric endoscopy shows pharmacobezoar extending from the fundus to the great curvature of the stomach after activated charcoal
decontamination with intact and broken tablets
von Düring et al. Journal of Medical Case Reports
(2019) 13:45
further observation, and she never showed any signs of
cardiovascular disturbance. Her treatment in the ICU
consisted of fentanyl 0–50 μg/h, enoxaparin 40 mg subcutaneously once daily, AC 30 g every 6 h, and glucosaline intravenous drip 1 L/day. Eight hours after ICU
admission, another abdominal x-ray (Fig. 2) confirmed a
persistent gastric tablet aggregate, and a second attempt
at gastroscopic extraction was performed. The patient
gradually awakened and was weaned off mechanical ventilation after 30 h. She was transferred to our psychiatric
unit on day 3 for further care. During her stay, she had
normal electrolytes, hemoglobin, and white blood cell
count. She never presented any hemodynamic instability,
had no QT interval prolongation on electrocardiogram
or any arrhythmias, and we had no clinical argument for
seizures. No therapeutic drug monitoring of clomipramine was performed at baseline or during the patient’s
hospital stay because she improved and recognized
having taken all the tablets from the empty blister
packs found at her home. She was hospitalized in our
local psychiatric hospital and discharged after 10 days.
Regular follow-up with her psychiatrist was uneventful
for the next 2 years.
Discussion
This patient was brought to our ER in a comatose
state after a massive drug intake. She was treated for
a gastric pharmacobezoar, and her evolution was
favorable. Despite bezoar removal, the treatment of
this patient was based essentially on supportive care.
Unlike some papers which suggest that tricyclic antidepressant (TCA) serum levels are needed to guide
therapy, we believe that this is not patient-oriented
Fig. 2 Abdominal x-ray confirming presence of gastric pharmacobezoar
Page 3 of 5
care because TCA serum levels correlate poorly with
outcome. In general, treatment should be based on
the known properties of the ingested substance(s), the
clinical presentations, and the organ failures. Serum
levels should be considered solely when they impact
the treatment (for example, paracetamol intoxication).
To our knowledge, this concept of care is rarely promoted but is essential and in line with the current
“less is more” movement of 21st-century medicine.
Several factors may have contributed to the pharmacobezoar formation in our patient, including
massive tablet ingestion (estimated at 181 tablets) and
clomipramine’s characteristics (Table 1). In addition
to their inhibition of norepinephrine and serotonin
reuptake, TCAs possess antihistaminic and anticholinergic effects [4, 5]. This latter can impair gastric motility, compromising gastric emptying and favoring
bezoar formation in cases where concomitant predisposing factors are present. Moreover, the clomipramine ingested by our patient was a sustained-release
formulation (Anafranil SR®) containing hypromellose,
a cellulose derivative. We suggest that this compound
may have formed a gel-like layer in the stomach and
could be responsible for the adhesion with other pills
when taken together, and that it thereby provoked an
aggregation resulting in a pharmacobezoar, in a
similar manner to cellulose acetate [6].
According to the GEMNet TCA overdose guidelines, AC may be considered within the first hour of
TCA ingestion to reduce drug absorption and
bioavailability, but there is no substantial evidence
that it is of benefit [5]. Multiple doses should not be
continued thereafter, owing to the risk of pulmonary
von Düring et al. Journal of Medical Case Reports
(2019) 13:45
Page 4 of 5
Table 1 Characteristics and risks related to overdose of the drugs implicated in this case
Drug name
Maximal oral daily
dosing (adult) 1
Half-life (adult)
Intoxication signs (non-exhaustive list) 2
Risk factors for bezoar’s
formation in the reported case
Clomipramine 250 mg/d
• Clomipramine: 19 - 37 h
• Tachycardia, hypotension, orthostasis
• Desmethylclomipramine
• PR, QRS, QTc intervals prolongation
(active metabolite): 54 – 77 h • Anticholinergic effects (e.g. blurred vision,
delirium, agitation, hallucinations, mydriasis,
sinus tachycardia, urinary retention)
• Seizures, sedation
Domperidone 30 mg/d
~7h
• QT interval prolongation, cardiac arrhythmia • Massive tablet ingestion
• Restlessness, drowsiness, insomnia, headache,
(i.e. 16 tablets, 160 mg)
confusion, dizziness and acute dystonic
reactions, akathisia, parkinsonian-like symptoms
• Diarrhea, dry mouth
Lorazepam
~ 12 h
• Sedation, dizziness, amnesia, ataxia, slurred
speech, lethargy
• Hypotension
• Hypothermia, respiratory failure
• Coma
10 mg/d
• Anticholinergic effect
• Sustained-release formulation
with hypromellose component
• Massive tablets ingestion
(i.e. 115 tablets, 8,625 g)
• Massive tablet ingestion
(i.e. 50 tablets, 125 mg)
References:
[1] Lexicomp mobile software, Lexi-Drugs, Hudson, Ohio: Wolters Kluwer Clinical Drug Information, Inc., July 14, 2016
[2] Lexicomp mobile software, Lexi-Tox, Hudson, Ohio: Wolters Kluwer Clinical Drug Information, Inc., July 14, 2016
aspiration due to the delayed gastric emptying. Some
authors suspect that AC itself could also exacerbate
pharmacobezoar formation by participating in the
adhesion process [1]. No specific antidote to TCA
exists.
Endoscopic gastric decontamination and pill removal
is the method of choice to manage a bezoar. However,
the repetition of required gastroscopies can result in
gastric hemorrhages and be deleterious. [7, 8]. In order
to fragment the pill aggregate for a proper retrieval, a
gastroscopic water-jet fragmentation can be performed
for aspiration or basket retrieval. A risk encountered
with this approach could be a larger release of active
drug from the conglomerate and an increased absorption associated with toxicity. Thus, its pros and cons,
as well as the patient’s clinical status, must be evaluated before proceeding. Some authors have used the
acid proprieties of soft drinks to dissolve a bezoar,
but to our reasoning this can only be used in non-imminently lethal cases with a type of bezoar that can
be dissolved by acid [9]. Surgical removal of the bezoar may be necessary in some cases but was not
considered for our patient, owing to the good evolution she demonstrated.
Seizures can occur with TCA overdose, usually
beginning early after ingestion, and are usually brief,
self-limited, generalized tonic-clonic seizures [10]. The
underlying mechanism could be related to the antagonist
effects of TCAs on the γ-aminobutyric acid type A
receptor. Our patient presented no sign of seizures during
hospitalization, probably in part due to the 125 mg of lorazepam she had ingested simultaneously. TCA can induce
marked cardiovascular deterioration [11, 12]. Rhabdomyolysis has also been described in rare cases, but the
mechanism remains unclear [13]. These complications
were not observed in our patient.
Conclusion
Our patient was admitted for a drug overdose of
slow-release clomipramine, lorazepam, and domperidone
resulting in a gastric pharmacobezoar, and she was treated
by AC and endoscopic gastric decontamination. The
massive tablet ingestion, as well as the sustained-release
form of clomipramine, contributed to bezoar formation
[1, 7]. Moreover, the anticholinergic properties of clomipramine may have decreased gastrointestinal motility,
counteracting the effect of domperidone.
In any case of drug overdose, a pharmacobezoar
should be suspected, especially when cellulose-coated
pills are ingested. Because severe poisoning due to delayed drug release from the gastric aggregate is a potential complication, detection by x-ray is a reasonable first
examination [3, 8]. Endoscopic evaluation is mandatory,
and management by endoscopic or surgical removal
should be considered rapidly.
Serum levels are not useful to guide management of
TCA intoxication because they correlate poorly with
clinical effects. They do not help to predict toxicity, even
if high serum levels are usually associated with severe
toxicity. A qualitative test may be performed to confirm
an exposure to a TCA if suspected. False-positive results
of urine TCA screenings are common.
In light of the wide variety of drugs often implicated in
drug overdoses, our patient’s case demonstrates why
patient-oriented care should not be based solely on the
pharmacological properties of the individual substances.
Several factors play a role in patient care: the combination
of known chemicals ingested, the clinical presentation
von Düring et al. Journal of Medical Case Reports
(2019) 13:45
corresponding to the pharmacological effect, and the
organ failure induced. It seems reasonable to assume that
our patient’s favorable outcome was due to the rapid
partial removal of the pharmacobezoar and to the AC
regimen, even if this latter treatment is controversial in
the case of bezoars.
Page 5 of 5
7.
8.
9.
10.
Abbreviations
AC: Activated charcoal; ER: Emergency room; ICU: Intensive care unit;
TCA: Tricyclic antidepressants
11.
12.
Acknowledgements
None.
13.
Funding
No funding was received for this study.
Availability of data and materials
All data generated or analyzed during this study are included in this
published article.
Authors’ contributions
SVD wrote the first draft of the manuscript. SVD and LC were in charge of
clinical patient care. CC was responsible for pharmacological knowledge and
guidance. CC and LC reviewed the manuscript. All authors read and
approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
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.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
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Author details
1
Department of Internal Medicine, Emergency Medicine and Critical Care
Medicine, Groupement Hospitalier de l’Ouest Lémanique (GHOL), Nyon
Hospital, Chemin Monastier 10, 1260 Nyon, Switzerland. 2Department of
Pharmacy, Pharmacie Interhospitalière de la Côte, Chemin du Crêt 2, 1110
Morges, Switzerland.
Received: 13 July 2018 Accepted: 16 January 2019
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Efeito da contaminação por saliva na resistência adesiva de braquetes cerâmicos utilizando uma resina ortodôntica hidrofílica
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Resumo Introdução: Alguns fatores, como a presença de saliva, são capazes de influenciar a adesão do braquete ao dente
durante o procedimento de colagem e podem causar falha da resistência adesiva. Objetivo: O objetivo deste
estudo foi avaliar a resistência ao cisalhamento de braquetes cerâmicos ortodônticos colados com Transbond XT
e Transbond Plus Color Change em esmalte de dentes bovinos, contaminado e não contaminado por saliva, além
de analisar o local da falha adesiva. Material e método: Sessenta incisivos bovinos foram divididos aleatoriamente
em quatro grupos (n=15), de acordo com o material de colagem e a presença de contaminação: Grupo 1
(G1): colagem com Transbond XT na ausência de contaminação; Grupo 2 (G2): colagem com Self Etching Pimer
e Transbond Color Change na ausência de contaminação; Grupo 3 (G3): colagem com Transbond XT na presença
de contaminação, e Grupo 4 (G4): colagem com Self Etching Primer e Transbond Color Change na presença de
contaminação. Resultado: O teste não paramétrico de Kruskal-Wallis mostrou que G1 diferiu estatisticamente
(p<0,05) de G2 e G3. Não houve diferença estatística significativa entre os demais grupos. O índice de adesivo
remanescente (IAR) variou entre 2 e 3 no G1 e entre 0 e 1 nos outros grupos. Conclusão: A contaminação por
saliva diminui a resistência adesiva ao cisalhamento de braquetes cerâmicos colados com a resina hidrofóbica
Transbond XT convencional. Por outro lado, a utilização da resina hidrofílica Transbond Plus Color Change
associada ao Self Etching Primer, em ambiente contaminado por saliva, confere resistência adesiva adequada
para o seu uso clínico. Descritores: Adesivo; braquetes ortodônticos; resistência ao cisalhamento; saliva. REVISTA DE ODONTOLOGIA DA UNESP REVISTA DE ODONTOLOGIA DA UNESP ARTIGO ORIGINAL Rev Odontol UNESP. 2018 May-June; 47(3): 131-136
Doi: https://doi.org/10.1590/1807-2577.02818 Rev Odontol UNESP. 2018 May-June; 47(3): 131-136
Doi: https://doi.org/10.1590/1807-2577.02818 Efeito da contaminação por saliva na resistência adesiva de
braquetes cerâmicos utilizando uma resina ortodôntica hidrofílica Luís Filipe Siu LONa*, Patrícia Pigato SCHNEIDERa, Dirceu Barnabé RAVELIa,
Denise Constance NASCIMENTOb, Odilon GUARIZA-FILHOb Luís Filipe Siu LONa*, Patrícia Pigato SCHNEIDERa, Dirceu Barnabé RAVELIa,
Denise Constance NASCIMENTOb, Odilon GUARIZA-FILHOb aUNESP – Universidade Estadual Paulista, Faculdade de Odontologia de Araraquara, Araraquara, SP, Brasil
bPUCPR – Pontifícia Universidade Católica do Paraná, Escola de Ciências da Vida, Curitiba, PR, Brasil Abstract Introduction: Some fator, such as the presence of saliva, are able to influence the adhesion of the bracket to the
tooth enamel during the orthodontic bonding procedure and may cause the adhesive failure. Objective: The aim
of this study was to evaluate the shear strength of orthodontic ceramic brackets bonded with Transbond XT
and Transbond Plus Color Change to bovine teeth, under saliva contamination and the adhesive failure mode. Material and method: Sixty bovine permanent incisors were randomly divided into 4 groups (n=15) according to
bonding material and saliva contamination: Group 1 (G1) bonded with Transbond XT with no saliva contamination;
Group 2 (G2) bonded with Self Etching Primer and Transbond Plus Color Change with no saliva contamination;
Group 3 (G3) bonded with Transbond XT with saliva contamination and Group 4 (G4) bonded with Transbond
Plus Color Change with saliva contamination. Result: The Kruskal-Wallis non-parametric test (p<0,05) was used
and showed that G1 differed statistically from G2 and G3. There was no statistical difference between the other
groups. The adhesive remnant index (ARI) ranged between 2 and 3 in G1 and between 0 and 1 score in the other
groups. Conclusion: Saliva contamination decreases the shear bond strength of ceramic brackets bonded with
conventional Transbond XT hydrophobic resin. On the other hand, the Transbond Plus Color Change hydrophilic
resin associated with a Self Etching Primer, under saliva contamination, provide a sufficient shear bond strength,
suitable for their clinical use. INTRODUÇÃO descalcificações, trincas e fraturas. Os dentes foram armazenados
para desinfecção em Cloramina-T 0,5%, durante sete dias. Após este
período, os tecidos moles foram removidos com o auxílio de curetas
periodontais (Duflex, Juiz de Fora, Brasil). As raízes dentárias foram
seccionadas no terço médio e a polpa dentária foi removida com
lima endodôntica Hedstron (Maillefer, Ballaigues, Suíça). Após esse
preparo, os dentes foram armazenados em água destilada, a 4°C,
trocada semanalmente. Foi com Newman1, em 1965, que surgiu a ideia da técnica do
condicionamento ácido para a fixação de braquetes ortodônticos,
substituindo o uso de bandas ortodônticas em todos os dentes. Isto se tornou possível devido ao conceito do condicionamento
ácido do esmalte introduzido por Buonocore2. O condicionamento ácido do esmalte permite uma melhor
penetração do adesivo nas microporosidades produzidas pela
desmineralização do esmalte e, consequentemente, aumenta a área
de superfície de contato, favorecendo a capacidade de molhamento
e um melhor escoamento do sistema adesivo3. Os sistemas adesivos
tradicionais utilizados em Ortodontia apresentam propriedades
hidrofóbicas e requerem superfícies secas para ter um desempenho
clínico aceitável4. Previamente à colagem, foi realizada profilaxia com taça de
borracha e pasta de pedra pomes (SS White, Rio de Janeiro, Brasil)
com micromotor em baixa rotação, durante 10 segundos, na região
da futura colagem (superfície mais plana do terço incisal vestibular
da coroa). Utilizou-se uma taça de borracha para cada cinco dentes,
para padronização do procedimento. Em seguida, os dentes foram lavados com jatos de água destilada
da seringa tríplice por 20 segundos e secos com ar comprimido isento
de umidade ou óleo, por 20 segundos. Após a profilaxia, os 60 corpos
de prova foram divididos aleatoriamente em quatro grupos (n=15)
e braquetes cerâmicos monocristalinos Radiance, slot retangular
0,022”, prescrição de Roth (American Orthodontics, Sheboygan,
WI, USA) para incisivos inferiores, com área da base de 10,63 mm2,
foram colados nas coroas dentárias, conforme o Quadro 1. Alguns fatores, como a presença de fluidos bucais (saliva, fluidos
gengivais e sangue), são capazes de influenciar negativamente a qualidade
da adesão do braquete ao dente durante o procedimento de colagem,
causando falha na interface adesiva esmalte dentário-braquete3,4. Devido à dificuldade de isolamento, as áreas mais críticas para
esta contaminação são a região dos segundos molares e as regiões
próximas ao tecido gengival, principalmente durante a colagem
cirúrgica de acessórios ortodônticos em dentes a serem tracionados5-7. INTRODUÇÃO No G1 (utilizado como grupo controle), o esmalte foi condicionado
com ácido fosfórico 37% por 15 segundos, lavado por 10 segundos
e seco por 10 segundos. Seguiu-se a aplicação do adesivo Transbond
XT primer (3M/Unitek, EUA), a inserção da Transbond XT
(3M/Unitek, EUA) na base do braquete, com o posicionamento sobre
a porção mais plana da porção incisal da coroa. Para padronizar
a espessura da resina, foi aplicada uma carga de 400 gf de pressão
com dinamômetro (Morelli, Sorocaba, Brasil) e os excessos da resina
escoada foi realizada com uma sonda exploradora. Os braquetes
foram fotopolimerizados com o aparelho fotopolimerizador, de
luz halógena, Optilux, 500 (Demetron Kerr, Danbury, CT), com
irradiância de 600 mW/cm2 por 10 segundos, com o feixe de luz
incidindo através dos braquetes cerâmicos, a uma distância de 5 mm
entre o feixe de luz e o braquete, conforme instruções do fabricante. Diante das dificuldades encontradas no procedimento de colagem
e dos materiais utilizados em Ortodontia, surgiram os sistemas
adesivos hidrofílicos, que possibilitaram uma colagem ortodôntica
mais segura, mesmo em condições de umidade ou contaminação6,7. A partir de então, desenvolveram-se novos sistemas adesivos
hidrofílicos e primers autocondicionantes, reunindo os passos
do condicionamento ácido e primer em frasco único; assim, tais
sistemas foram desenvolvidos com o objetivo de reduzir os passos
clínicos deste procedimento e, consequentemente, diminuir as
fontes de contaminação8. Entretanto, o uso destes sistemas adesivos
autocondicionantes com as resinas convencionais, hidrofóbicas,
reduziria grande parte desta capacidade. Desta forma, na tentativa de
reduzir os problemas de contaminação, os fabricantes introduziram
o Transbond Color Change (3M Unitek, St. Paul, MN, EUA), um
compósito resinoso com características hidrofílicas. No G2, utilizou-se um primer acidificado autocondicionante
(Self Etching primer, SEP, 3M/Unitek, EUA), o qual foi mantido
em contato com o esmalte por 10 segundos. Seguiu-se a inserção
da Transbond Plus Color Change (3M/Unitek, EUA) na base do
braquete, que foi posicionado na porção central do esmalte sob
pressão de 400 gf e fotopolimerizado por 10 segundos. A crescente procura de pacientes adultos por tratamento
ortodôntico, em busca de correção da oclusão e estética, aumentou
também o interesse pelo uso de braquetes cerâmicos. Neste contexto,
este trabalho tem por objetivo avaliar a resistência ao cisalhamento
de braquetes cerâmicos colados com a resina hidrofóbica Transbond
XT (3M Unitek, St. Paul, MN, EUA) e a resina hidrofílica Transbond
Plus Color Change (3M Unitek, St. Descriptors: Adhesives; orthodontic brackets; shear strength; saliva. Descriptors: Adhesives; orthodontic brackets; shear strength; saliva. Este é um artigo publicado em acesso aberto (Open Access) sob a licença Creative Commons Attribution, que permite uso,
distribuição e reprodução em qualquer meio, sem restrições desde que o trabalho original seja corretamente citado. Rev Odontol UNESP. 2018 May-June; 47(3): 131-136 Lon, Schneider, Raveli et al. 132
132/1 INTRODUÇÃO Paul, MN, EUA) ao esmalte
de dentes bovinos, previamente contaminado por saliva, além de
analisar os locais da falha adesiva. No G3, após o condicionamento com ácido fosfórico a 37%,
durante 15 segundos, a lavagem e a secagem, conforme descrito
no G1, foi aplicado o adesivo Transbond XT primer (3M/Unitek,
EUA). Em seguida, a área de colagem foi contaminada com saliva Quadro 1. Divisão dos grupos
Grupo
Contaminação
Sistema adesivo
1
não
Transbond XT Primer + Transbond XT
2
não
Self Etching Primer + Transbond Color
3
saliva
Transbond XT Primer + Transbond XT
4
saliva
Self Etching Primer + Transbond Color Quadro 1. Divisão dos grupos
Grupo
Contaminação
Sistema adesivo
1
não
Transbond XT Primer + Transbond XT
2
não
Self Etching Primer + Transbond Color
3
saliva
Transbond XT Primer + Transbond XT
4
saliva
Self Etching Primer + Transbond Color MATERIAL E MÉTODO Este estudo foi aprovado pelo Comitê de Ética no Uso de
Animais (CEUA) da Pontifícia Universidade Católica do Paraná
(PUCPR), com o parecer n.º 204/07. Para sua realização, foi
utilizada uma amostra de 60 incisivos inferiores permanentes
bovinos, recém-extraídos, com as coroas intactas e ausência de Rev Odontol UNESP. 2018 May-June; 47(3): 131-136 Efeito da contaminação por saliva... 133
/136 no ensaio de cisalhamento (Figura 1J). Os corpos de prova ficaram
armazenados em água destilada à temperatura de 37°C por 24 horas. artificial Salivan (Apsen Farmacêutica, São Paulo, SP, Brasil) sobre
a superfície do esmalte, seguido do posicionamento do braquete
utilizando a resina Transbond XT (3M/Unitek, EUA), com uma
pressão de 400 gf e fotopolimerização por 10 segundos. A resistência adesiva (RA) foi avaliada em um ensaio de
cisalhamento, realizado 24 horas após a colagem, na máquina
universal de ensaios EMIC DL 500 (Emic Equipamentos e Sistemas
de Ensaio Ltda., São José dos Pinhais, Brasil) (Figura 1K), a uma
velocidade constante de 0,5 mm/min. Uma célula de carga de
50 kgf foi conectada ao computador, que registrou a resistência ao
cisalhamento em MPa de cada corpo de prova, considerando-se
a área da base de cada braquete (RA= Força/Área) (Figura 1L). No G4, após a aplicação do primer acidificado autocondicionante
(Self Etching primer, SEP, 3M/Unitek, EUA), foi aplicada saliva artificial
sobre a superfície, seguido da colagem do braquete com a resina
Transbond Plus Color Change (3M/Unitek, EUA), da remoção dos
excessos e da fotopolimerização, como descrito nos grupos 1, 2 e 3. Utilizou-se um dispositivo de aço inoxidável com um fio retangular
0,021” × 0,025” (Morelli, Sorocaba, Brasil) soldado em 90° em
relação à sua base, para garantir a perpendicularidade do braquete
em relação ao plano horizontal (Figuras 1A e 1B). Os dentes foram
fixados no dispositivo por uma ligadura elástica (Tp Orthodontics,
La Port, IN, USA) colocada nas aletas do braquete, prendendo-o a
este fio retangular (Figuras 1C e 1D). Após a fixação, a raiz dentária
foi envolvida por um anel metálico (2,5 cm de diâmetro e 2,0 cm
de altura) (Figuras 1E, 1F e 1G), que foi preenchido com resina
acrílica autopolimerizável (Jet, São Paulo, Brasil) (Figura 1H). MATERIAL E MÉTODO Desta forma, a superfície de colagem ficou perpendicular ao plano
horizontal (Figura 1I) e paralela à direção de aplicação da força, Após a descolagem dos braquetes, a superfície do esmalte e os
braquetes foram examinados com uma lupa estereoscópica Leica
Zoom 2000 (Leica, Wetzlar, Alemanha) com 20 vezes de aumento,
para verificar o Índice de Adesivo Remanescente (IAR). O adesivo
remanescente foi graduado de acordo com o método utilizado por
Årtun e Bergland9, variando de 0 a 3. A graduação (0) indica que não
há material aderido à superfície de esmalte do dente; (1) indica que
menos da metade do material ficou aderido à superfície de esmalte
do dente; (2) indica que mais da metade do material ficou aderido à
superfície de esmalte do dente; (3) indica que todo o material ficou
aderido, inclusive a impressão da malha do braquete. Figura 1 - Sequência da montagem dos corpos de prova. (A) e (B) dispositivo para a inclusão dos dentes; (C) e (D) dente preso no fio
0,021” × 0,025” com ligadura elástica; (E), (F) e (G) anel metálico de inclusão posicionado para o preenchimento com acrílico autopolimerizável;
(H) preenchimento com o acrílico; (I) e (J) corpo de prova com o braquete perpendicular à base e paralelo à direção da força de cisalhamento;
(K) máquina universal de ensaios EMIC DL 500; (L) ensaio da RA ao cisalhamento. 150×129 mm (300 × 300 DPI). Figura 1 - Sequência da montagem dos corpos de prova. (A) e (B) dispositivo para a inclusão dos dentes; (C) e (D) dente preso no fio
0,021” × 0,025” com ligadura elástica; (E), (F) e (G) anel metálico de inclusão posicionado para o preenchimento com acrílico autopolimerizável;
(H) preenchimento com o acrílico; (I) e (J) corpo de prova com o braquete perpendicular à base e paralelo à direção da força de cisalhamento;
(K) máquina universal de ensaios EMIC DL 500; (L) ensaio da RA ao cisalhamento. 150×129 mm (300 × 300 DPI). Figura 1 - Sequência da montagem dos corpos de prova. MATERIAL E MÉTODO (A) e (B) dispositivo para a inclusão dos dentes; (C) e (D) dente preso no fio
0,021” × 0,025” com ligadura elástica; (E), (F) e (G) anel metálico de inclusão posicionado para o preenchimento com acrílico autopolimerizável;
(H) preenchimento com o acrílico; (I) e (J) corpo de prova com o braquete perpendicular à base e paralelo à direção da força de cisalhamento;
(K) máquina universal de ensaios EMIC DL 500; (L) ensaio da RA ao cisalhamento. 150×129 mm (300 × 300 DPI). Lon, Schneider, Raveli et al. Rev Odontol UNESP. 2018 May-June; 47(3): 131-136 134
/ Tabela 2. Frequência de distribuição do Índice de Adesivo Remanescente – IAR (%)
Grupos
n
Contaminação
Resina
IAR (%)
0
1
2
3
G1
20
sem
Transbond XT
10
10
40
40
G2
20
sem
Transbond Plus
45
40
10
5
G3
20
saliva
Transbond XT
90
10
0
0
G4
20
saliva
Transbond Plus
60
30
10
0
IAR - Índice de Adesivo Remanescente: 0= nenhuma resina aderida ao dente; 1= menos de 50% de resina aderida ao dente; 2= mais de 50% de resina aderida ao dente
e 3= toda a resina aderida ao dente inclusive a malha do braquete. Tabela 1. Estatística descritiva da resistência adesiva (MPa)
Grupo
n
Resina
Contaminação
Média
Mediana
Desvio Padrão
G1
15
Transbond XT
sem
6,65
6,25a
1,63
G2
15
Transbond Plus
sem
4,81
4,26b
1,22
G3
15
Transbond XT
saliva
4,24
2,85b
2,85
G4
15
Transbond Plus
saliva
4,78
4,37ab
2,13
Letras iguais indicam diferença estatisticamente não significante entre os grupos (p<0,05). Tabela 1. Estatística descritiva da resistência adesiva (MPa)
Grupo
n
Resina
Contaminação
Média
Mediana
Desvio Padrão
G1
15
Transbond XT
sem
6,65
6,25a
1,63
G2
15
Transbond Plus
sem
4,81
4,26b
1,22
G3
15
Transbond XT
saliva
4,24
2,85b
2,85
G4
15
Transbond Plus
saliva
4,78
4,37ab
2,13
Letras iguais indicam diferença estatisticamente não significante entre os grupos (p<0,05). Tabela 1. Estatística descritiva da resistência adesiva (MPa) Letras iguais indicam diferença estatisticamente não significante entre os grupos (p<0,05). Tabela 2. MATERIAL E MÉTODO Frequência de distribuição do Índice de Adesivo Remanescente – IAR (%)
Grupos
n
Contaminação
Resina
IAR (%)
0
1
2
3
G1
20
sem
Transbond XT
10
10
40
40
G2
20
sem
Transbond Plus
45
40
10
5
G3
20
saliva
Transbond XT
90
10
0
0
G4
20
saliva
Transbond Plus
60
30
10
0
IAR - Índice de Adesivo Remanescente: 0= nenhuma resina aderida ao dente; 1= menos de 50% de resina aderida ao dente; 2= mais de 50% de resina aderida ao dente
e 3= toda a resina aderida ao dente inclusive a malha do braquete. Tabela 2. Frequência de distribuição do Índice de Adesivo Remanescente – IAR (%) Remanescente: 0= nenhuma resina aderida ao dente; 1= menos de 50% de resina aderida ao dente; 2= mais de 50% de resina aderida ao dent
da ao dente inclusive a malha do braquete. DISCUSSÃO O condicionamento ácido do esmalte inadequado, a saliva, o
sangue e os fluidos gengivais são os principais fatores que podem
interferir na colagem direta de braquetes ortodônticos ao esmalte
dentário e diminuir a RA. Os adesivos e resinas ortodônticas
tradicionais são extremamente sensíveis a estes contaminantes,
prejudicando seu desempenho clínico3,10,11. ANÁLISE ESTATÍSTICA Na avaliação do Índice de Adesivo Remanescente, o grupo 1
apresentou os escores do IAR variando entre 2 e 3, e nos demais
grupos, os escores predominantes do IAR variaram entre 0 e 1,
sugerindo pouco adesivo remanescente aderido ao esmalte nos
grupos 2, 3 e 4. A frequência do IAR é apresentada na Tabela 2. A análise estatística foi feita com o software Statistical Package
for Social Science 13.0 for Windows (SPSS, Inc., Chicago, IL) e
utilizaram-se o teste de Kolmogorov-Smirnov e o teste de Levene
para verificação de normalidade e homogeneidade de variâncias,
respectivamente. Como a amostra não apresentou normalidade e
nem homogeneidade, a diferença entre os grupos foi examinada
por meio do teste de comparações múltiplas não paramétrico de
Kruskal-Wallis. O nível de significância adotado foi de 5%. RESULTADO Uma vez que dois dos quatros grupos não apresentaram
normalidade dos dados e homogeneidade de variância entre si,
utilizou-se, para comparar os quatro grupos, o teste não paramétrico
de Kruskal-Wallis, o qual demonstrou existir diferenças estatísticas
nos valores médios da RA. A desmineralização dos prismas de esmalte, que ocorre
no condicionamento ácido do esmalte, permite uma retenção
micromecânica pelo escoamento e penetração do adesivo nas
porosidades resultantes da ação do ácido sobre o esmalte12. Os sistemas adesivos ortodônticos convencionais foram
desenvolvidos para serem utilizados em ambiente sem contaminação
por umidade, proporcionando uma resistência adesiva apropriada10,11. Embora alguns estudos13-15 tenham mostrado que a contaminação por
umidade é um fator importante na diminuição da RA de braquetes
ortodônticos, outros estudos não encontraram diferença na RA
frente à contaminação16-18. Estes resultados diferentes podem ser
atribuídos aos diferentes sistemas adesivos utilizados nos estudos. O teste de comparação múltipla não paramétrica de Kruskal-Wallis
mostrou diferença estatisticamente significante entre pelo menos dois
grupos (p<0,05). O grupo 1 apresentou diferenças estatisticamente
significantes quando comparado com os grupos 2 (Self Ecthing
primer com Transbond color change sem contaminação) e o grupo
3 (Transbond XT primer com Transbond XT com contaminação),
não existindo diferenças entre os demais. Observou-se que o valor
mediano de (RA) do grupo 1 (Transbond XT primer com Transbond
XT sem contaminação) foi maior do que o valor mediano de (RA)
dos grupos 2 e 3, e não apresentou diferença estatisticamente
significante em relação ao G4. Os grupos 2, 3 e 4 não apresentaram
diferenças significantes entre si. A Tabela 1, a seguir, apresenta as
estatísticas descritivas da (RA), segundo o grupo. Visando melhorar a colagem de braquetes e acessórios ortodônticos
em meio úmido, a indústria odontológica vem desenvolvendo
novos adesivos hidrofílicos para que, além de diminuírem os
passos clínicos na rotina profissional, sejam resistentes às forças
ortodônticas e de mastigação19. Rev Odontol UNESP. 2018 May-June; 47(3): 131-136 Efeito da contaminação por saliva... 135
/136 é que, mesmo tomando estes cuidados, pode haver, de forma
inadvertida, alguma contaminação. Entre os materiais desenvolvidos pela indústria, destacam-se
os adesivos autocondicionantes hidrofílicos e a resina Transbond
Color Change (hidrofílico). Uma importante vantagem dos adesivos
autocondicionantes é que o adesivo penetra em toda profundidade
do esmalte condicionado, promovendo uma boa retenção mecânica20. Este trabalho, portanto, apresenta grande relevância, uma vez que
apresenta resultados que podem ser utilizados de forma imediata na
rotina clínica. CONCLUSÃO A contaminação por saliva diminui a resistência adesiva
ao cisalhamento de braquetes cerâmicos colados com a resina
hidrofóbica Transbond XT convencional. Mesmo ao utilizar estes adesivos associados às resinas hidrofílicas,
os autores deste trabalho não descartam a necessidade de realizar
a colagem de braquetes com cuidados exigidos pela técnica, como
utilização de rolinhos de algodão no fundo de vestíbulo, uso de
sugador, secagem dos dentes e uso de afastadores bucais. A questão A utilização da resina hidrofílica Transbond Plus Color Change
associada ao Self Etching Primer, em ambiente contaminado por
saliva, confere resistência adesiva adequada para o uso clínico. RESULTADO Pacientes com hipersalivação, por exemplo, podem
ser beneficiados com estes materiais. Este estudo teve como objetivo estudar se estes adesivos
autocondicionantes são seguros para utilizar na clínica ortodôntica,
em circunstancias de meio ambiente úmido. Observou-se que
não houve diferença estatística entre o G1 (controle) Transbond
XT- controle, resina convencional, braquetes colados em meio
seco, com o G4, em que se utilizou o adesivo autocondicionante
com a resina hidrofílica Transbond Color Change, em ambiente
contaminado por saliva. Para manter a integridade do esmalte dentário, a falha adesiva
ideal na descolagem de braquetes deve ocorrer na interface
braquete-adesivo22. As falhas adesivas que ocorrem na interface
esmalte-adesivo podem aumentar o risco de fratura do esmalte23. Os escores do IAR idealizados por Årtun e Bergland9 têm a
finalidade de quantificar o material remanescente sobre o esmalte,
mostrando o local em que ocorreu a falha no teste de cisalhamento. Neste trabalho, os grupos que tiveram contaminação de saliva
mostraram a predominância do IAR entre 0 e 1, concordando com
estudos anteriores24,25, que também avaliaram os escores do IAR de
braquetes ortodônticos colados sob contaminação de água ou saliva. Estes resultados concordam com outros estudos16-18,21, comprovando
que estes materiais apresentam adequada performance, mesmo
quando há contaminação. Estes resultados proporcionam segurança
ao clínico, já que o ambiente bucal é extremamente úmido e, mesmo
com cuidados de controle da saliva, pode ocorrer esta contaminação
durante o procedimento de colagem de braquetes. Outros autores13-15
encontraram resultados conflitantes com os deste estudo. REFERÊNCIAS 2. Buonocore MG. A simple method of increase the adhesion of acrylic filling materials to enamel surfaces. J Dent Res. 1955 Dec;34(6):849-53. http://dx.doi.org/10.1177/00220345550340060801. PMid:13271655. 2. Buonocore MG. A simple method of increase the adhesion of acrylic filling materials to enamel surfaces. J Dent Res. 1955 Dec;34(6):849-53. http://dx.doi.org/10.1177/00220345550340060801. PMid:13271655. 3. Rajagopal R, Padmanabhan S, Gnanamani J. A comparison of shear bond strength and debonding characteristics of conventional, moisture-
insensitive, and self-etching primers in vitro. Angle Orthod. 2004 Apr;74(2):264-8. http://dx.doi.org/10.1043/0003-3219(2004)074<0264:AC
OSBS>2.0.CO;2. PMid:15132455. 4. Hormati AA, Fuller JL, Denehy GE. Effects of contamination and mechanical disturbance on the quality of acid-etched enamel. J Am Dent
Assoc. 1980 Jan;100(1):34-8. http://dx.doi.org/10.14219/jada.archive.1980.0033. PMid:6985623. 5. Schaneveldt S, Foley TF. Bond strength comparison of moisture insensitive primers. Am J Orthod Dentofacial Orthop. 2002 Sep;122(3):267-
73. http://dx.doi.org/10.1067/mod.2002.126594. PMid:12226607. 6. Oztoprak MO, Isik F, Sayinsu K, Arun T, Aydemir B. Effect of blood and saliva contamination on shear bond strength of brackets bonded
with 4 adhesives. Am J Orthod Dentofacial Orthop. 2007 Feb;131(2):238-42. http://dx.doi.org/10.1016/j.ajodo.2005.02.035. PMid:17276865. 7. Sfondrini MF, Gatti S, Scribante A. Effect of blood contamination on shear bond strength of orthodontic brackets and disinclusion buttons. Br J Oral Maxillofac Surg. 2011 Jul;49(5):404-8. http://dx.doi.org/10.1016/j.bjoms.2010.06.011. PMid:20655131. 8. Carvalho RM. Adesivos dentinários: fundamentos para aplicação clínica. Rev Dent Rest. 1998;1(2):60-96. 9. Årtun J, Bergland S. Clinical trials with crystal growth conditioning as an alternative to acid-etch enamel pretreatment. Am J Orthod. 1984
Apr;85(4):333-40. http://dx.doi.org/10.1016/0002-9416(84)90190-8. PMid:6231863. 10. Silverman E, Cohen M, Demke RS, Silverman M. A new light-cured glass ionomer cement that bonds brackets to teeth without etching
in the presence of saliva. Am J Orthod Dentofacial Orthop. 1995 Sep;108(3):231-6. http://dx.doi.org/10.1016/S0889-5406(95)70014-5. PMid:7661137. 11. Grandhi RK, Combe EC, Speidel TM. Shear bond strength of stainless steel orthodontic brackets with a moisture-insensitive primer. Am J
Orthod Dentofacial Orthop. 2001 Mar;119(3):251-5. http://dx.doi.org/10.1067/mod.2001.110988. PMid:11244419. 11. Grandhi RK, Combe EC, Speidel TM. Shear bond strength of stainless steel orthodontic brackets with a moisture-insensitive primer. Am J
Orthod Dentofacial Orthop. 2001 Mar;119(3):251-5. http://dx.doi.org/10.1067/mod.2001.110988. PMid:11244419. 11. Grandhi RK, Combe EC, Speidel TM. Shear bond strength of stainless steel orthodontic brackets with a moisture-insensitive primer. Am J
Orthod Dentofacial Orthop. 2001 Mar;119(3):251-5. http://dx.doi.org/10.1067/mod.2001.110988. PMid:11244419. Rev Odontol UNESP. 2018 May-June; 47(3): 131-136 Lon, Schneider, Raveli et al. 136
136/ 12. el-Kalla IH, Garcia-Godoy F. Saliva contamination and bond strength of single-bottle adhesives to enamel and dentin. Am J Dent. 1997
Apr;10(2):83-7. PMid:9545895. 12. el-Kalla IH, Garcia-Godoy F. CONFLITOS DE INTERESSE Os autores declaram não haver conflitos de interesse. REFERÊNCIAS Xie J, Powers JM, McGuckin RS. In vitro bond strength of two adhesives to enamel and dentin under normal and contaminated conditions. Dent Mater. 1993 Sep;9(5):295-9. http://dx.doi.org/10.1016/0109-5641(93)90046-S. PMid:7995480. 25. Cacciafesta V, Sfondrini MF, De Angelis M, Scribante A, Klersy C. Effect of water and saliva contamination on shear bond strength of brackets
bonded with conventional, hydrophilic, and self-etching primers. Am J Orthod Dentofacial Orthop. 2003 Jun;123(6):633-40. http://dx.doi. org/10.1016/S0889-5406(03)00198-7. PMid:12806342. REFERÊNCIAS Saliva contamination and bond strength of single-bottle adhesives to enamel and dentin. Am J Dent. 1997
Apr;10(2):83-7. PMid:9545895. 13. Bishara SE, VonWald L, Laffoon JF, Warren JJ. Effect of a self-etch primer/adhesive on the shear bond strength of orthodontic brackets. Am
J Orthod Dentofacial Orthop. 2001 Jun;119(6):621-4. http://dx.doi.org/10.1067/mod.2001.113269. PMid:11395706. 13. Bishara SE, VonWald L, Laffoon JF, Warren JJ. Effect of a self-etch primer/adhesive on the shear bond strength of orthodontic brackets. Am
J Orthod Dentofacial Orthop. 2001 Jun;119(6):621-4. http://dx.doi.org/10.1067/mod.2001.113269. PMid:11395706. 14. Bishara SE, Oonsombat C, Ajlouni R, Laffoon JF. Comparison of the shear bond strength of 2 self-etch primer/adhesive systems. Am J
Orthod Dentofacial Orthop. 2004 Mar;125(3):348-50. http://dx.doi.org/10.1016/j.ajodo.2003.04.010. PMid:15014413. 15. Romano FL, Tavares SW, Nouer DF, Consani S, Borges de Araújo Magnani MB. Shear bond strength of metallic orthodontic brackets bonded
to enamel prepared with Self-Etching Primer. Angle Orthod. 2005 Sep;75(5):849-53. http://dx.doi.org/10.1043/0003-3219(2005)75[849:SB
SOMO]2.0.CO;2. PMid:16285044. 16. Sirirungrojying S, Saito K, Hayakawa T, Kasai K. Efficacy of using self-etching primer with a 4-META/MMA-TBB resin cement in bonding
orthodontic brackets to human enamel and effect of saliva contamination on shear bond strength. Angle Orthod. 2004 Apr;74(2):251-8. http://dx.doi.org/10.1043/0003-3219(2004)074<0251:EOUSPW>2.0.CO;2. PMid:15132453. 17. Retamoso LB, Collares FM, Ferreira ES, Samuel SM. Shear bond strength of metallic brackets: influence of saliva contamination. J Appl Oral
Sci. 2009 May-Jun;17(3):190-4. http://dx.doi.org/10.1590/S1678-77572009000300011. PMid:19466249. 18. Maia SR, Cavalli V, Liporonti PC, do Rego MA. Influence of saliva contamination on the shear bond strength of orthodontic brackets bonded
with self-etching adhesive systems. Am J Orthod Dentofacial Orthop. 2010 Jul;138(1):79-83. http://dx.doi.org/10.1016/j.ajodo.2008.08.035. PMid:20620837. 19. Katona TR, Long RW. Effect of loading mode on bond strength of orthodontic brackets bonded with 2 systems. Am J Orthod Dentofacial
Orthop. 2006 Jan;129(1):60-4. http://dx.doi.org/10.1016/j.ajodo.2004.09.020. PMid:16443480. 20. Buyukyilmaz T, Usumez S, Karaman AI. Effect of self-etching primers on bond strength -- are they reliable? Angle Orthod. 2003 Feb;73(1):64-
70. http://dx.doi.org/10.1043/0003-3219(2003)073<0064:EOSEPO>2.0.CO;2. PMid:12607857. 21. Deprá MB, Almeida JX, Cunha TMA, Lon LFS, Retamoso LB, Tanaka OM. Effect of saliva contamination on bond strength with a hydrophilic
composite resin. Dental Press J Orthod. 2013 Feb;18(1):63-8. http://dx.doi.org/10.1590/S2176-94512013000100015. PMid:23876951. 22. Liu JK, Chung CH, Chang CY, Shieh DB. Bond strength and debonding characteristics of a new ceramic bracket. Am J Orthod Dentofacial
Orthop. 2005 Dec;128(6):761-5, quiz 802. http://dx.doi.org/10.1016/j.ajodo.2004.03.041. PMid:16360918. 23. Bishara SE, Gordan VV, VonWald L, Jakobsen JR. Shear bond strength of composite, glass ionomer, and acid primer adhesive systems. Am
J Orthod Dentofacial Orthop. 1999 Jan;115(1):24-8. http://dx.doi.org/10.1016/S0889-5406(99)70312-4. PMid:9878954. 24. *AUTOR PARA CORRESPONDÊNCIA Luis Filipe Siu Lon, UNESP – Universidade Estadual Paulista, Faculdade de Odontologia de Araraquara, Rua Humaitá, 1680,
1º andar, Centro, 14801-903 Araraquara, SP, Brasil, e-mail: filipelon@hotmail.com Luis Filipe Siu Lon, UNESP – Universidade Estadual Paulista, Faculdade de Odontologia de Araraquara, Rua Humaitá, 1680,
1º andar, Centro, 14801-903 Araraquara, SP, Brasil, e-mail: filipelon@hotmail.com Recebido: Fevereiro 27, 2018
Aprovado: Maio 15, 2018
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Providing surgery in a war-torn context: the Médecins Sans Frontières experience in Syria
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Conflict and health
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Providing surgery in a war-torn context:
the Médecins Sans Frontières experience
in Syria Miguel Trelles1, Lynette Dominguez1, Katie Tayler-Smith2*, Katrin Kisswani3, Alberto Zerboni4, Thierry Vandenborre4,
Silvia Dallatomasina3, Alaa Rahmoun5 and Marie-Christine Ferir3 Trelles et al. Conflict and Health (2015) 9:36
DOI 10.1186/s13031-015-0064-3 Trelles et al. Conflict and Health (2015) 9:36
DOI 10.1186/s13031-015-0064-3 Open Access * Correspondence: katietaylersmith@gmail.com
2MSF-OCB, Operational Research Unit, MSF-Luxembourg, Luxembourg,
Luxembourg
Full list of author information is available at the end of the article © 2015 Trelles et al. Open Access 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 Background: Since 2011, civil war has crippled Syria leaving much of the population without access to healthcare. Various field hospitals have been clandestinely set up to provide basic healthcare but few have been able to provide
quality surgical care. In 2012, Medecins Sans Frontieres (MSF) began providing surgical care in the Jabal al-Akrad region
of north-western Syria. Based on the MSF experience, we describe, for the period 5th September 2012 to 1st January
2014: a) the volume and profile of surgical cases, b) the volume and type of anaesthetic and surgical procedures
performed, and c) the intraoperative mortality rate. Methods: A descriptive study using routinely collected MSF programme data. Quality surgical care was assured
through strict adherence to the following minimum standards: adequate infrastructure, adequate water and sanitation
provisions, availability of all essential disposables, drugs and equipment, strict adherence to hygiene requirements and
universal precautions, mandatory use of sterile equipment for surgical and anaesthesia procedures, capability for blood
transfusion and adequate human resources. Results: During the study period, MSF operated on 578 new patients, of whom 57 % were male and median age
was 25 years (Interquartile range: 21–32 years). Violent trauma was the most common surgical indication
(n-254, 44 %), followed by obstetric emergencies (n-191, 33 %) and accidental trauma (n-59, 10 %). In total,
712 anaesthetic procedures were performed. General anaesthesia without intubation was the most common
type of anaesthesia (47 % of all anaesthetics) followed by spinal anaesthesia (25 %). A total of 831 surgical
procedures were performed, just over half being minor/wound care procedures and nearly one fifth, caesarean
sections. There were four intra-operative deaths, giving an intra-operative mortality rate of 0.7 %. Conclusions: Surgical needs in a conflict-afflicted setting like Syria are high and include both combat and non-combat
indications, particularly obstetric emergencies. Provision of quality surgical care in a complex and volatile setting like
this is possible providing appropriate measures, supported by highly experienced staff, can be implemented that allow
a specific set of minimum standards of care to be adhered to. This is particularly important when patient outcomes - as
a reflection of quality of care - are difficult to assess. Keywords: Quality surgical care, Conflict settings, Syria, Minimum standards of care Study setting Jabal al-Akrad is a rural mountainous region in north-
west Syria close to the Turkish border, with a population
of about 150,000. As a result of the civil war, the region
has seen extensive fighting between armed groups and the
Syrian army and also between different armed groups. y
y
g
p
The MSF field hospital was set up and functional by
the end of August 2012. Initially the hospital was orga-
nised within a cave, but in November 2012, due to inse-
curity (the position of the cave was considered to be too
close to a frontline and military position) and limited
space, the field hospital was moved and re-established in
a permanent brick structure (an abandoned chicken
farm) located 6 km from the Turkish border. The
provision of surgical care was shifted from the cave to
the chicken farm in just one day. Several days prior to
this shift, a new surgical tent was erected in the chicken
farm and a proportion of the emergency/ surgical care
supplies and pharmacy stock was transferred to the
chicken farm. On the day of the move, the operating
table and operating lamp were shifted from the cave to
the chicken farm together with the anaesthetist (in the
first convoy) and the surgeon and emergency physician
(in the second convoy); there was only a 90 min window
during which surgical activities had to be put on hold. The remaining supplies at the cave were transferred to
the chicken cave over the following two days. Various field hospitals have been clandestinely set up
to provide some level of healthcare [9, 10] but very few
have managed to establish the capacity to provide quality
surgical care. Médecins Sans Frontières (MSF), an international, in-
dependent, humanitarian medical organisation, provides
medical assistance to vulnerable populations, including
those affected by conflicts. Surgical care is an integral
part of its work. Shortly after the conflict in Syria esca-
lated into a full-scale civil war in 2012, MSF-OCB
(Operational Centre Brussels) intervened in the Jabal al-
Akrad region of north-western Syria, where it estab-
lished a field hospital that included a surgical centre. Despite major challenges, MSF-OCB managed to pro-
vide surgery for nearly 600 patients. To date, there have been few published studies report-
ing on the provision of surgical activities in conflict
settings. * Correspondence: katietaylersmith@gmail.com
2MSF-OCB, Operational Research Unit, MSF-Luxembourg, Luxembourg,
Luxembourg
Full list of author information is available at the end of the article © 2015 Trelles et al. Open Access 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. Page 2 of 8 Trelles et al. Conflict and Health Study design This was a descriptive study using routinely collected
MSF programme data. Background and organised, but the study only reported on 28 surgi-
cal patients. The ‘how’ of providing quality surgical care
for larger numbers of patients presenting with different
surgical indications in a complex war-torn context like
Syria has not been formally reported on. Surgical care is an integral part of any healthcare system
and yet in low and middle income countries (LMICs)
access to such care is often limited [1, 2]. When such
contexts are in turn afflicted by conflict, the situation
becomes even more dire: not only is access further ham-
pered, but this at a time when the surgical needs of the
population are likely to be greater (war-related injuries
in addition to non-war related conditions such as obstet-
ric emergencies and infections) [3]. Based on the MSF-OCB experience of providing surgi-
cal care in the Jabal al-Akrad region of Syria between
September 2012 and January 2014, this study thus aims
to report on how quality surgical care can be provided
in a complex conflict setting and what volume and type
of surgical activities are feasible. Specific objectives were
to report on a) the volume and profile of surgical cases
seen, b) the volume and type of anaesthetic and surgical
procedures performed and c) the intraoperative mortal-
ity rate. Since 2011, Syria has been crippled by a brutal civil
war resulting in what has been described by the United
Nations (UN) as the worst humanitarian crisis in recent
times. Approximately 220,000 people (mostly civilians)
are estimated to have died since the conflict began and
it is estimated that 12.2 million people are in need of hu-
manitarian assistance, 7.6 million of whom are internally
displaced [4]. Intense fighting and complicated relief
operations due to political barriers have left millions of
Syrians without access to essential services [5], access to
health services being the greatest challenge of all [6]. Access to basic health care, including surgical care, is
severely hampered by restricted ability to move, the
destruction and breakdown of health infrastructure,
shortage of medical supplies, lack of human resources
and threats of kidnappings and killings by different
armed groups on medical staff [7, 8]. Reports estimate
that more Syrians have died due to health complications
resulting from inadequate health care services than as a
direct consequence of the conflict [4]. Study setting Those that have, have generally reported on the
typology of the surgical care provided [3, 11], remote
models of care [12], the feasibility of performing specific
surgical procedures [13, 14], and the surgical manage-
ment and related outcomes for specific conditions
[15, 16]. One study from Syria [17] described how a field
hospital, which included surgical activities, was set up MSF surgical activities MSF adapted a standardised design for organising its
surgical complex and implemented standardised proce-
dures for surgical activities and training based on in-
ternal policy guidelines. The surgical centre comprised Page 3 of 8 Trelles et al. Conflict and Health these conditions. There were seven pre-requisites: i) ad-
equate infrastructure, (including protection from the
external environment and appropriate electricity and
lighting), ii) adequate water and sanitation provisions,
(waste management being a key priority), iii) availability of
all essential disposables, drugs and equipment, iv) strict
adherence to hygiene requirements and universal precau-
tions, v) mandatory use of sterile equipment for surgical
and anaesthesia procedures, vi) capability for blood trans-
fusion, and vii) adequate human resources in quantity and
quality. In order to meet these minimum standards in the
context of Syria, various measures had to be taken. Some
of the key measures included: i) adapting the standard
field hospital design - this to meet the functional/medical
needs of the hospital, ensure the ‘safe’ circuit of patients in
terms of hygiene standards and accommodate for the
challenges around lack of space, lack of infrastructure and
lack of security; ii) ensuring that highly experienced
personnel were in place to oversee the design and timely
implementation of these technical adaptations (medical
staff - who could clearly identify the needs of the hospital -
and logistics staff with extensive experience in emergency
hospital design and set-up) and iii) establishing a clandes-
tine supply line from Turkey to ensure a regular supply of
logistical materials, fuel and medical supplies. of one operating room (OR) in the form of an inflatable
tent, together with all the necessary supportive units in-
cluding an emergency department (with a resuscitation,
treatment and observation area), a pre- and post-operative
ward, a sterilisation unit, a basic laboratory and blood
transfusion capacity. Surgical activities were overseen by a team of experi-
enced international staff (one surgeon, one anaesthetist,
one emergency doctor and one OR nurse) and sup-
ported by a number of local nurses (recruited from the
local community and trained accordingly to assist with
surgical activities). Staff turnover among local personnel
was minimal while international staff were rotated on a
monthly basis - the whole team being replaced by a new
one. On several occasions, international staff members,
including those in the surgical team, had to be evacuated
because of security constraints. Data collection and analysis For this analysis, a surgical intervention was defined as
any intervention performed in the OR that required
local, regional and / or general anaesthesia. One surgical
intervention corresponded with one patient entrance
into the OR. The data collection tool in place was
designed such that a maximum of three surgical proce-
dures could be recorded per surgical intervention. Data
on all surgical interventions were entered into a standar-
dised logbook and transcribed into an electronic data-
base (Microsoft Excel). These data were then aggregated
at MSF-OCB headquarters and reviewed for complete-
ness and accuracy. Data pertaining to this study were
sourced from the electronic database and included the
following variables: date of surgical intervention, patient
gender, patient age, the American Society of Anaesthe-
siologists (ASA) score (a classification system for de-
scribing a patient’s pre-operative physical status, see
Table 2), indication for surgery (grouped as accidental
trauma, violent trauma, obstetric care and other), type
of
anaesthesia
used
(general
anaesthesia
without
intubation, general anaesthesia with intubation, spinal Emergency care, including surgery, was available 24 h
a day every day, and all care and treatment was provided
free of charge. Appropriate triage systems were put in
place to try to ensure a more efficient use of limited re-
sources and to better manage heavy caseloads during
busy times. During times of ‘normal’ care, the South
African Triage scale was used in the emergency de-
partment and the process of triage was task-shifted
to nursing staff so that the few available doctors
could concentrate their efforts on treating patients
once triaged; during times of a mass casualty inci-
dent (MCI), START (simple triage and rapid treat-
ment) [19] was implemented - a triage method used
to quickly classify victims during a MCI based on
the severity of their injury. MSF surgical activities On three occasions, the
evacuation period was 15 days or longer and during
these evacuations periods surgical activities were tem-
porarily suspended. During these times, the hospital
remained open and patient care and management was
overseen by local health personnel. An important part of the MSF intervention included
setting up an effective referral system with appropriate
services in Turkey. If a patient arrived at the field hos-
pital in a critical condition that could not be surgically
managed because of resource constraints, damage con-
trol surgery [18] was performed and the patient was re-
ferred to Turkey. Patients with head trauma were also
referred. Furthermore, by providing orthopaedic surgical
materials (mainly external fixators) to a nearby Syrian
hospital with the human resource capacity to provide
orthopaedic surgery, MSF was able to refer some of its
orthopaedic cases to this hospital. Study population
Th
d
l The study population included all new patients who
underwent any surgical intervention at the MSF field
hospital in Jabal al-Akrad between 5th September 2012
and 1st January 2014. Table 1 Measures taken by MSF to ensure the provision of quality surgical care in Jabal Akkrad, Syria
Essential conditions for the provision of quality
surgical care
Measures taken to achieve these conditions in the cave and chicken farm Table 1 Measures taken by MSF to ensure the provision of quality surgical care in Jabal Akkrad, Syria
Essential conditions for the provision of quality
surgical care
Measures taken to achieve these conditions in the cave and chicken farm • The standard design of the field hospital was adapted to meet the functional/medical
needs of the hospital, to ensure the ‘safe’ circuit of surgical patients (in terms of
hygiene standards) and to accommodate for the challenges around lack of quality space,
lack of infrastructure and lack of security, all the while ensuring that quality care of care
would not be compromised. Essential for the design and timely implementation of these
technical solutions was the presence of highly experienced personnel (medical staff who
could clearly identify the functional needs of the hospital and logistics staff with extensive
experience in emergency hospital design and set-up). Adequate infrastructure, including protection from
the external environment and appropriate
electricity and lighting. Adequate water and sanitation provisions, waste
management being a key priority g
y
g
• An inflatable tent served as the operating theatre in order to ensure a minimum level
of cleanliness. To ensure protection from environmental factors (such as rain, wind,
dust and falling debris from the roof of the cave), plastic sheets formed a cover for
the floor of the tent and wooden frames lined with plastic sheets formed the roof
of the tent. The same features were put in place once the field hospital relocated
to an abandoned chicken farm. • An entirely new electrical wiring system was installed, respecting all necessary electrical
safety measures. In addition, two generators were installed (one as back up), to ensure
a reliable electricity supply during power cuts. • An entirely new electrical wiring system was installed, respecting all necessary electrical
safety measures. In addition, two generators were installed (one as back up), to ensure
a reliable electricity supply during power cuts. y
y
• Establishing the field hospital in the abandoned chicken farm in particular, required a
huge coordinated global effort between the field team in Syria, headquarter teams in
Brussels and a team based in Turkey: the field team selected the site (the chicken farm)
and then sent photos to the emergency team in Brussels who, together with the
logistics team, provided the team based in Turkey with a list of all the required materials. Adequate water and sanitation provisions, waste
management being a key priority Table 1 Measures taken by MSF to ensure the provision of quality surgical care in Jabal Akkrad, Syria
Essential conditions for the provision of quality
surgical care
Measures taken to achieve these conditions in the cave and chicken farm The Turkey based team obtained these materials through local procurement and then
made the necessary arrangements to transport these materials across the Turkish border
into Syria. While these preparations went on, logistics staff in the field set about designing
the new hospital. This coordinated effort enabled the hospital to be set up within 3–4
days of relocating from the cave. • A clandestine supply line from Turkey was essential for obtaining the logistical equipment
and materials needed to set up the field hospital Adequate water and sanitation provisions, waste
management being a key priority • Minimum water requirements were pre-set at 100 L water/anaesthesia and 40–60 L water/person/
day in the IPD. At the cave, three water points were installed: one for emergency care, one for
scrubbing, and one for sterilisation; at the abandoned chicken farm, a 30,000 L water tank was
already in place. To avoid potential contamination of water with previously used toxic chemicals
at the farm, the tank was emptied, cleaned, disinfected with chlorine and filled again. • A new canal system was installed in just four days and water points were installed in seven
areas of care-triage, consultation & emergency care, resuscitation, the inpatient department,
scrubbing, sterilization, and laboratory. • Minimum water requirements were pre-set at 100 L water/anaesthesia and 40–60 L water/person/
day in the IPD. At the cave, three water points were installed: one for emergency care, one for
scrubbing, and one for sterilisation; at the abandoned chicken farm, a 30,000 L water tank was
already in place. To avoid potential contamination of water with previously used toxic chemicals
at the farm, the tank was emptied, cleaned, disinfected with chlorine and filled again. A
l
ll d
f
d
d
ll d p
g
• A new canal system was installed in just four days and water points were installed in seven
areas of care-triage, consultation & emergency care, resuscitation, the inpatient department,
scrubbing, sterilization, and laboratory. • Several easy-to-clean tiled toilet units with hand washing points were installed and connected
up to a large soak-away pit built outside (not accessible to patients or visitors). g
y
• Surgical activities produce large quantities of organic, liquid and dangerous waste and there
should be proper segregation of the waste material produced. Minimum standards of surgical care Table 1 summarises the pre-requisites considered by
MSF to be essential for performing quality surgical inter-
ventions in Jabal al-Akrad and the measures taken to meet Page 4 of 8 Page 4 of 8 Trelles et al. Conflict and Health (2015) 9:36 Mandatory use of sterile equipment for surgical
and anaesthesia procedures Blood transfusion capability Ethics approval
h
d anaesthesia, local/ regional anaesthesia, or combined an-
aesthesia), type of surgical procedure (minor, wound care,
caesarean section, visceral surgery, orthopaedic surgery,
other) and intra-operative outcome (dead/alive). The study met the Medecins Sans Frontieres Ethics re-
view Board (Geneva, Switzerland) approved criteria for
studies of routinely collected data. The study was con-
ducted as a retrospective analysis of routine programme
data, and thus informed consent was not sought from
study subjects. No patient Identifying information was
included in the final analysis or reported on. Intraoperative mortality was defined as any death oc-
curring during the induction of anaesthesia, the inter-
vention itself, or the immediate recovery period (i.e., the
time during which a patient is monitored and managed
in a post-anaesthetic recovery unit [20]). Data were analysed in STATA/IC version 11.0 (Stata
corporation, Texas 77845, USA) using summary statistics. Anaesthetic and surgical procedures performed Anaesthetic and surgical procedures performed
Table 4 shows the type of anaesthetic and surgical proce-
dures performed. In total, 712 anaesthetic procedures
were performed on the 578 patients (one anaesthetic per
patient entrance into the OR: 578 new patient entrances
and 134 re-entrances for surgical re-interventions). Gen-
eral anaesthesia without intubation was the most com-
mon type of anaesthesia (used in 47 % of surgical
interventions), followed by spinal anaesthesia (25 %). Spinal procedures were used for 84 % of Caesarean sec-
tions (data not shown in the Table). Class 3:
→Patient with systemic disease severe enough to limit
activity but not incapacitating →Patient’s health: disease of more than one body system
or one major system A total of 831 surgical procedures were performed on the
578 patients. Just over half of these were minor-wound pro-
cedures and nearly one fifth, caesarean sections. Volume and profile of surgical cases p
g
Between 5th September 2012 and 1st January 2014, MSF
performed surgery on 578 new patients. Table 3 shows
the profile of these patients. Males comprised 57 % of
the cases and the median overall age was 25 years (Inter-
quartile range: 21–32 years). The majority of patients
had an ASA score of 1 indicating good pre-operative
physical status. Violent trauma was the most common
surgical indication (n-254, 44 %); the remaining indica-
tions for surgery were non-violent in type, most com-
monly being obstetric emergencies (n-191, 33 %) and
accidental trauma (n-59, 10 %). Table 2 American Society of Anaesthesiologists (ASA) score
Class 1:
→Patient in apparent good health notwithstanding his
surgical problem Class 1:
→Patient in apparent good health notwithstanding his
surgical problem →Limitations on activity: none →Limitations on activity: none →Excluded: persons at extremes of age (very young, very old) Class 2:
→Patient with mild systemic disease (e.g., mild hypertension,
mild-moderate anaemia) →Patient’s health: disease of one body system →Status of underlying disease: well controlled →Limitations on activity: none Table 1 Measures taken by MSF to ensure the provision of quality surgical care in Jabal Akkrad, Syria
Essential conditions for the provision of quality
surgical care
Measures taken to achieve these conditions in the cave and chicken farm While operating from the cave,
there was only space enough for one waste pit which therefore had to serve as a refuse for
all of the different types of waste. Once the hospital relocated to the abandoned chicken farm,
a waste-management zone with an appropriate number of pits was built and waste could
be segregated according to protocol. Availability of all essential disposables, drugs and
equipment • A regular supply of drugs, material and equipment is an essential MSF pre-requisite in order
to guarantee no ruptures in care. A clandestine supply line from Turkey ensured this, with
supplies being transported across the Turkish border either by truck (with support of the
Turkish Red Crescent) or hand-carried. • Hydro-alcoholic solution was made readily available. Strict adherence to hygiene requirements and
universal precautions y
y
• A clean sufficient water supply and sanitation provisions were ensured (as outlined above). • In the absence of water hot enough to adequately clean dirty laundry, chlorine was added
to the cold water supplying the washing machine. Mandatory use of sterile equipment for surgical
and anaesthesia procedures • In the cave, dry-heat sterilization was installed y
• At the abandoned chicken farm, a gas barrel was installed in a separate and protected
ventilated place, and linked to the heater of the autoclave inside the building. To generate
the correct circuit of activities (from dirty, to clean, and finally to sterile) the sterilization
service was provided in a large space, with two wicket gates for the “entry” and “exit” of materia Blood transfusion capability • In the cave, there was no blood bank in place. Even so, several blood transfusions were
performed when indicated if suitable donors could be identified on the spot. • Once the surgical center moved to the abandoned chicken farm, a blood bank was set
up with transfusion therapy restricted to life saving indications. Page 5 of 8 Trelles et al. Conflict and Health Table 1 Measures taken by MSF to ensure the provision of quality surgical care in Jabal Akkrad, Syria (Continued) Table 1 Measures taken by MSF to ensure the provision of quality surgical care in Jabal Akkrad, Syria (Continued) Table 1 Measures taken by MSF to ensure the provision of quality surgical care in Jabal Akkrad, Syria (Continued)
Adequate human resources in quantity and
quality
• Human resource requirements focused on sufficiency in numbers and sufficiency in skill level. • Logisticians had to be recruited to organise the surgical centre, while surgical and aesthesia
practitioners were needed to perform the required surgical procedures. • A recruitment call was made for expatriate staff with previous experience of working in war
torn settings. The technical referents devised a simplified design for the surgical centre and
then up to 70 Syrian staff (plumbers, carpenters, bricklayers etc.) were employed to rehabilitate
the abandoned chicken farm into a hospital with an operating theatre (taking just four days). Adequate human resources in quantity and
quality
• Human resource requirements focused on sufficiency in numbers and sufficiency in skill level. • Logisticians had to be recruited to organise the surgical centre, while surgical and aesthesia
practitioners were needed to perform the required surgical procedures. • Human resource requirements focused on sufficiency in numbers and sufficiency in skill level. • Logisticians had to be recruited to organise the surgical centre while surgical and aesthesia • Human resource requirements focused on sufficiency in numbers and sufficiency in skill level. • Logisticians had to be recruited to organise the surgical centre, while surgical and aesthesia
practitioners were needed to perform the required surgical procedures Adequate human resources in quantity and
quality Human resource requirements focused on sufficiency in numbers and sufficiency in skill level. • Logisticians had to be recruited to organise the surgical centre, while surgical and aesthesia
practitioners were needed to perform the required surgical procedures. • A recruitment call was made for expatriate staff with previous experience of working in war
torn settings. The technical referents devised a simplified design for the surgical centre and
then up to 70 Syrian staff (plumbers, carpenters, bricklayers etc.) were employed to rehabilitate
the abandoned chicken farm into a hospital with an operating theatre (taking just four days). Intra-operative mortality Fi-
nally, data on patient referrals to Turkey were not routinely
collected and therefore could not be reported on. Despite
th
li it ti
th
t d
i
b
f i
th t
Table 3 Profile of all new surgical patients managed at the MSF
field hospital in Jabal al-Akrad, Syria (5th September 2012-1st
January 2014)
Variable
n (%)
Total
578
Sex
Female
250 (43)
Male
328 (57)
Age (years)
0–14
50 (9)
15–29
332 (57)
30–44
145 (25)
≥45
51 (9)
Median [IQR]
25 (21–32)
ASA score
1
483 (84)
2
69 (12)
3
19 (3)
4
6 (1)
5
1 (0.2)
Surgical indication
Violent trauma
254 (44)
Obstetric emergencies
191 (33)
Accidental trauma
59 (10)
Non-traumatic and non-obstetric pathologies
74 (13)
IQR Interquartile range, ASA American Society of Anaesthesiologists
Table 4 Type of anaesthesia and surgical procedures performed
at the MSF field hospital in Jabal al-Akrad, Syria (5th September
2012-1st January 2014)
Variable
n (%)
Type of anaesthetic procedures (n-712)
General without intubation
337 (47)
Spinal
179 (25)
General with intubation
122 (17)
Local/regional
53 (7)
Combined
21 (3)
Type of surgical procedures (n-831)
Minor/ Wound care
450 (54)
Caesarean-sections
153 (18)
Visceral surgery
114 (14)
Orthopaedic/specialised surgery
64 (8)
Gynaeco-obstetric surgerya
50 (6)
aExcluding caesarean sections and reporting of observational studies [22]. There were a
number of study limitations. First, the only surgical out-
come assessed was intra-operative mortality. Other useful
indicators might have included post-operative infection
rates, other post-operative complications and peri-operative
mortality. Unfortunately however, these could not be com-
prehensively assessed due to major contextual constraints
in patient follow-up. Second, given the inherent limitations
of an observational study like this, we cannot directly attri-
bute the low intra-operative mortality rate to the measures
implemented to ensure a minimum standard of care. Fi-
nally, data on patient referrals to Turkey were not routinely
collected and therefore could not be reported on. Despite
these limitations, the study raises a number of issues that
merit discussion, particularly in relation to the provision of
quality surgical care in conflict settings. pancreatic tears. Of the 154 newborns delivered by
Caesarean section, 6 (4 %) died. pancreatic tears. Of the 154 newborns delivered by
Caesarean section, 6 (4 %) died. pancreatic tears. Of the 154 newborns delivered by
Caesarean section, 6 (4 %) died. Intra-operative mortality There were four intra-operative deaths among the 578
patients, equating to an intra-operative mortality rate of
0.7 %. The four deaths were among cases injured by
bomb blasts: one with multiple severe traumas, one with
a severe hip fracture, one with a lung laceration and
haemo-peritoneum, and one with inferior vena cava and Page 6 of 8 Trelles et al. Conflict and Health pancreatic tears. Of the 154 newborns delivered by
Caesarean section, 6 (4 %) died. Table 3 Profile of all new surgical patients managed at the MSF
field hospital in Jabal al-Akrad, Syria (5th September 2012-1st
January 2014)
Variable
n (%)
Total
578
Sex
Female
250 (43)
Male
328 (57)
Age (years)
0–14
50 (9)
15–29
332 (57)
30–44
145 (25)
≥45
51 (9)
Median [IQR]
25 (21–32)
ASA score
1
483 (84)
2
69 (12)
3
19 (3)
4
6 (1)
5
1 (0.2)
Surgical indication
Violent trauma
254 (44)
Obstetric emergencies
191 (33)
Accidental trauma
59 (10)
Non-traumatic and non-obstetric pathologies
74 (13)
IQR Interquartile range, ASA American Society of Anaesthesiologists pancreatic tears. Of the 154 newborns delivered by
Caesarean section, 6 (4 %) died. Table 3 Profile of all new surgical patients managed at the MSF
field hospital in Jabal al-Akrad, Syria (5th September 2012-1st
January 2014)
Variable
n (%)
Total
578
Sex
Female
250 (43)
Male
328 (57)
Age (years)
0–14
50 (9)
15–29
332 (57)
30–44
145 (25)
≥45
51 (9)
Median [IQR]
25 (21–32)
ASA score
1
483 (84)
2
69 (12)
3
19 (3)
4
6 (1)
5
1 (0.2)
Surgical indication
Violent trauma
254 (44)
Obstetric emergencies
191 (33)
Accidental trauma
59 (10)
Non-traumatic and non-obstetric pathologies
74 (13)
IQR Interquartile range, ASA American Society of Anaesthesiologists pancreatic tears. Of the 154 newborns delivered by
Caesarean section, 6 (4 %) died. Disc ssion
and reporting of observational studies [22]. There were a
number of study limitations. First, the only surgical out-
come assessed was intra-operative mortality. Other useful
indicators might have included post-operative infection
rates, other post-operative complications and peri-operative
mortality. Unfortunately however, these could not be com-
prehensively assessed due to major contextual constraints
in patient follow-up. Second, given the inherent limitations
of an observational study like this, we cannot directly attri-
bute the low intra-operative mortality rate to the measures
implemented to ensure a minimum standard of care. Discussion When it cannot be
upheld, we would strongly assert that such surgery should
not be undertaken; this is when a good referral sys-
tem to higher level care becomes a key responsibility
of the surgical team. Third, while recognising the limitations inherent with
an observational study like ours, together with the fact
that our only outcome measure was restricted to intra-
operative mortality, the constraints of this particular
context make it very difficult to suggest a more suitable
study design and to propose that additional outcome
measures should have been assessed. When circum-
stances
allow, we would
strongly
recommend that
indicators such as post-operative infection rates, post-
operative complications and peri-operative mortality be
measured in order to better assess the overall effect of a
surgical intervention. In the context of Syria however
this was not possible due to a number of factors, includ-
ing: i) the need to discharge patients as early as possible-
because of both the limited number of hospital beds and
the high security risk of keeping patients at the hospital -
thus limiting in-patient monitoring time, ii) patients
being unable to return or be referred back to the hos-
pital for follow-up due to insecurity and transportation
constraints, and iii) patient tracing and follow-up via cell
phone not being possible due to the disruption of tele-
phone signal. Finally, based on MSF’s experience of providing surgi-
cal care in Syria, there are a number of lessons learnt
and practice implications that could help to inform other
actors who set out to provide surgical care in conflict
settings like Syria. First, the demands are high: surgical
centres often need to be erected in a short time frame
and usually from scratch, and temporary structures are
often the only feasible option which poses a number of
challenges particularly in relation to ensuring a mini-
mum quality of care. Second, pertaining to the principle
of ‘do no harm’ we would strongly recommend that sur-
gical activities only be offered if a specific complement
of minimum measures can be implemented to assure
quality of care (adequate infrastructure, a clean and suf-
ficient water supply, effective sterilisation, comprehen-
sive guidelines and protocols etc.). This is only likely to
be possible when innovative, adapted and well-organised
ways of working can be implemented, supported by
highly experienced staff. Discussion In a conflict setting with extreme security constraints,
where access to surgical care is severely restricted, MSF
was able to adapt its modus-operandi and implement a
model of emergency surgical care with good outcomes. Quality care was assured through strict adherence to a
specific set of minimum standards of care and, over a
16 month period, MSF operated on nearly 600 patients -
mainly young adults comprising slightly more males
than females. Violent trauma was the most common rea-
son for surgery, followed by obstetric emergencies which
accounted for one third of all cases. Minor/wound care
surgery made up just over half of all the surgical proce-
dures performed, and Caesarean sections one fifth. The
intra-operative mortality rate was low at 0.7 %. First, although surgical care in conflict settings has
often been considered to be primarily indicated for war
victims, our study shows that in addition to combat-
related cases, a large number of cases are non-combat
related, tending to represent the regular burden of surgi-
cal disease found in the general population e.g., obstet-
rical emergencies and infections. This corroborates the
findings of other studies from conflict settings [3, 11]. In
fact, non-combat related surgical needs are likely to be
even greater during times of conflict due to the popula-
tion being more exposed to factors such as poor hygiene,
infectious diseases and poor access to obstetric care [23]. In this regard it is essential that surgical teams who
come to operate in such settings come prepared (i.e.,
have an appropriate team of surgeons and the neces-
sary surgical equipment) to manage both combat
and non-combat related surgical cases such as ob-
stetric emergencies. The strengths of this study were that it included i) a
large number of patients, ii) routine collection of data in
the field hospital was thorough and standardised, and iii)
the study adhered to the STROBE (Strengthening the
Reporting of Observational Studies in Epidemiology)
guidelines [21] and sound ethics principles for conducting Page 7 of 8 Trelles et al. Conflict and Health (2015) 9:36 and “outcomes” [24].” Structure” describes the factors that
affect the context in which care is delivered (e.g., hospital
buildings, equipment, human resources); “process” de-
scribes the transactions between patients and healthcare
providers during healthcare delivery (i.e., those factors that
influence how healthcare is delivered); and “outcomes” re-
fers to the effects of healthcare on the health status of pa-
tients/populations. Discussion The minimum standards of surgical
care endorsed by MSF ensure that all the necessary “struc-
tural” elements are in place for maximising quality of care. Meanwhile, MSF ensures that important ‘process’ factors
are upheld through comprehensive and standardised pro-
tocols for all levels of care, appropriate record keeping
and comprehensive data collection systems. In conflict
settings, such as Syria, where outcome measurement - as
a reflection of quality of care - is often going to be a chal-
lenge, we would advocate that when minimum standards
of care are rigorously endorsed, and guidelines and proto-
cols strictly adhered to, we can reasonably assume that pa-
tients are receiving good quality care and that the results
of our activities are satisfactory. This rationale is sup-
ported by the fact that across different MSF surgical
projects (where there are notable differences in context,
surgical caseload, and surgical capacity, but where the
same minimum standards of care are strictly adhered to),
intra-operative mortality and post-operative rates are con-
sistently low [25]. Second, although we cannot attribute the low intra-
operative mortality seen in our study to the surgical inter-
vention itself, our outcomes are extremely favourable
when compared to the outcomes reported by another
study conducted in a clandestine field hospital in Syria
[17]. In this study, among the 28 patients operated on -
the most common procedures being vascular procedures,
orthopaedic procedures and abdominal exploration - two
patients died intra-operatively (giving an intraoperative
mortality rate of 7 %). Although we can only speculate,
the significantly lower intra-operative mortality rate seen in
our study may be due to a number of factors including i)
patients dying before arriving at the MSF hospital, ii) better
quality of surgical care in the MSF hospital, and/or iii) less
severe cases making up the MSF surgical caseload (severe
cases being referred to Turkey for management). This latter
point raises an important issue - namely the importance of
surgical teams recognising when they do not have the cap-
acity to manage particular cases. Patients can pay a high
price for sub-standard surgical care - post-operative infec-
tions, disability and even death - and thus the principle of
‘do no harm’ should prevail at all times. Discussion Regional Analysis Syria - Part A: Syria - October 2014. [http://www.acaps. org/img/reports/p-140404-ras-part-i—finalpdf] Accessed 10 July 2015 5. SNAP. Regional Analysis Syria - Part A: Syria - October 2014. [http://www.acaps. f
f org/img/reports/p-140404-ras-part-i—final.pdf]. Accessed 10 July 2015. 6. Coutts A, Fouad FM. Response to Syria’s health crisis—poor and
di
t d L
t 2013 381 2242 3 6. Coutts A, Fouad FM. Response to Syria’s health crisis—poor and
uncoordinated. Lancet. 2013;381:2242–3. 6. Coutts A, Fouad FM. Response to Syria’s health crisis—poor and 6. Coutts A, Fouad FM. Response to Syria’s health crisis—poor and
uncoordinated. Lancet. 2013;381:2242–3. uncoordinated. Lancet. 2013;381:2242–3. 7. Coutts A, McKee M, Stuckler D. The emerging Syrian health crisis. Lancet. 2013;381(9865):e6–7. 7. Coutts A, McKee M, Stuckler D. The emerging Syrian health crisis. Lancet. 2013;381(9865):e6–7. 8. Gulland A. Medical students perform operations in Syria’s depleted health
system. BMJ. 2013;346:f3107. y
9. Kaldor M. In defence of New wars. Stability: Intern J Secur Dev. 2013;2:3–16. Authors’ contributions
MT LD KTS
d KK 18. Jaunoo SS, Harji DP. Damage control surgery. Int J Surg. 2009;7:110–3. MT, LD, KTS and KK were involved with the study conception and design. MT, LD, AZ, TV, SD and MCF were involved with implementation. MT and
LD were involved with the acquisition of the data. MT, KTS and KK were
involved with the analysis and all co-authors were involved with the
interpretation of the data. KTS, MT and LD wrote the first draft of the paper
which was reviewed by all co-authors who contributed significantly to the
intellectual content. All co-authors approved the final manuscript and agreed
to be accountable for all aspects of the work behind the study. 19. Kahn C, Schultz C, Miller C, Anderson CL. Does START triage work?: an
outcomes assessment after a disaster. Ann Emerg Med. 2009;54:424–30. 20. Association of Anaesthetists of Great Britain and Ireland. Imm
anaesthesia recovery 2013. Anaesthesia. 2013;68:288–97. 21. Von Elm E, Altman DG, Egger M. The Strengthening the Reporting of
Observational Studies in Epidemiology (STROBE) statement: guidelines for
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ethics is indispensable for good-quality operational research. Public Health
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Th
i The surgical project in Syria was funded by MSF-Operational Centre Brussels,
and all authors of this study were supported by the same. 25. Alvarado O, Trelles M, Tayler-Smith K, Joseph H, Gesline R, Wilna TE, et al. Orthopaedic surgery in natural disaster and conflict settings: how can
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ting like Syria are high and include both combat and a
large number of non-combat indications, particularly ob-
stetric emergencies. Provision of quality surgical care in
a volatile and complex setting like this is possible pro-
viding appropriate measures, supported by highly experi-
enced staff, can be implemented that allow a specific set
of minimum standards of care to be adhered to. This is
particularly important when patient outcomes - as a re-
flection of quality of care - are difficult to monitor and
assess. 11. Chu K, Trelles M, Ford N. Rethinking surgical care in conflict. Lancet. 2010;375:262–3. 12. Chu K, Ford N, Trelles M. Providing surgical care in Somalia: a model of task
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low-velocity penetrating head injuries. Indications and management
protocol based upon more than 8 years follow-up of 99 cases from
Iran–Iraq conflict. Surg Neurol. 2003;60:105–11. 14. Amirjamshidi A, Abbassioun K, Rahmat H. Minimal debridement or
simple wound closure as the only surgical treatment in war victims with
low-velocity penetrating head injuries. Indications and management
protocol based upon more than 8 years follow-up of 99 cases from
Iran–Iraq conflict. Surg Neurol. 2003;60:105–11. 15. Splavski B, Vrankovic D, Blagus G, Mursic B, Rukovanjski M. Early
management of war missile spine and spinal cord injuries: experience
21 cases. Injury. 1996;27:699–702. 15. Splavski B, Vrankovic D, Blagus G, Mursic B, Rukovanjski M. Early
management of war missile spine and spinal cord injuries: experience with
21 cases. Injury. 1996;27:699–702. j y
16. Zangana AM. Penetrating liver war injury: a report on 676 cases, after
Baghdad invasion and Iraqi civilian war April 2003. Adv Med Dent Sci. 2007;1:10–4. 16. Zangana AM. Penetrating liver war injury: a report on 676 cases, after
Baghdad invasion and Iraqi civilian war April 2003. Adv Med Dent Sci. 2007;1:10–4. 1.
Bickler S, Ozgediz D, Gosselin R, Weiser T, Spiegel D, Hsia R, et al. Key
concepts for estimating the burden of surgical conditions and the unmet
need for surgical care. World J Surg. 2010;34:374–80. Discussion management of war missile spine and spinal cord injuries:
21 cases. Injury. 1996;27:699–702. 16. Zangana AM. Penetrating liver war injury: a report on 676
Baghdad invasion and Iraqi civilian war April 2003. Adv Me
2007;1:10–4. 17. Hasanin A, Mukhtar A, Mokhtar A, Radwan A. Syrian revolu
hospital under attack. Am J Disaster Med. 2013;8(4):259–65
18. Jaunoo SS, Harji DP. Damage control surgery. Int J Surg. 20
19. Kahn C, Schultz C, Miller C, Anderson CL. Does START triag
outcomes assessment after a disaster. Ann Emerg Med. 20
20. Association of Anaesthetists of Great Britain and Ireland. Im
anaesthesia recovery 2013. Anaesthesia. 2013;68:288–97. 21. Von Elm E, Altman DG, Egger M. The Strengthening the Re
Observational Studies in Epidemiology (STROBE) statemen
reporting observational studies. Bull World Health Organ. 2
22. Edginton M, Enarson D, Zachariah R, Reid T, Satyanarayana
ethics is indispensable for good-quality operational researc
Action. 2012;2:21–2. 23. Sidel VW, Levy BS. The health impact of war. Int J Inj Cont
2008;15:189–95. 24. Donabedian A. Evaluating the quality of medical care. Milb
Q. 1996;44:166–203. 25. Alvarado O, Trelles M, Tayler-Smith K, Joseph H, Gesline R,
Orthopaedic surgery in natural disaster and conflict setting
quality care be ensured? Int Orthop. 2015;39:1901–8. 26
B kl J FM Ni k
JW
S h
b J R d
d AD M care. Finally, a certain level of preparedness is essential, in-
cluding having i) policies on minimum standards accord-
ing to best practice pre-established, ii) standardised
protocols for all levels of care (safety, infection control,
surgical etc., iii) standardised monitoring and evaluation
tools in place so that reliable data can be collected
[26]- including, where possible, patient follow-up data
(infection rates, complications, peri-operative mortality)
and data on patient referrals, iv) a pool of surgical
personnel to recruit from, and v) the surgical capacity to
manage both war-wounded and non-trauma emergency
cases, such as obstetrical emergencies. 2. McQueen KA, Parmar P, Kene M, Broaddus S, Casey K, Chu K, et al. Burden
of surgical disease: strategies to manage an existing public health
emergency. Prehosp Disaster Med. 2009;24 Suppl 2:228–31. 3. Chu K, Havet P, Ford N, Trelles M. Surgical care for the direct and indirect
victims of violence in the eastern democratic Republic of Congo. Confl
Health. 2010;4:1–6. 4. European Commission’s Humanitarian Aid and Civil Protection department
(ECHO). ECHO Factsheet-Syria crisis-July 2015. [http://ec.europa.eu/echo/files/aid/
countries/factsheets/syria_en.pdf]. Accessed 8 July 2015. y
p
y
5. SNAP. Discussion Third, beyond the provision of
surgical activities, appropriate referral networks should
ideally be identified where possible, so that patients who
cannot be managed due to resource constraints can be re-
ferred for appropriate care. Related to this, complimentary
support (e.g., the supply of specific surgical materials) to
other actors in the field who have the human resource
capacity to provide general and more specialised surgery
can improve overall access to a greater scope of surgical The difficulties around post-operative monitoring and
patient follow-up are added justification for MSF’s insist-
ence that minimum standards of surgical care be strictly
adhered to. According to the concepts outlined in the
Donabedian Model, quality of healthcare can be consid-
ered in relation to three domains: “structure,” “process,” Trelles et al. Conflict and Health (2015) 9:36 Page 8 of 8 Page 8 of 8 2. McQueen KA, Parmar P, Kene M, Broaddus S, Casey K, Chu
of surgical disease: strategies to manage an existing public
emergency. Prehosp Disaster Med. 2009;24 Suppl 2:228–31
3. Chu K, Havet P, Ford N, Trelles M. Surgical care for the dire
victims of violence in the eastern democratic Republic of C
Health. 2010;4:1–6. 4. European Commission’s Humanitarian Aid and Civil Protection
(ECHO). ECHO Factsheet-Syria crisis-July 2015. [http://ec.europa
countries/factsheets/syria_en.pdf]. Accessed 8 July 2015. 5. SNAP. Regional Analysis Syria - Part A: Syria - October 2014. [ht
org/img/reports/p-140404-ras-part-i—final.pdf]. Accessed 10 Ju
6. Coutts A, Fouad FM. Response to Syria’s health crisis—poo
uncoordinated. Lancet. 2013;381:2242–3. 7. Coutts A, McKee M, Stuckler D. The emerging Syrian healt
2013;381(9865):e6–7. 8. Gulland A. Medical students perform operations in Syria’s d
system. BMJ. 2013;346:f3107. 9. Kaldor M. In defence of New wars. Stability: Intern J Secur
10. Attar S. Field hospitals in Syria. Lancet. 2014;383:303. 11. Chu K, Trelles M, Ford N. Rethinking surgical care in conflic
2010;375:262–3. 12. Chu K, Ford N, Trelles M. Providing surgical care in Somalia
shifting. Confl Health. 2011;5:1–5. 13. Tajsic NB, Husum H. Reconstructive surgery including freef
be performed in low-resource settings: experiences from a
scenario. J Trauma Inj Infect CritCare. 2008;65:1463–7. 14. Amirjamshidi A, Abbassioun K, Rahmat H. Minimal debride
simple wound closure as the only surgical treatment in wa
low-velocity penetrating head injuries. Indications and ma
protocol based upon more than 8 years follow-up of 99 c
Iran–Iraq conflict. Surg Neurol. 2003;60:105–11. 15. Splavski B, Vrankovic D, Blagus G, Mursic B, Rukovanjski M. Author details
1
d y
26. Burkle Jr FM, Nickerson JW, von Schreeb J, Redmond AD, McQueen KA,
Norton I, et al. Emergency surgery data and documentation reporting forms
for sudden-onset humanitarian crises, natural disasters and the existing
burden of surgical disease. Prehosp Disaster Med. 2012;27:577–82. 1Médecins Sans Frontières-Operational Centre Brussels (MSF-OCB), Surgery,
Anaesthesia, Gynaecology, and Emergency Medicine Unit, Brussels, Belgium. 2MSF-OCB, Operational Research Unit, MSF-Luxembourg, Luxembourg,
Luxembourg. 3MSF-OCB, Emergency pool, Brussels, Belgium. 4MSF-OCB,
Logistics department, Brussels, Belgium. 5MSF-OCB, MSF Syria project, Latakia
Governorate, Syria. Received: 21 August 2015 Accepted: 8 November 2015 References 1. Bickler S, Ozgediz D, Gosselin R, Weiser T, Spiegel D, Hsia R, et al. Key
concepts for estimating the burden of surgical conditions and the unmet
need for surgical care. World J Surg. 2010;34:374–80.
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The different responses of rice genotypes to heat stress associated with morphological, chlorophyll and yield characteristics
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◦C and moreover, sustained high temperature for more than a week cause severe damage (Melo et al., 2021). As an important cereal crop, rice productivity might decrease globally by about 3.2% per each degree-Celsius increase in global mean temperature (Zhao et al., 2017). As the rice crop is concerned, the optimum temperature is 33°C (Jagadish et al., 2007). Depending on the specific stage of rice development, heat stress reduces number of tillers/panicles, decreases grain number/plant and lower grain weight, thus negatively impacting yield formation, improving rice crop tolerance to heat stress in terms of sustaining yield stability under high day temperature, high night temperature, or combined high day and night temperature will bolster future food security (Xu et al., 2020). Therefore, high temperatures over 35 °C encourage spikelets sterility and unfilled grain (Ghazy, 2012). Moreover, high temperature affects rice plant growth, for instance, during seed germination, high temperature may slow down or totally inhibit germination, depending on plant species and the intensity of the stress (Yoshida, 1981). Also, high temperature over 35 °C may harmfully affect photosynthesis, water relations, membrane stability and primary and secondary metabolites (Yoshida, 1981; Naveed et al., 2020). Moreover, throughout plant ontogeny, enhanced the expression of heat shock proteins, other stress-related proteins, and reactive oxygen species accumulation (Wahid et al., 2007). Plant tolerance to heat can be divided into escape (successful reproduction before the onset of severe stress), avoidance (maintenance of a high plant water status during stress e.g. caused by stomatal closure, reduced leaf area, senescence of older leaves, etc. or by increased root growth) and tolerance (the maintenance of plant function at limited water availability and/or the recovery of plant water status and plant function after stress) may involve osmotic adjustments, but may also be the result of rigid cell walls or small cells (Kondamudi et al., 2012). The heat stress response depends on plant species and genotypes (Wahid et al., 2007), so very important to identify the tolerant genotypes based on their morphological and physiological characters under heat conditions. Heat stress dominantly reduced fertility when drought and heat stress co-occurred and the pollen count on the stigma reduced in Moroberekan, therefore, not all drought-tolerant cultivars are tolerant to heat stress by default at sensitive stages however, heat stress seems to be the dominant stress at reproductive stage (Costa et al., 2021). The phenotypic correlation among some heat morphological characteristics and their contribution to grain yield under heat conditions were directly or indirectly expected in rice (Barnabás et al., 2008). The thousand-grain weight and spikelet fertility showed a positive and significant at probability level 0.05 association with yield plant-1under heat conditions at the genotypic level. Paddy yield had a strong correlation with grain number panicle-1, days to maturity and 1000-grain weight (Gaballah and Abu El-Ezz, 2019). Theeffect of heat stress at the seedling and tillering stages of rice led to no panicle development in the cultivars (Azameti et al., 2021). The objective of this study was to evaluate the impact of high temperatures stress on yield and related parameters ofrice, to identify rice genotypes tolerance for heat stress, estimate the correlation coefficient based on agro-morphological characteristics and applicability of stress index under high heat stress conditions. Sakha -Kafr EL-SheikhMonthDate20182019Max.Min.RH %Max.Min.RH %May10-Jan29.071763.4929.417.264.120-Nov29.961861.430.318.46220-3031.0520.558.9231.420.759.5June10-Jan29.9619.851.7830.320.252.320-Nov31.0521.866.331.422.266.420-3029.4621.4163.529.821.863.6July10-Jan31.0521.4168.231.421.868.320-Nov33.132273.133.52273.220-3035.6124.665.13624.665.2August10-Jan35.926.267.553626.368.220-Nov35.625.566.0735.725.666.720-3033.923.368.253423.368.9September10-Jan35.123.365.5735.223.366.220-Nov34.323.866.634.423.866.720-3034.524.361.334.624.361.4EL-Kharga -New ValleyMay10-Jan3721.226.236.42126.320-Nov37.320.525.137.420.525.220-3039.223.824.140.423.824.1June10-Jan39.925.822.34025.922.320-Nov38.924.626.23924.626.320-3038.723.527.138.823.527.2July10-Jan38.523.728.238.623.728.320-Nov40.623.627.140.723.627.220-3043.225.121.143.325.221.1August10-Jan45.229.122.245.329.222.220-Nov44.130.426.144.230.526.220-3039.926.730.94026.831September10-Jan40.525.431.140.625.531.220-Nov41.125.831.241.225.931.320-3041.525.827.141.625.927.2The soil properties in the two locations are presented in Table 3. The soil samples of 0-15 cm depth were collected from selected plots to determine the chemical and physical properties. The soil samples were air-dried, crushed and passed through 2 mm sieve to analyse organic carbon and total N content. Soil organic carbon was analysed by Walkley-Black method (Allison, 1965). One g air-dried sample (passed through 0.5 mm sieve) was taken in a 500 ml Erlenmeyer flask. Ten ml 1N K2Cr2O7and 20 ml conc. H2SO4was added to the flask and react for 30 minutes after which 200 ml distilled water and 10 ml H3PO4was added. One ml orthophenanthroline indicator was added to the flask and was titrated with about 0.5N FeSO4solution. Blank titration was run to calculatethe strength of the FeSO4solution. Total nitrogen was determined by Microkjeldahl instrument, 0.5 g air-dried soil sample was digested in 3 ml conc. H2SO4in the presence of 0.5 g of digestion mixture (50: 10: 1 K2SO4: CuSO4.5H2O: metallic selenium). Thedigested sample was distillate with 40% NaOH and the distillate was collected in 4% boric acid containing three drops of mixed indicator (bromocresol green and methyl red), which was then titrated with 0.05 N H2SO4. C: N ratio was calculated by dividing the results of organic carbon by total nitrogen (Bremner, 1965). All measurements of organic carbon and total nitrogen were done in triplicate. Soil pH in water was measured from a soil: water ratio 1: 2.5 using glass electrode method (Peech, 1965). Twenty gram of air-dried soil sample was taken in 100 ml of plastic bottle and 50 ml of distilled water was added. The suspension was stirred with a glass-rod at regular interval for 30 minutes. A glass electrode pH meter calibrated with buffer pH 7.0 and 4.0 andthe pH of soil suspension was measured. The measurement was done in triplicate. Olsen-P was extracted with 0.5M sodium bicarbonate (pH 8.5) (Olsen et al., 1954). P content in the extract was determined using ascorbic acid as reducing agent by a spectrophotometer. Available potassium (NH4OAc-K) was extracted with neutral 1N ammonium acetate (Hanway and Heidel, 1952) and estimated by a flam photometer. Source of varianceYears(Y)Environments (E)E x YBlocks (EY)Genotypes (G)G x YG x EG x E x YPoolederrorTotaldf111815151515120191Days to heading (day)3.19 ns19865.47 **2.09 ns2.48278.58 **1.587 ns118.78 **2.21 *1.17Chlorophyll content (mg dS-1)2.09 ns4101.13 **10.05 ns2.5735.67 **0.58 ns46.58 **1.05 ns1.06Leaf rolling0.08ns963.02**0.08ns0.665.57**0.15 ns5.82 **0.150 ns0.41Plant height (cm)42.13 ns17557.31**2.19 ns0.57183.52**1.39**157.05**2.29**0.56Panicle length (cm)2.46 ns810.76**1.07 ns0.66.33**0.62 ns20.03**0.21 ns0.59Number of tillers plant-17.27 ns2442.59**1.17 ns1.211.22**0.28 ns20.60**0.45 ns0.87Number of panicles plant-14.15 ns2647.74**1.56 ns1.8821.20**0.62 ns16.77**1.19 ns0.81100-grain weight (g)0.02 ns6.70**0.02 ns0.0010.57**0.01 ns0.45**0.02 ns0.01Sterility percentage (%)1.85 ns56123.33**21.16 ns2.77706.06**0.77 ns1058.23**0.78ns0.91Grain yieldplant-1 (g)30.59 ns185803.54**0.30 ns2.24227.03**1.05 ns220.00**1.41 *0.82(ns) = Not Significant, (*) = Significant at 0.05 and (**) = Significant at 0.01 level of probabilityThe interaction between genotypes and EnvironmentsRegarding the interaction in Table 5, the results indicated that, the shortest days to heading genotypes were observed in ‘Sakha 102’ under heat conditions while the longest days to heading genotypes were recorded with ‘Egyptian Yasmine’ and ‘Sakha 101’ under natural conditions. The highest values of chlorophyll content were found in ‘Sakha 103’ under natural conditions and the minimum values were found in ‘Giza177’ under heat conditions. Concerning leaf rolling, the minimum mean values were achieved from ‘Giza 178’, ‘Giza 179’ and ‘Sakha 107’ under heat stress conditions, nevertheless, the highest values were obtained with ‘Giza 177’, ‘Giza 182’, ‘Sakha 101’, ‘Sakha 102’ and ‘Sakha 103’ under heat stress conditions. The highest panicles length was recorded with ‘Egyptian Yasmine’ under natural conditions and the minimum values were achieved with ‘Sakha 105’ under heat stress conditions.The interaction between rice genotypes and environments for grain yield and its related characters were shown in Table 6. The maximum values of panicles number plant-1were achieved with ‘Hybrid 2’ under natural conditions; however, the minimum values were obtained with ‘Sakha 103’ under heat conditions. The heaviest 100-grain weight was recorded with ‘Egyptian Yasmine’ and ‘Sakha 104’ under natural conditions and the minimum grains weight was obtained with ‘Giza 178’, ‘Sakha 103’, ‘Sakha 104’ and ‘Sakha 106’ under heat stress. The minimum values of sterility percentage were recorded with ‘Sakha 101’, ‘Sakha 103’ and ‘Sakha107’ under natural conditions, therefore the highest percentage was obtained from ‘Sakha 106’ under heat stress. Additionally, the highest mean values of grain yield plant-1were obtained from ‘Hybrid 2’ under natural conditions, however, the minimum values were obtained with ‘Sakha 102’ and ‘Giza 177’ under heat conditions. We would like to acknowledge the
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Antifungal and Antioxidant Properties of Chitosan Polymers Obtained from Nontraditional Polybius henslowii Sources
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Marine drugs
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Received: 27 January 2019; Accepted: 24 March 2019; Published: 22 April 2019 Abstract:
Chitin was extracted from Polybius henslowii, a swimming crab, captured in large
quantities throughout the Portuguese coast by purse seine vessels as bycatch. After standard chitin
extraction procedures, water-soluble chitosan products were obtained via two different methods:
(1) N-acetylation with the addition of acetic anhydride and (2) a reaction with hydrogen peroxide. The chemical structure and molecular weight of chitosan derivatives, water-soluble chitosan (WSC)
and chitooligosaccharides (COS), were confirmed by Fourier Transform Infrared Spectroscopy (FT-IR)
and gel permeation chromatography (GPC). Antioxidant and metal chelation activities were evaluated,
and the growth inhibition capacity was tested on four phytopatogens. The chitooligosaccharides
from pereopods (pCOS) and shell body parts (sCOS) inhibited all fungal species tested, particularly
Cryphonectria parasitica with 84.7% and 85.5%, respectively. Both radical scavenging and antifungal
activities proved to be dose-dependent. Chitooligosaccharides with a low molecular weight (2.7,
7.4, and 10.4 Kg·mol−1) showed the highest activity among all properties tested. These results
suggested that chitosan derivatives from P. henslowii raw material could potentially be used against
phytopathogens or as ingredient in cosmetics and other products related to oxidative stress. Keywords: Polybius henslowii; marine resources; chitosan; chitooligosaccharides; antifungal activity;
antioxidant activity Article
Antifungal and Antioxidant Properties of Chitosan
Polymers Obtained from Nontraditional
Polybius henslowii Sources Francisco Avelelas 1, André Horta 2, Luís F.V. Pinto 3,4
, Sónia Cotrim Marques 2,5
,
Paulo Marques Nunes 1, Rui Pedrosa 1 and Sérgio Miguel Leandro 1,* Francisco Avelelas 1, André Horta 2, Luís F.V. Pinto 3,4
, Sónia Cotrim Marques 2,5
,
Paulo Marques Nunes 1, Rui Pedrosa 1 and Sérgio Miguel Leandro 1,* 1
MARE—Marine and Environmental Sciences Centre, ESTM, Instituto Politécnico de Leiria,
2520-641 Peniche, Portugal; franciscoavelelas@gmail.com (F.A.); paulo.nunes@ipleiria.pt (P.M.N.);
rpedrosa@ipleiria.pt (R.P.) p
p
p
2
MARE—Marine and Environmental Sciences Centre, Instituto Politécnico de Leiria,
2520-641 Peniche, Portugal; andre.horta@ipleiria.pt (A.H.); sonia.cotrim@ipleiria.pt (S.C.M.) 3
BioCeramed, S.A., Rua José Gomes Ferreira nº 1 - Armazém D 2660-360 São Julião do Tojal, Portuga
info@bioceramed.com 4
CENIMAT/I3N, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia FC
Universidade Nova de Lisboa, Campus da Caparica, 2829-516 Caparica, Portugal
5
Instituto Português do Mar e da Atmosfera (IPMA) Rua Alfredo Magalhães Ramalho, 6,
1449-006 Lisboa, Portugal g
*
Correspondence: sleandro@ipleiria.pt; Tel.: +351-262-783-607; Fax: +351-262-783-088 Received: 27 January 2019; Accepted: 24 March 2019; Published: 22 April 2019 marine drugs marine drugs 1. Introduction Fungal pathogens are responsible for huge economic losses worldwide. These pathogens may cause
damage to roots, crowns, stems, leaves, and fruits of a large range of economically important plants. In Portugal, several fungi species, such as Phytophthora cinnamomi, Botrytis cinerea, Cryphonectria parasitica,
and Heterobasidion annosum cause considerable damage in cork oak forests [1], vegetable crops [2], chestnut
trees [3], pines, and conifers [4], respectively. Since chemical fungicides such as sulfur dioxide [5] raise
health concerns, the agrochemical industry has been searching for less toxic products. www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, 239; doi:10.3390/md17040239 2 of 15 Mar. Drugs 2019, 17, 239 Chitosan, a straight-chain polymer of glucosamine and N-acetylglucosamine [6] has emerged
as a promising antimicrobial material [7]. As they are obtained from chitin present in crustacean’s
exoskeleton and fungi cell walls, chitosan products are biocompatible and biodegradable, with a wide
range of applications such as in wastewater treatment, food, cosmetics, agrochemicals, cell culture,
textiles, and medical devices [8]. Chitosan also exhibits antioxidant activity [9,10] and, therefore, could
also be used as a replacement for synthetic antioxidants such as butylated hydroxytoluene (BHT),
butylated hydroxy-anisole (BHA), and tert-butylhydroquinone (TBHQ) [11]. In recent years, the valorization of fisheries discards by-products has received much attention due
to the increasing awareness of its economic potential and environmental impacts [12]. Polybius henslowii,
a benthopelagic species is found at depths between 0 and 650 m along the eastern Atlantic coasts from
Ireland and Britain to the Alborán Sea [13,14] and Morocco [15]. Despite its benthic habit, it also has
periodic pelagic phases when larges swarms move along the surface to coastal waters, gathering at high
densities from 0 to 14812 individuals per ha [16], with strong interannual oscillations [17]. Although being
an extremely abundant marine resource, it is not presently subject to commercial use, and approximately
1240 tonnes/year (mean values from 2004 to 2009) are discarded annually [18]. In Portugal, it is captured as
bycatch during the purse seining of Sardina pilchardus [18], having a destructive impact on the fishing nets. As such, it is regarded as a plague by the fisherman and not as a potential source of economic incomes. The objective of the present study was to determine if this bycatch could be converted into a
value-added product by chitin extraction and chitosan production. The products were characterized by
gel permeation chromatography (GPC) and FT-IR, and their antioxidant, metal chelation, and antifungal
properties were evaluated. 2.2. Chitin Extraction Optimization The conventional demineralization process of crustacean waste uses strong acids such as
hydrochloric acid (HCl). This chemical treatment can result in changes to the physiochemical
properties of chitin (the hydrolysis of the chitin chains that reduces the average molecular weight of the
biopolymer), produces harmful effluent wastewater, and contributes to the cost of the chitin purification
process [26]. A mineral-free chitin with a very low ash content is usually required for applications with
a very low impurity tolerance, such as biomedical and nutrition products [8]. Therefore, three different
HCl concentrations were tested to minimize chitin chain damage and acid use. The mineral content in pereopods and shell samples after treatment with HCl was different
regarding calcium carbonate removal (Table 2). The lowest ash content was found for the pereopods
and shells after treatment with 1 M HCl (Table 2). Nevertheless, all treatments (0.5, 0.75, and 1 M)
promoted a high ash removal. Table 2. The ash and protein contents of segmented body parts of P. henslowii after treatment with 1 M,
0.75 M, and 0.5 M HCl and NaOH. Mean value (± SD). NaOH/HCl
Shells Samples
Protein Content (%)
Protein Removal (%)
Ash Content (%)
Ash Removal (%)
1 M
2.0 ± 0.12
96.1 ± 0.25
0.8 ± 0.01
98.2 ± 0.02
0.75 M
2.3 ± 0.14
95.35 ± 0.28
1.0 ± 0.05
97.8 ± 0.11
0.5 M
2.29 ± 0.15
95.39 ± 0.31
1.2 ± 0.17
97.3 ± 0.38
NaOH/HCl
Pereopods Samples
Protein Content (%)
Protein Removal (%)
Ash Content (%)
Ash Removal (%)
1 M
1.2 ± 0.12
92.2 ± 0.78
0.4 ± 0.19
99.1 ± 0.4
0.75 M
1.5 ± 0.07
90.5 ± 0.48
0.5 ± 0.01
98.9 ± 0.8
0.5 M
1.8 ± 0.06
88.2 ± 0.43
0.7 ± 0.05
98.6 ± 0.1 Table 2. The ash and protein contents of segmented body parts of P. henslowii after treatment with 1 M,
0.75 M, and 0.5 M HCl and NaOH. Mean value (± SD). For an efficient chitin extraction, the associated proteins should be removed at a second stage. Deproteinization carried out with sodium hydroxide (NaOH) at elevated temperatures promoted the
removal of protein from crab wastes. Residual protein was determined after each wash in order to
evaluate the efficiency of each concentration applied. After treatment with NaOH, the protein values
were highest in shells and pereopods treated with 0.5 M NaOH. 2.1. Polybius henslowii Characterization The biochemical composition in terms of protein, minerals, lipids, and chitin of the swimming
crab Polybius henslowii exoskeleton is shown in Table 1. Table 1. The characterization of dried P. henslowii expressed as a percentage of dried weight (% of DW). Mean value (± SD). Table 1. The characterization of dried P. henslowii expressed as a percentage of dried weight (% of DW). Mean value (± SD). Raw Material
Protein (%)
Ash (%)
Lipids (%)
Chitin (%)
Shell
32.1 ± 6.68
44.5 ± 0.57
13.2 ± 0.25
9.7 ± 0.57
Pereopods
16.6 ± 1.21
49.3 ± 5.86
1.6 ± 0.14
11.4 ± 0.19 Raw Material
Protein (%)
Ash (%)
Lipids (%)
Chitin (%)
Shell
32.1 ± 6.68
44.5 ± 0.57
13.2 ± 0.25
9.7 ± 0.57
Pereopods
16.6 ± 1.21
49.3 ± 5.86
1.6 ± 0.14
11.4 ± 0.19 The shell samples showed a higher percentage of protein and lipids than pereopods. The ash
percentage, directly correlated with the calcium carbonate in the exoskeleton, was similar in both
body parts. The protein and ash results are in accordance with those reported in previous studies [19],
suggesting a higher yield of ash than protein, lipids, or chitin from the crab samples (about 16.6% for
protein and 66.6% for ash content). The protein values are in line with the literature [20] for Alaska king
crab (from 16.3 to 20.7%) but are lower than those reported for other crab species such as the Metacarcinus
magister [21], Callinectes sapidus [22], and Carcinus maenas [23]. Previous studies [23] suggested that
differences in the diet of crab at the harvesting site influences their biochemical composition, which
could contribute to differences reported in the protein and lipid contents. Previously published studies on crab species reported values of chitin from 14 to 28% of their total
dry weight [24]. The pereopods of the swimming crab showed a higher chitin content than the shells. The chitin values shown in Table 1 are lower than those from other published studies [19,25]; however,
authors [25] suggested that differences in chitin yield could be related to such factors as harvest year
and/or shell storage duration, which could explain the low yield. 3 of 15 Mar. Drugs 2019, 17, 239 2.2. Chitin Extraction Optimization The residual protein concentration
was higher when 0.5 and 0.75 M of NaOH were used, rather than 1 M NaOH (Table 2). g
(
)
A high-quality chitosan product should have less than 1% protein [27]. Moreover, a complete
removal of protein is desirable, since it allows a higher solubility of chitosan after deacetylation [28]. 2.3. Chitosan and Water-Soluble Chitosan Characterization 2.3. Chitosan and Water-Soluble Chitosan Characterization The data for chitosan samples characterization in terms of yield, dynamic viscosity, degree of
deacetylation, and molecular weight are shown in Table 3. The degree of deacetylation (DD) of chitosan
products was determined by infrared spectroscopy analysis. The ratio A1320/A1420 was slightly different
between the samples. The degree of deacetylation of chitosan obtained from shell chitin was 95.1 ±
0.01%, while the samples from pereopods were 91 ± 0.04% deacetylated. The difference is probably
due to the longer deacetylation process of 7 h, the time needed to allow a complete dissolution in 1%
acetic acid. Therefore, chitosan from pereopods resulted in a higher Mw (378 kg.mol−1) and dynamic
viscosity (749.2 ± 62.7 cP). Since the average Mw and dynamic viscosity are closely related, the decrease
in these values is consistent with a prolonged reaction time. Previous work [29] demonstrated that a
longer exposure to NaOH during deacetylation resulted in a decrease in Mw and dynamic viscosity. The yield of chitosan as a percentage of the crab dry weight from both body parts also showed lower 4 of 15 Mar. Drugs 2019, 17, 239 values when compared with those from other studies [30], a consequence of the small amount of chitin
extracted from the initial raw material. Table 3. Chitosan yield (%), dynamic viscosity (cP), deacetylation degree (DD%), and molecular
weight (kg.mol−1) obtained from Polybius henslowii raw material. pWSC—Pereopods water-soluble
chitosan; pCOS—pereopods chitooligosaccharides; sWSC—shells water-soluble chitosan; sCOS—shells
chitooligosaccharides. Mean value (± SD). Chitosan Products
Yield (%)
Dynamic Viscosity (cP)
DD (%)
Mw (kg·mol−1)
Pereopods chitosan
9.7 ± 0.62
749.2 ± 62.69
94.3 ± 0.04
378.2 ± 78.00
pWSC
-
-
62 ± 0.53
404.0 ± 45.00
pCOS
-
-
93.3 ± 0.04
7.4 ± 1.20
Shells chitosan
8.0 ± 0.24
417.2 ± 94.99
95.1 ± 0.01
247.0 ± 31.20
sWSC
-
-
55.0 ± 3.21
279.0 ± 33.00
sCOS
-
-
95.0 ± 0.62
2.7 ± 0.40
Commercial chitosan
-
-
87.0
780.0
ccWSC
-
-
57.0 ± 0.83
775.0 ± 42.00
ccCOS
-
-
86.0 ± 1.4
10.4 ± 0.70 Chitosan is only soluble in acidic solutions, which limits its applications. The solution viscosity is
usually also quite high, which makes it difficult to prepare highly concentrated solutions. In contrast,
chitooligosaccharides and other water-soluble chitosan derivatives allow highly concentration solution
with low viscosities [31]. 2.3. Chitosan and Water-Soluble Chitosan Characterization In the present study, hydrogen peroxide proved to be an efficient tool for chitosan degradation
due to the formation of reactive hydroxyl radicals by the dissociation of hydrogen peroxide [32]. The results in Table 3 show the production of chitooligosaccharides (COS) from both segmented body
parts with 7.4 kg.mol−1 for pCOS and 2.7 kg.mol−1 for sCOS, as well as for commercial chitosan
chitooligosaccharides (ccCOS) with 10.4 ± 0.7 kg.mol−1. Due to the decrease in molecular weight
and degree of deacetylation, these products showed a good solubility in distilled water as previously
observed by other authors [33]. Besides COS, another derivative soluble in water (WSC) was prepared through the N-acetylation
of degraded chitosan. The water-soluble chitosan (WSC) products had a molecular weight range
of 378 to 404 kg.mol−1 in pWSC, of 247 to 279 kg.mol−1 in sWSC, and of 780.0 to 775 Kg.mol−1 in
ccWSC. According to previous studies [34], the reason for the increased solubility of chitosan was
the destruction of intramacromolecular and interchain hydrogen bonds, which alters the secondary
structure of chitosan, decreasing its crystallinity and unfolding its molecular chains. Also, the degree
of deacetylation decreased likely due to the acetylation reaction induced by the acetic anhydride acting
as a source of acetyl group for the amines. 2.4. DPPH Radical Scavenging Activity Since chitosan chains have active hydroxyl and amino groups that can react with free radicals
[38], the scavenging activity of chitosan may be due to the reaction between the free radicals and
protonated amino groups [38]. Several researchers suggested that the scavenging mechanism of
chitosan was based on the reaction of hydroxyl and superoxide anion radicals with active hydrogen
atoms in chitosan to form a stable macromolecule radical. In the chitosan structure, there are three
hydrogen sources, at the C−2 (NH2), C−3 (OH) and C−6 (OH) positions respectively [39]. The present
results support this theory, suggesting that the number of free amino groups is definitely important
to a good antioxidant performance, since a high degree of deacetylation resulted in chitosans (COS)
with better antioxidant properties. Other studies revealed the contribution of a prolonged N-
deacetylation and its impact on the scavenging activities through the production of highly
deacetylated products [40,41]. Once again, this seems to be in line with our findings, when compared
the chitooligosaccharides (DD: 86−93%) and water-soluble chitosan (DD: 55−62%), proving that
i
ibl
j
f
f
f
di
l
i
i i
Since chitosan chains have active hydroxyl and amino groups that can react with free radicals [38],
the scavenging activity of chitosan may be due to the reaction between the free radicals and protonated
amino groups [38]. Several researchers suggested that the scavenging mechanism of chitosan was based
on the reaction of hydroxyl and superoxide anion radicals with active hydrogen atoms in chitosan
to form a stable macromolecule radical. In the chitosan structure, there are three hydrogen sources,
at the C−2 (NH2), C−3 (OH) and C−6 (OH) positions respectively [39]. The present results support this
theory, suggesting that the number of free amino groups is definitely important to a good antioxidant
performance, since a high degree of deacetylation resulted in chitosans (COS) with better antioxidant
properties. Other studies revealed the contribution of a prolonged N-deacetylation and its impact on
the scavenging activities through the production of highly deacetylated products [40,41]. Once again,
this seems to be in line with our findings, when compared the chitooligosaccharides (DD: 86−93%) and
water-soluble chitosan (DD: 55−62%), proving that amino groups are a possible major factor for free
radical scavenging activity. amino groups are a possible major factor for free radical scavenging activity. 2.4. DPPH Radical Scavenging Activity One important mechanism of antioxidation involves scavenging of hydrogen radicals. DPPH (2,2-diphenyl-1-picrylhydrazyl) has a hydrogen-free radical with a characteristic absorption at
517 nm, allowing its detection as the purple color of the DPPH solution fades rapidly when it reacts
with proton radical scavengers [35]. The DPPH radical scavenging activity of WSC, COS, and ascorbic acid is shown in Figure 1. At 1 mg·mL−1, all chitosan products exhibited the highest scavenging ability. The scavenging ability
proved to be dose-dependent. 5 of 15
5 of 14 Mar. Drugs 2019, 17, 239
Mar. Drugs 2019, 17, x g
,
,
r. Drugs 2019, 17, x
5 of 14
Figure 1. The scavenging ability of water-soluble chitosan (WSC), chitooligosaccharides (COS), and
ascorbic acid on 1,1-diphenyl-2-picrylhydrazyl radicals: The values are means of eight replicates ±
standard errors. Figure 1. The scavenging ability of water-soluble chitosan (WSC), chitooligosaccharides (COS),
and ascorbic acid on 1,1-diphenyl-2-picrylhydrazyl radicals: The values are means of eight replicates ±
standard errors. Figure 1. The scavenging ability of water-soluble chitosan (WSC), chitooligosaccharides (COS), and
ascorbic acid on 1,1-diphenyl-2-picrylhydrazyl radicals: The values are means of eight replicates ±
standard errors
Figure 1. The scavenging ability of water-soluble chitosan (WSC), chitooligosaccharides (COS),
and ascorbic acid on 1,1-diphenyl-2-picrylhydrazyl radicals: The values are means of eight replicates ±
standard errors. pCOS, sCOS, and ccCOS showed the highest scavenging activity. The WSC products showed a
lower activity than COS at all concentrations tested (Figure 1). Also, no differences were noted
between products obtained from P. henslowii and the commercial product. However, compared to the
i
bili
f
bi
id
ll
d
h d l
l
pCOS, sCOS, and ccCOS showed the highest scavenging activity. The WSC products showed
a lower activity than COS at all concentrations tested (Figure 1). Also, no differences were noted
between products obtained from P. henslowii and the commercial product. However, compared to the
scavenging ability of ascorbic acid, all products had lower values. scavenging ability of ascorbic acid, all products had lower values. Previous studies proved that the antioxidant activity of chitosan depends on the molecular
i ht
d th d
f d
t l ti
[36 37]
Previous studies proved that the antioxidant activity of chitosan depends on the molecular weight
and the degree of deacetylation [36,37]. weight and the degree of deacetylation [36,37]. 2.4. DPPH Radical Scavenging Activity In addition, previous investigations have revealed that the DPPH radical scavenging activity of
chitosan increased with a decreasing molecular weight (MW) [36,41]. According to previous studies
[36], a high-MW chitosan (WSC) would have a lower mobility than a low-MW chitosan (COS). Consequently, this would increase the possibility of inter- and intramolecular bonding of the high-
MW chitosan molecules; thus, the chance of exposure of their amine groups might be restricted. An
approximate 20% scavenging ability with 1 mg·mL−1 of chitosan from crustaceans has been reported
i
l
[41]
In addition, previous investigations have revealed that the DPPH radical scavenging activity
of chitosan increased with a decreasing molecular weight (MW) [36,41]. According to previous
studies [36], a high-MW chitosan (WSC) would have a lower mobility than a low-MW chitosan
(COS). Consequently, this would increase the possibility of inter- and intramolecular bonding of the
high-MW chitosan molecules; thus, the chance of exposure of their amine groups might be restricted. An approximate 20% scavenging ability with 1 mg·mL−1 of chitosan from crustaceans has been reported
previously [41]. previously [41]
The results obtained in the current study suggest that the degradation of chitosan by hydrogen
peroxide enhanced not only the solubility but also its antioxidant activity, supporting the use of P. henslowii as raw material for the manufacturing of chitosan products towards antioxidant
The results obtained in the current study suggest that the degradation of chitosan by hydrogen
peroxide enhanced not only the solubility but also its antioxidant activity, supporting the use of
P. henslowii as raw material for the manufacturing of chitosan products towards antioxidant applications. applications.
2.5. Superoxide Radical (O2−) Scavenging Ability 2.5. Superoxide Radical (O2−) Scavenging Ability
Superoxides are radicals of which unpaired electrons are on oxygen. Despite their limited
chemical reactivity they can form more dangerous species including singlet oxygen hydrogen
Superoxides are radicals of which unpaired electrons are on oxygen. Despite their limited
chemical reactivity, they can form more dangerous species, including singlet oxygen, hydrogen
peroxide, and hydroxyl radicals in living organisms [42,43]. Further, superoxides were also known to Mar. Drugs 2019, 17, 239 6 of 15 indirectly initiate lipid peroxidation as a result of H2O2 formation, creating precursors of hydroxyl
radicals [44]. tly initiate lipid peroxidation as a result of H2O2 formation, creating precursors of hydroxyl
s [44]. In the present study a superoxide radical scavenging assay was based on the capacity of
water-soluble chitosan (WSC) and chitooligosaccharides (COS) to inhibit the reduction of nitro blue
tetrazolium (NBT). Figure 2 summarizes the scavenging effects of WSCs and COSs produced from P. henslowii raw
material (pereopods and shells) and commercial chitosan on superoxide radicals within a concentration
range from 0.0625 to 1 mg·mL−1. All the products scavenged superoxide in a concentration-dependent
manner. The figure showed that chitooligosaccharides (COSs) had the highest scavenging activity
towards superoxide anion radicals with concentrations above 0.25 mg·mL−1. Again, no differences
were reported between products obtained from P. henslowii and commercial samples for both WSCs and
COSs. At 0.0625 mg·mL−1, all samples proved a low scavenging activity with values changing from
9.2 ± 1.9% (ccWSC) to 16.1 ± 2.9% (pCOS). On the other hand, EDTA proved much higher superoxide
scavenging values (65.1 ± 3.6%) within the same concentrations (0.0625 mg·mL−1). While testing the
highest concentration at 1 mg·mL−1, the pCOS, sCOS, and ccCOS samples clearly exhibited a higher
scavenging activity (61.9 ± 7%, 60.1 ± 5.2%, and 57.5 ± 4.1%, respectively) when compared to pWSC,
sWSC, and ccWSC (28.7 ± 2.7%, 31.9 ± 4.5%, and 28.5 ± 1.5%, respectively)
Mar. Drugs 2019, 17, x
6 of 14
to indirectly initiate lipid peroxidation as a result of H2O2 formation, creating precursors of hydroxyl
radicals [44]. In the present study a superoxide radical scavenging assay was based on the capacity of water-
soluble chitosan (WSC) and chitooligosaccharides (COS) to inhibit the reduction of nitro blue
tetrazolium (NBT) Figure 2. The scavenging ability of water-soluble chitosan (WSC), chitooligosaccharides (COS), and
ascorbic acid on superoxide radical: The values are means of eight replicates ± standard errors. Figure 2. applications.
2.5. Superoxide Radical (O2−) Scavenging Ability The scavenging ability of water-soluble chitosan (WSC), chitooligosaccharides (COS),
and ascorbic acid on superoxide radical: The values are means of eight replicates ± standard errors. Figure 2. The scavenging ability of water-soluble chitosan (WSC), chitooligosaccharides (COS), and
ascorbic acid on superoxide radical: The values are means of eight replicates ± standard errors
Figure 2. The scavenging ability of water-soluble chitosan (WSC), chitooligosaccharides (COS),
and ascorbic acid on superoxide radical: The values are means of eight replicates ± standard errors. Figure 2 summarizes the scavenging effects of WSCs and COSs produced from P. henslowii raw
material (pereopods and shells) and commercial chitosan on superoxide radicals within a
concentration range from 0.0625 to 1 mg·mL−1. All the products scavenged superoxide in a
concentration-dependent manner. The figure showed that chitooligosaccharides (COSs) had the
highest scavenging activity towards superoxide anion radicals with concentrations above 0.25
mg·mL−1. Again, no differences were reported between products obtained from P. henslowii and
commercial samples for both WSCs and COSs. At 0.0625 mg·mL−1, all samples proved a low
scavenging activity with values changing from 9.2 ± 1.9% (ccWSC) to 16.1 ± 2.9% (pCOS). On the
other hand, EDTA proved much higher superoxide scavenging values (65.1 ± 3.6%) within the same
concentrations (0.0625 mg·mL−1). While testing the highest concentration at 1 mg·mL−1, the pCOS,
sCOS and ccCOS samples clearly exhibited a higher scavenging activity (61 9 ± 7% 60 1 ± 5 2% and
Previously published studies [45–47] suggested a relationship between molecular weight and
the ability to scavenge superoxide anions. Compared with chitosan, chitooligosaccharides have very
short chains and the ability to form intramolecular hydrogen bonds (O3–O5 and N2–O6) decreases,
which means that the hydroxyl and amino groups are activated, being helpful to the reaction with
superoxide anions. This fact may be related once again to the formation of strong intermolecular
and intramolecular hydrogen bonds that reduced the reactivity of hydroxyl and amino groups in
the polymer chains. Other authors [48] proved the influence of hydroxyl and amino groups in the
scavenging process, showing lower scavenging values for chitosan-thiamine pyrophosphate (CS-TPP)
and chitosan-hydroxybenzotriazole (CS-HOBt) when compared to chitosan-acetate and chitosan-EDTA. According to the author, it might be due to the higher ability of TPP and HOBt to bond with the
hydroxyl and amino groups of chitosan, blocking the reaction with the superoxide. 57.5 ± 4.1%, respectively) when com
28 5
1 5%
i
l )
2.6. Chelating Ability on Ferrous Ions 28.5 ± 1.5%, respectively)
Previously published studies [45–47] suggested a relationship between molecular weight and
the ability to scavenge superoxide anions. Compared with chitosan, chitooligosaccharides have very
short chains and the ability to form intramolecular hydrogen bonds (O3–O5 and N2–O6) decreases,
The generation of radicals can be retarded by chelation of ferrous ions [49], being chitosan and
chitosan derivatives reported as significant chelators [50]. This is why chitosan can be considered as a
potential natural antioxidant to prolong food shelf life [40]. 7 of 15
7 of 14 Mar. Drugs 2019, 17, 239
Mar Drugs 2019 17 x Figure 3 shows the ferrous-ion chelating ability of WSCs and COSs produced from P. henslowii
raw material (pereopodes and shells) and from commercial chitosan within a concentration range from
0.0625 to 1 mg·mL−1. Figure 3 shows the ferrous-ion chelating ability of WSCs and COSs produced from P. henslowii
raw material (pereopodes and shells) and from commercial chitosan within a concentration range
from 0.0625 to 1 mg·mL−1. Figure 3. The chelating ability of water-soluble chitosan (WSC), chitooligosaccharides (COS), and
ethylenediaminetetraacetic acid (EDTA) on ferrous ions: The values are means of eight replicates ±
Figure 3. The chelating ability of water-soluble chitosan (WSC), chitooligosaccharides (COS),
and ethylenediaminetetraacetic acid (EDTA) on ferrous ions: The values are means of eight replicates ±
standard errors. Figure 3. The chelating ability of water-soluble chitosan (WSC), chitooligosaccharides (COS), and
ethylenediaminetetraacetic acid (EDTA) on ferrous ions: The values are means of eight replicates ±
Figure 3. The chelating ability of water-soluble chitosan (WSC), chitooligosaccharides (COS),
and ethylenediaminetetraacetic acid (EDTA) on ferrous ions: The values are means of eight replicates ±
standard errors. standard errors. In general, the chelation ability of all WSCs with concentrations ranging from 0.0625 to 1
mg·mL−1 was relatively low. On the contrary, EDTA exhibited an excellent ferrous ion-chelating
capacity of approximately 74.6 ± 4.1% at a concentration of 0.0625 mg·mL−1. Nevertheless, all products
proved a high chelation ability within 1 mg·mL−1, showing COSs the highest activities, from 40.7 ±
10.1% (sCOS) to 45.2 ± 10% (pCOS). When tested at 0.5 and 1 mg·mL−1, COSs almost presented a two-
times-higher chelating power than WCSs. Also, no differences were seen between sCOS, pCOS, and
ccCOS. Still, EDTA proved to be much more potent at 1 mg·mL−1, chelating all ferrous ions (100%). Other authors [51] reported similar activities for EDTA when compared to the N-alkylated
disaccharide chitosan derivative. 57.5 ± 4.1%, respectively) when com
28 5
1 5%
i
l )
2.6. Chelating Ability on Ferrous Ions Despite the close relation of the chitosan metal ion absorption
capability to its amino acid content and their distribution, other factors such as affinity for water, pH,
In general, the chelation ability of all WSCs with concentrations ranging from 0.0625 to 1 mg·mL−1
was relatively low. On the contrary, EDTA exhibited an excellent ferrous ion-chelating capacity
of approximately 74.6 ± 4.1% at a concentration of 0.0625 mg·mL−1. Nevertheless, all products
proved a high chelation ability within 1 mg·mL−1, showing COSs the highest activities, from 40.7 ±
10.1% (sCOS) to 45.2 ± 10% (pCOS). When tested at 0.5 and 1 mg·mL−1, COSs almost presented a
two-times-higher chelating power than WCSs. Also, no differences were seen between sCOS, pCOS,
and ccCOS. Still, EDTA proved to be much more potent at 1 mg·mL−1, chelating all ferrous ions (100%). Other authors [51] reported similar activities for EDTA when compared to the N-alkylated disaccharide
chitosan derivative. Despite the close relation of the chitosan metal ion absorption capability to its
amino acid content and their distribution, other factors such as affinity for water, pH, temperature,
and crystallinity also affected the ion-chelating activity [52,53]. p
y
y
p
temperature, and crystallinity also affected the ion-chelating activity [52,53]. In addition, the ion-chelating activity of chitosan seems to be strongly affected by the degree of
acetylation, with the fully acetylated chitosan showing very little chelating activity [54]. Similar
findings were reported in the present study, revealing higher degrees of deacetylation (COSs) and a
In addition, the ion-chelating activity of chitosan seems to be strongly affected by the degree of
acetylation, with the fully acetylated chitosan showing very little chelating activity [54]. Similar findings
were reported in the present study, revealing higher degrees of deacetylation (COSs) and a stronger
effect on metal chelation abilities, when compared to low deacetylated products (WSCs). stronger effect on met
2.7. Antifungal Assay The effect of COS and WSC products (concentration ranging from 0.0125 to 0.1 mg·mL−1)
on the growth of Heterobasidion annosum (a), Phytophthora cinnamomi (b), Cryphonectria parasitica (c),
and Botrytis cinerea (d): The values are means of eight replicates ± standard error. (d) (c) Figure 4. The effect of COS and WSC products (concentration ranging from 0.0125 to 0.1 mg·mL−1) on
he growth of Heterobasidion annosum (a), Phytophthora cinnamomi (b), Cryphonectria parasitica (c), and
Botrytis cinerea (d): The values are means of eight replicates ± standard error. Figure 4. The effect of COS and WSC products (concentration ranging from 0.0125 to 0.1 mg·mL−1)
on the growth of Heterobasidion annosum (a), Phytophthora cinnamomi (b), Cryphonectria parasitica (c),
and Botrytis cinerea (d): The values are means of eight replicates ± standard error. Furthermore, the results showed that the capability to inhibit fungal growth was clearly higher
or COS from both segmented body parts, especially the pereopods, than for WSC products. While
P. cinnamomi exhibited a lower vulnerability towards all chitosans, B. cinerea and C. parasitica were
highly inhibited by COS. B. cinerea sensitivity to chitosan has been reported previously [56]. Furthermore, the results showed that the capability to inhibit fungal growth was clearly
higher for COS from both segmented body parts, especially the pereopods, than for WSC products
While P. cinnamomi exhibited a lower vulnerability towards all chitosans, B. cinerea and C. parasitica
were highly inhibited by COS. B. cinerea sensitivity to chitosan has been reported previously [56]. Furthermore, the results showed that the capability to inhibit fungal growth was clearly higher
for COS from both segmented body parts, especially the pereopods, than for WSC products. While
P. cinnamomi exhibited a lower vulnerability towards all chitosans, B. cinerea and C. parasitica were
highly inhibited by COS. B. cinerea sensitivity to chitosan has been reported previously [56]. Furthermore, the results showed that the capability to inhibit fungal growth was clearly
higher for COS from both segmented body parts, especially the pereopods, than for WSC products. While P. cinnamomi exhibited a lower vulnerability towards all chitosans, B. cinerea and C. parasitica
were highly inhibited by COS. B. cinerea sensitivity to chitosan has been reported previously [56]. Several mechanisms have been proposed for the antimicrobial action of chitosan. stronger effect on met
2.7. Antifungal Assay It has been
suggested that chitosan may inhibit microbial growth by acting as a chelating agent rendering metals,
trace elements, or essential nutrients unavailable for the organism to grow at a normal rate [57]. This
could be caused by the accumulation of chitosan precipitates on the membrane surface, as the
physiological pH in microbial cells is around neutral [58,59]. On the contrary, water soluble chitosan
derivatives, due to their solubility in neutral solutions, would be unable to form such a layer, and
therefore, no antimicrobial activity would be expected. Several mechanisms have been proposed for the antimicrobial action of chitosan. It has been
suggested that chitosan may inhibit microbial growth by acting as a chelating agent rendering
metals, trace elements, or essential nutrients unavailable for the organism to grow at a normal
rate [57]. This could be caused by the accumulation of chitosan precipitates on the membrane surface,
as the physiological pH in microbial cells is around neutral [58,59]. On the contrary, water soluble
chitosan derivatives, due to their solubility in neutral solutions, would be unable to form such a layer,
and therefore, no antimicrobial activity would be expected. Another hypothesis explains the activity of chitosan as being based on the electrostatic
interaction of the protonated amino groups with the negatively charged cell wall surface of the
targeted microorganisms, which can lead to the disruption of the cell wall and, therefore, to its death
[60]. The importance of protonation has been reported by several studies [61–63], which proved that
only positively charged chitosan’s are able to inhibit microbial growth. Another hypothesis explains the activity of chitosan as being based on the electrostatic interaction
of the protonated amino groups with the negatively charged cell wall surface of the targeted
microorganisms, which can lead to the disruption of the cell wall and, therefore, to its death [60]. The importance of protonation has been reported by several studies [61–63], which proved that only
positively charged chitosan’s are able to inhibit microbial growth. Moreover, the degree of deacetylation certainly plays an important role not only on the
antioxidant activity but also in antifungal activity, since the products with a higher degree of
deacetylation (COS), exhibited a higher growth inhibition. stronger effect on met
2.7. Antifungal Assay 2.7. Antifungal Assay
The molecular weight of chitosan is known to influence its ability to inhibit the activity of several
fungal species [55]. The antifungal activity of WSC and COS products towards the plant pathogens
B. cinerea, H. annosum, P. cinnamomi, and C. parasitica is shown in Figure 4. The concentrations of
chitosan ranged from 0.0125 to 0.1 mg·mL−1. The antifungal activity of all chitosan products proved
to be dose-dependent, but despite the observed inhibitory effect, none of the tested fungal species
were completely inhibited by COS or WSC at the highest concentrations. However, pCOS and sCOS
showed a good inhibition against Cryphonectria parasitica. Overall, pCOS exhibited a higher activity
The molecular weight of chitosan is known to influence its ability to inhibit the activity of several
fungal species [55]. The antifungal activity of WSC and COS products towards the plant pathogens
B. cinerea, H. annosum, P. cinnamomi, and C. parasitica is shown in Figure 4. The concentrations of
chitosan ranged from 0.0125 to 0.1 mg·mL−1. The antifungal activity of all chitosan products proved to
be dose-dependent, but despite the observed inhibitory effect, none of the tested fungal species were
completely inhibited by COS or WSC at the highest concentrations. However, pCOS and sCOS showed
a good inhibition against Cryphonectria parasitica. Overall, pCOS exhibited a higher activity against all
other tested species than sCOS, pWSC, and sWSC. Mar. Drugs 2019, 17, 239
Mar. Drugs 2019, 17, x 8 of 15
8 of 14 (a)
(b)
(c)
(d)
Figure 4. The effect of COS and WSC products (concentration ranging from 0.0125 to 0.1 mg·mL−1) on
the growth of Heterobasidion annosum (a), Phytophthora cinnamomi (b), Cryphonectria parasitica (c), and
Botrytis cinerea (d): The values are means of eight replicates ± standard error. Figure 4. The effect of COS and WSC products (concentration ranging from 0.0125 to 0.1 mg·mL−1)
on the growth of Heterobasidion annosum (a), Phytophthora cinnamomi (b), Cryphonectria parasitica (c),
and Botrytis cinerea (d): The values are means of eight replicates ± standard error. (a) (b) (b) (a) (a) (b)
(d) ( )
(c) ( )
( )
(c)
(d)
Figure 4. The effect of COS and WSC products (concentration ranging from 0.0125 to 0.1 mg·mL−1) on
the growth of Heterobasidion annosum (a), Phytophthora cinnamomi (b), Cryphonectria parasitica (c), and
Botrytis cinerea (d): The values are means of eight replicates ± standard error. Figure 4. 3.1. Biochemical Characterization of Raw Materials Swimming crab, Polybius henslowii was by-catched by fishing vessels when capturing Sardina
pilchardus along the west coast of Peniche (39◦24´22.19´´N, 9◦35´50.51´´O), Portugal. Dead organisms
were first boiled, then dried in an oven at 100 ◦C for 2 days, and segmented into shell and pereopods. The raw material was powdered into particles with diameters ranging from 150 to 500 µm. The ash
content was determined by initially drying the raw material in an oven at 100 ◦C for 6h. The dried
samples were placed in a furnace at 530 ◦C for 20 h, and the remaining material was weighed after
cooling in a desiccator. The microbiuret method was used for protein assays [66], and the values were
compared with a standard curve established with known concentrations of bovine serum albumin (BSA). Free fat was extracted from the raw material by Soxhlet using ether as the solvent [67]. Three replicates
were conducted for each sample. stronger effect on met
2.7. Antifungal Assay Again, the number of free amino groups
seems to influence bioactivities Also it is possible that chitosan’s antifungal activity is caused by its
Moreover, the degree of deacetylation certainly plays an important role not only on the antioxidant
activity but also in antifungal activity, since the products with a higher degree of deacetylation (COS),
exhibited a higher growth inhibition. Again, the number of free amino groups seems to influence 9 of 15 Mar. Drugs 2019, 17, 239 bioactivities. Also, it is possible that chitosan’s antifungal activity is caused by its chains, which interact
with the cell’s membrane and inhibits the intracellular functions [64]. Recently published studies [65]
suggested that interactions between low molecular weight chitosans, DNA, RNA, and protein could
partly explain the effects of chitosan on translation efficiency. In addition, other studies showed that
the effectiveness of chitosan did not depend solely on the chitosan formulation but also on the type of
fungi [61]. This is in accordance with our findings, since both pathogens from the phylum Ascomycota
(Botrytis cinerea and Cryphonectria parasitica) were highly inhibited at 0.1 mg·mL−1 when compared to
the remaining species, Heterobasidion annosum (phylum Basidiomycota) and Phytophthora cinnamomi
(phylum Heterokontophyta). 3.2. Chitin Extraction and Deacetylation Chitin was isolated according to Reference [19] with some modifications. Demineralization was
carried out with three different concentrations of HCl in order to optimize the extraction of calcium
carbonate: 0.5, 0.75, and 1 M at 21◦C for 30 min (ratio of 1:30, w/v). After washing with distilled water
and drying in an incubator for 24 h at 40 ◦C, the samples were subjected to different concentrations of
sodium hydroxide (0.5 M, 0.75 M, and 1 M of NaOH; ratio of 1:15, w/v), thereby removing the organic
matter. The shells and pereopods were incubated at 70 ◦C for 2 h in a water bath and washed with
distilled water until a neutral pH was achieved. The demineralization and deproteinization efficiencies
were respectively determined through the ash content and microbiuret assay measurements. The chemical deacetylation of chitin was achieved by subjecting chitin to 12 M of NaOH solution at
120 ◦C for 3 h (pereopods) and 7 h (shells). After the alkali treatment, chitosan was collected and washed
with distilled water to remove NaOH residues until a neutral pH was reached. These conditions
yielded chitosan products from both body parts, which exhibited a complete dissolution in acetic acid
(1%, v/v) at 25 ◦C. Three batches were performed in each step, therefore having a minimum of three replicates
per sample. 3.6. Superoxide Radical (O2−) Scavenging Activity The superoxide scavenging ability of all water-soluble chitosan (WSC) and chitooligosaccharide
(COS) samples was assessed by the method of Reference [71]. This assay was based on the reduction
of nitro blue tetrazolium (NBT) in the presence of NADH and phenazine methosulphate (PMS)
under aerobic condition. The 3.00 mL reaction mixture contained 50 µL of lM NBT, 150 µL of l M
nicotinamide adenine dinucleotide (NADH), and Trisbuffer (0.02 M, pH 8.0). The samples were added
with concentrations ranging from 0.0625 to 1.0 mg·mL−1. The reaction started by adding 15 µL of
lM phenazine methosulfate (PMS). After incubation at room temperature for 5 min, 300 µL of each
reaction were transferred to a 96-well plate, and the absorbance was recorded at 560 nm against a blank
in microplate reader (Biotek, Vermont, USA). EDTA was used as a positive control. The capability of
scavenging to superoxide radical was calculated using the following Equation (2): (2) Scavenging effect (%) = (1 −Asample/Acontrol) × 100
(2) 3.4. Chitosan Product Characterization The dynamic viscosity was determined in triplicate with a rotational Haake viscotester 7 plus at
(20 ± 1) ◦C after the samples were dissolved in 1% (v/v) acetic acid. The molecular weight (Mw) of
chitosan products was determined by gel permeation chromatography (GPC) [68] with some variations. A Varian PL aquagel-OH MIXED bed column (Varian, France) 8-µm column was used. The samples
(approx. 5 mg) were dissolved in 1 mL of eluent (AcOH/AcONa buffer pH 4.5, Panreac Spain) and
filtered through a microsyringe filter prior to injection. The flow rate was 1 mL/min. A calibration
curve was obtained by using Varian pullulan polysaccharides certified standards (Varian, France) at
the same chromatographic conditions. FTIR spectroscopy (Bruker FTIR-ATR spectrophotometer) was
employed to determine the degree of acetylation (DA) [69]. The spectra of all samples were recorded
in KBr pellets (Sigma Aldrich, Steinheim, Germany) by the accumulation a minimum of 64 scans with
a resolution of 4 cm−1. 3.3. Chitooligosaccharides and Water-Soluble Chitosan Production Water-soluble chitosan (WSC) was prepared according to a previously published procedure [34],
through N-acetylation with acetic anhydride (Ac2O). One gram of chitosan was dissolved in 25 mL of
2.8 % acetic acid, and 25 mL of ethanol were added. Acetic anhydride was charged after a complete
dissolution of chitosan and left shaking for 4 h. The reaction mixture was precipitated with ethanol
and dried at 55 ◦C for 24 h. The production of chitooligosaccharides (COS) was derived by adding hydrogen peroxide (H2O2) [32]. One gram of chitosan was dissolved in 20 mL of 2% (w/w) acetic acid. After a complete dissolution, 5.5%
H2O2 was added to the mixture and incubated at 37 ◦C for 4 h. Then, chitooligosaccharide products 10 of 15 Mar. Drugs 2019, 17, 239 were precipitated with ethanol and dried at 55 ◦C for 24 h. The same procedure was carried out with the
commercial chitosan (cc) product (Altakitin S.A., Portugal) in order to compare its physical and biochemical
properties. All samples were performed in triplicate. 3.5. Scavenging of 1,1-diphenyl-2-picrylhydrazyl Radicals The chitosan scavenging of 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) was determined
according to a previously described method [70]. DPPH (Sigma Aldrich, Steinheim, Germany) solution
was prepared at 0.1 mM with methanol. A volume of 10 µL of each sample was added to 990 µL of a
DPPH solution. The sample concentrations varied from 0.0625 to 1 mg·mL−1. The reaction mixture
was shaken vigorously and stored in the dark at room temperature for 30 min. The absorbance was
read at 517 nm in a microplate reader (Biotek, Vermont, USA). All samples were run in triplicate. Ascorbic acid was used for comparison. The free radical scavenging activity was calculated by the
following Equation (1): Scavenging activity (%) = (1 −Abssample/Abscontrol) × 100
(1) (1) 3.7. Chelating Ability on Ferrous Ions The ferrous ion-chelating potential of all chitosan products (WSC and COS) was investigated
according to the method of Decker and Welch [72]. Each WSC and COS sample (0.0625–1.0 mg·mL−1) in
a 0.2% acetic acid solution was mixed with 0.1 mL of FeCl2 (2 mM) and 3.7 mL of methanol. The reaction
was initiated by adding 2.0 mL ferrozine (5 mM), shaken vigorously and incubated for 10 min in the
dark at room temperature. The ferrous ion-chelating ability was determined by the absorbance at
562 nm against a blank in a microplate reader (Biotek, Vermont, USA). EDTA was used as a positive Mar. Drugs 2019, 17, 239
11 of 15 11 of 15 Mar. Drugs 2019, 17, 239 control. The ability of WSC and COS to chelate ferrous ions was calculated according to the following
Equation (3): control. The ability of WSC and COS to chelate ferrous ions was calculated according to the following
Equation (3):
Chelating activity (%) = (1 −Abssample/Abscontrol) × 100
(3) (3) Chelating activity (%) = (1 −Abssample/Abscontrol) × 100 Chelating activity (%) = (1 −Abssample/Abscontrol) × 100 3.8. Effect of Chitosan on Mycelial Growth Cryphonectria parasitica (DSMZ 62626), Phytophthora cinnamomi (DSMZ 62654), Botrytis cinerea
(DSMZ 4709), and Heterobasidion annosum (DSMZ 1531) were all purchased from the Leibniz Institut
DSMZ (German Collection of Microorganisms and Cells Cultures), Germany. The four strains were
grown for 5 days at 28 ◦C on potato dextrose agar (PDA) plates. The antifungal assessment of the
chitosan products was conducted through the mycelial radial growth inhibition technique on a PDA
medium according to El Ghaouth et al. [73]. Mycelial discs 4 mm in diameter were cut from the margins
of the initial colony and placed on PDA plates containing different concentrations of chitosan, ranging
from 0.0125 to 0.1 mg·mL−1. Control plates of PDA were prepared without chitosan. The PDA plates
were incubated at (23 ± 2) ◦C for 7 days. The mycelial radial growth was measured when the control
colony had grown to the edge of the plate. The diameter of each fungal colony prepared in triplicate
was measured in mm. and the activity was expressed as the inhibition of mycelial growth. Conflicts of Interest: The authors declare no conflict of interest. 4. Conclusions This study shows that P. henslowii captured as bycatch could be applied as a source of chitosan
derivatives. The swimming crab characterization clearly showed that, considering its composition,
different segments of its body could be applied to several biotechnological applications such as feed
supplements, adding value to the raw material. Moreover, the bioavailability of this marine resource,
the optimization of chemical processes (through solvent reuse), and, considering all production costs,
the final price could be highly competitive compared to the current market offer. The results suggested
that the chitosan derivatives, mainly chitooligosaccharides obtained through processing of P. henslowii
raw material, could be effectively employed as an ingredient in cosmetics or functional products in
order to decrease oxidative stress. In general, the chitosan derivatives produced from the raw material
of P. henslowii remarkably inhibited the tested phytopathogenic fungi, mainly Cryphonectria parasitica. In addition, these samples produced an interesting inhibitory activity when compared with commercial
chitosan derivatives. However, in order to promote the use of this raw material, extensive research
on its characterization (RNM) and other applications (antibacterial activity and allergenic tests) must
be addressed. Author Contributions: R.P., S.M.L., and S.C.M. were responsible for the design of the study. L.F.V.P. and P.M.N. were responsible for the characterization of chitosan samples. A.H. carried out the antifungal assays. F.A. was
responsible for the extraction of chitin and the production of chitosan derivatives. S.C.M. evaluated the antioxidant
activity. R.P., S.M.L., and F.A. were responsible for the interpretation of the data. F.A. and S.M.L. drafted the
article. All authors revised the paper critically for important intellectual content and gave final approval to the
version to be published. Funding:
This research was funded by GAC Oeste (PROMAR—European Fisheries Fund) through the
project Pilado add value, under the FP7, grant agreement no. 278612 and by Fundação para a Ciência
e Tecnologia (FCT), through the strategic project UID/MAR/04292/2019 granted to MARE, the Integrated
Programme of SR&TD “Smart Valorization of Endogenous Marine Biological Resources Under a Changing
Climate” (Centro-01-0145-FEDER-000018), co-funded by Centro 2020 programme, Portugal 2020, European Union
through the European Regional Development Fund and the grant awarded to SCM (SFRH/BPD/110400/2015). References Exp. Med. Biol. 2014, 824, 5–17. [PubMed] rdá, C.; Sánchez, C.; Climent, B.; Vázquez, A.; Iradi, A.; El Amrani, F.; Bediaga, A.; Sáez, G.T. Oxidative stress
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system. Biochem. Biophys. Res. Commun. 1975, 63, 463–468. References [CrossRef] 72. Decker, E.A.; Welch, B. Role of ferritin as a lipid oxidation catalyst in muscle food. J. Agric. Food Chem. (USA)
1990, 38, 674–677. [CrossRef] 73. El Ghaouth, A.; Arul, J.; Grenier, J.; Asselin, A. Antifungal activity of chitosan on two post-harvest
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https://hal.science/hal-03556308/document
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English
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Hydrogen steelmaking. Part 1: Physical chemistry and process metallurgy
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Matériaux & techniques/Matériaux et techniques
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cc-by
| 5,863
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To cite this version: Fabrice Patisson, Olivier Mirgaux, Jean-Pierre Birat. Hydrogen steelmaking. Part 1:
Physical
chemistry and process metallurgy. Matériaux et Techniques, 2021, 109 (3-4), pp.303. 10.1051/mat-
tech/2021025. hal-03556308 Distributed under a Creative Commons Attribution 4.0 International License Received: 27 October 2021 / Accepted: 14 December 2021 Abstract. Pushed to the forefront by the objective to drastically reduce the CO2 emissions from the steel
industry, a new steelmaking route based on hydrogen and electricity is the subject of a great deal of attention and
numerous R&D projects. The first step is to chemically reduce iron ore with H2, which is produced by electrolysis
of water with low-carbon electricity, and then to transform the direct reduced iron into steel in an electric arc
furnace. The second step is a conventional one, similar to that used for scrap recycling. The first step is similar to
the so-called direct reduction process but would use pure electrolytic H2 instead of the H2–CO syngas obtained
from natural gas reforming. In this paper, we first show how the reduction by pure H2 takes place at the
microscopic level of the iron oxide grains and pellets. The three-step (hematite-magnetite-wüstite-iron)
reduction occurs successively in time and simultaneously in the pellets. Secondly, a sophisticated kinetic model
of the reduction of a single pellet based on the experimental findings is described. Lastly, we present a
mathematical model for the simulation of the reduction by pure H2 in a shaft furnace, which can be very useful for
the design of a future installation. The main results are that using pure hydrogen, the reduction kinetics are
faster and can end with full metallization, the direct reduction process would be simpler, and the shaft furnace
could be squatter. The gains in terms of CO2 emissions are quantified (85% off) and the whole route is compared
to other zero-carbon solutions in Part 2. HAL Id: hal-03556308
https://hal.science/hal-03556308v1
Submitted on 3 Feb 2022 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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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 Matériaux & Techniques 109, 303 (2021)
© F. Patisson et al., 2022
https://doi.org/10.1051/mattech/2021025 Matériaux & Techniques 109, 303 (2021)
© F. Patisson et al., 2022
https://doi.org/10.1051/mattech/2021025 1 Introduction agreement and the Green Deal Strategy of the European
Commission, soon duplicated in other regions of the world,
were the major reasons for a change of speed. This led to an
explosion of project proposals, in response to financing
opportunities for research and further industrial imple-
mentation opening on this topic, especially in the EU (HEU
program, Innovation Fund, etc.). Those current projects
are extensively described in Part 2 of this 2-paper series [9]. Even reputable newspapers with a very large audience are
interested in this evolution [10]. Given its volume (1.88 billion tons of steel produced in 2019
[1]) and its demand for fossil energy, mainly coal, the steel
industry is one of the world’s leading emitters of CO2 (7% of
global anthropogenic emissions [2]). However, this situa-
tion is not new and the steel sector has been investigating
low-carbon solutions for producing steel, including hydro-
gen-based ones, for the last 60 years at least [3,4]. Many
programs were conducted in the 2000s decade, including
several large international ones: the ULCOS (Ultra-low
CO2 steelmaking) program in Europe [5,6] and the CO2
Breakthrough program at world scale [7,8] were the most
important. The
2010s
were
a
trough
period,
when the R&I efforts slowed down significantly, both
because of the crisis and of the need for financing
institutions to reexamine and refocus their support. More
recently, a consensus emerged about the need to act with
determination to control climate change: the COP21 Paris The whole hydrogen route to steel was first defined and
evaluated in the ULCOS program (Fig. 1). The basic idea is to use hydrogen as a reductant for the
important step that consists in reducing the iron ore into
metallic iron, thus releasing harmless H2O instead of CO2,
and to use carbon-lean electricity as much as possible. Of
course H2 cannot be produced from natural gas as usual. Water
electrolysis
using
carbon-lean
electricity
was
selected as the best process to get the huge amount of
“green”hydrogenneeded(70 000 m3
STP/hfor1 Mtsteel/year). The DRI (direct reduced iron) is transformed into steel in
an electric arc furnace, while the end of the route is
identical to that used to produce secondary steel from
recycled scrap. Matériaux
&Techniques ,
https://doi.org/10.1051/mattech/2021025 Overview, state of the art, recent developments and future trends regarding Hydrogen
route for a green steel making process, edited by Ismael Matino and Valentina Colla Available online at:
www.mattech-journal.org Available online at:
www.mattech-journal.org REGULAR ARTICLE ☆ESTEP H2GreenSteel Web-Workshop, Virtual, 7th, 21st, 28th
May and 11th June 2021
* e-mail: fabrice.patisson@univ-lorraine.fr 1 Introduction ☆ESTEP H2GreenSteel Web-Workshop, Virtual, 7th, 21st, 28th
May and 11th June 2021
* e-mail: fabrice.patisson@univ-lorraine.fr This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 2 Fig. 1. ULCOS hydrogen-based route to steel [11]. Fig. 1. ULCOS hydrogen-based route to steel [11]. The main challenges of this route are in our opinion (1)
to know how to design and operate a shaft furnace under
pure hydrogen, (2) to know how to use a carbon-free DRI in
the electric arc furnace, and (3) to be able to produce
massively and at low cost green hydrogen. Point (3) is
clearly crucial and is discussed in Part 2 [9]. Note that the
abundance of large electrolyser projects is a precursor. Point (2) probably deserves specific investigations. It
should be noted, however, that the current trend to
manufacture and use DRI with 4% carbon is based
primarily on economic considerations. The addition of
carbon in the electric furnace is a daily practice. Point (1) is
the main focus of this paper. We will discuss it from the
perspective
of
laboratory
studies
and
mathematical
modeling. The most advanced current projects, such as
HYBRIT [12], are already approaching it from the point of
view of reduced-scale industrial demonstration. than 20 000 articles were devoted to it since 1900 [11]. This
reflects the economic importance of this reduction, which is
the first and central step in the production of steel. Without
attempting to examine all of this work here, we will recall
here the relevant features. The reduction of hematite to iron, whether by CO or by
H2, takes place in two or three stages, depending on
whether the temperature is above or below 570 °C. Hematite Fe2O3 is first reduced to magnetite Fe3O4, then
to wüstite FeyO, a non-stoichiometric compound, and
finally to iron. 1 Introduction 3Fe2O3 þ CO ¼ 2Fe3O4 þ CO2;
ð1Þ
Fe3O4 þ 16
19 CO ¼ 60
19 Fe0:95O þ 16
19 CO2;
ð2Þ
Fe0:95O þ CO ¼ 0:95Fe þ CO2;
ð3Þ
3Fe2O3 þ H2 ¼ 2Fe3O4 þ H2O;
ð4Þ
Fe3O4 þ 16
19 H2 ¼ 60
19 Fe0:95O þ 16
19 H2O;
ð5Þ
Fe0:95O þ H2 ¼ 0:95Fe þ H2O:
ð6Þ ð1Þ ð2Þ Of course, this route is clearly different from the
conventional
BF-BOF
(Blast
Furnace-Basic
Oxygen
Furnace) route, and also different from the existing DR-
EAF (Direct Reduction-Electric Arc Furnace) route, which
uses a CO-H2 syngas, produced from natural gas. A
comparison of the energy, environmental and economic
performance of these routes, based on a cradle-to-gate life
cycle approach, was carried out in ULCOS and the main
results are presented in Part 2 [9]. A comparison with other
estimates in the literature is also given in [11]. The most
important result, which justifies the current focus on this
breakthrough route, is the estimation of its specific
greenhouse gas emissions: between 25 and 300 kgCO2eq/t
of steel, depending on the sources, compared to 1850 kg for
the classical route, i.e. more than 85% off. ð3Þ ð5Þ Fe0:95O þ H2 ¼ 0:95Fe þ H2O:
ð6Þ ð6Þ The so-called Chaudron or Baur–Glaessner diagram
(Fig. 2) shows the stability ranges of the different phases as
a function of temperature and oxidizing power of the gas. An important deduction is that, with CO, the reduction is
thermodynamically easier at low temperature than at high
temperature, the opposite being true with H2 (see the
arrows at 1000 °C in the diagram). At 800 °C, it is
equivalent. Moreover,
thermodynamics
indicates
us
(Tab. 1) that the first reduction is exothermic, and is
more so with CO, the second is endothermic, and is less so
with CO, while the third is exothermic with CO whereas it
is endothermic with H2. It will thus be necessary overall to
provide energy to reduce by H2, in addition to the energy
necessary to heat the materials. The present paper is organized as follows: first, the
physical-chemical aspects of the iron oxide reduction by
hydrogen are recalled, the features that can influence an
industrial process are highlighted, and a comparison with
CO is given; second, the findings obtained from mathe-
matical modeling, both on the pellet and reactor scales, are
discussed. 2 Chemical characteristics of the reduction
of iron ore by hydrogen Reaction
DrH800 °C (J mol1)
3Fe2O3 þ CO ¼ 2Fe3O4 þ CO2
–40 040
3Fe2O3 þ H2 ¼ 2Fe3O4 þ H2O
–6020
Fe3O4 þ CO ¼ 3FeO þ CO2
18 000
Fe3O4 þ H2 ¼ 3FeO þ H2O
46 640
FeO þ CO ¼ Fe þ CO2
–17 610
FeO þ H2 ¼ Fe þ H2O
16 410 Table 1. Heat values of the reduction reactions. In the case of the reduction of ore pellets, such as
industrial pellets, which are initially composed of small
hematite grains, we have shown [13] that the porous and
granular structure evolves with the reduction (Fig. 4). Initially, a slight porosity appears on the surface of the
grains when hematite is transformed into magnetite. From
magnetite to wüstite and then to iron, the same evolution
by increase in the microporosity continues if the reduction
is conducted at low temperature (less than 800 °C). But at
higher temperatures, the wüstite grains disintegrate and
the iron phase that forms around the wüstite grains is dense
(Fig. 4h). The iron spreads around the small wüstite grains,
which we called crystallites, and tends to join these
crystallites together. The size of the grains and pores
increases with the temperature and with the course of
reaction. Figure 5 shows an example of the increase in
porosity and mean pore size for a CVRD-DR (from Vale,
Brazil, Direct Reduction grade) pellet, before and after
reduction by H2 at 1000 °C. All these changes have of course
consequences on the reaction kinetics. The influence of temperature on the reduction kinetics
with H2 is complex as shown in Figure 6. From 600 to 900 °C, and from 1100 to 1200 °C, the
temperature accelerates the kinetics as expected. Between
900 and 1100 °C, however, we observe that the kinetics slow
down after about 70% conversion. We also note a final
slowdown for the temperature of 700 °C. These observa-
tions reflect the fact that several potentially-limiting
kinetic processes are at work during the wüstite-to-iron
conversion. According to [13], the kinetics take place in a
mixed regime (controlled by both chemical reaction and
intergranular
diffusion)
but
at
high
temperature
a reactant must reach the reaction site and the gaseous
product must be evacuated, generally by gas phase
diffusion. Diffusion of species in the solid phase can also
occur. 2 Chemical characteristics of the reduction
of iron ore by hydrogen The reduction reactions (1–6) are heterogeneous gas-
solid reactions. They take place at the interfaces between
gas and solid and between solids. In addition to chemical
processes
(adsorption,
reaction,
desorption),
physical
transport processes are also involved. Indeed, the gaseous Most iron ores are oxides, most often hematite Fe2O3, and
the reduction of this oxide to metallic iron by CO, by H2 or
by CO–H2 mixtures have been extensively studied. More F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 3 Fig. 2. Phase diagram of the iron phase domains as a function of
the oxidizing power of the gas and temperature, for the gas
mixtures CO–CO2 and H2–H2O. The arrows represent the driving
force for the wüstite-to-iron reduction. Fig. 3. Schematic representation of a pellet (a) and pores around
constituting grains (b) showing the transport processes associated
with the chemical reaction. Fig. 3. Schematic representation of a pellet (a) and pores around
constituting grains (b) showing the transport processes associated
with the chemical reaction. Fig. 2. Phase diagram of the iron phase domains as a function of
the oxidizing power of the gas and temperature, for the gas
mixtures CO–CO2 and H2–H2O. The arrows represent the driving
force for the wüstite-to-iron reduction. influence the course of the reduction and thus its overall
rate, even for identical conditions of temperature and
composition of the reducing gas. This explains the great
variety of behaviors, sometimes seeming contradictory,
reported in the literature. It is possible to obtain intrinsic
kinetic parameters but, in order to be applicable to real
systems, they must be associated with measurements of
physical and geometrical parameters and with a modeling
of the transport processes. Table 1. Heat values of the reduction reactions. Reaction
DrH800 °C (J mol1)
3Fe2O3 þ CO ¼ 2Fe3O4 þ CO2
–40 040
3Fe2O3 þ H2 ¼ 2Fe3O4 þ H2O
–6020
Fe3O4 þ CO ¼ 3FeO þ CO2
18 000
Fe3O4 þ H2 ¼ 3FeO þ H2O
46 640
FeO þ CO ¼ Fe þ CO2
–17 610
FeO þ H2 ¼ Fe þ H2O
16 410 Table 1. Heat values of the reduction reactions. F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 2 Chemical characteristics of the reduction
of iron ore by hydrogen This implies that the situation will be completely
different if one reduces dense grains of a pure oxide, a small
bed of powder of the same oxide, a lump of ore or even a
pellet (Fig. 3). The size of the grains, lumps or pellets, the
total porosity and the distribution of the pore sizes F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 4 Fig. 4. SEM (Scanning Electron Microscope) micrographs showing the morphological changes at the granular scale in CVRD-DR
pellets; (a) to (d): grains in different stages of a reduction at 800 °C; (d) to (g): iron grains after reduction at different temperatures; (h):
polished cross-section at a conversion degree of 65% of a sample reduced at 900 °C, the red arrow points to shrinking wüstite cores (dark
gray) surrounded with a spreading layer of iron (light gray), indicated by the blue arrow. All images by SE (secondary electrons),
except (h) BSE (backscattered electrons). Reducing gas used: 2 L/min H2–He (60–40 vol.%) in a thermobalance. Fig. 4. SEM (Scanning Electron Microscope) micrographs showing the morphological changes at the granular scale in CVRD-DR
pellets; (a) to (d): grains in different stages of a reduction at 800 °C; (d) to (g): iron grains after reduction at different temperatures; (h):
polished cross-section at a conversion degree of 65% of a sample reduced at 900 °C, the red arrow points to shrinking wüstite cores (dark
gray) surrounded with a spreading layer of iron (light gray), indicated by the blue arrow. All images by SE (secondary electrons),
except (h) BSE (backscattered electrons). Reducing gas used: 2 L/min H2–He (60–40 vol.%) in a thermobalance. Fig. 5. Pore size distribution of a CVRD-DR pellet before and
after reduction by H2 at 1000 °C. Fig. 6. Effect of temperature on the kinetics of reduction of one
CVRD pellet under H2 (40%H2 in He), measured by thermo-
gravimetry. Fig. 6. Effect of temperature on the kinetics of reduction of one
CVRD pellet under H2 (40%H2 in He), measured by thermo-
gravimetry. Fig. 6. Effect of temperature on the kinetics of reduction of one
CVRD pellet under H2 (40%H2 in He), measured by thermo-
gravimetry. Fig. 5. Pore size distribution of a CVRD-DR pellet before and
after reduction by H2 at 1000 °C. F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 5 Fig. 7. 2 Chemical characteristics of the reduction
of iron ore by hydrogen Reduction of CVRD pellets under CO–H2 mixtures in a thermobalance [18]. Fig. 7. Reduction of CVRD pellets under CO–H2 mixtures in a thermobalance [18]. wüstite stage, the grains subdivide into crystallites. At the
grain scale, the reactions are topochemical, with a
shrinking core. transport step by solid phase diffusion through the dense
iron layer is also involved. The latter is more difficult
through the Fe-g austenite (formed above 912 °C) than
through the Fe-a ferrite, which explains the final kinetic
slowdown at 950 and 1000 °C. These particular behaviors
can of course have repercussions on the performance of
industrial shaft furnaces and in particular on the final
degree of metallization obtained. It is thus a more sophisticated model than the shrinking
core pellet models, with one or three interfaces, often used
in the literature. It should be noted here that experimen-
tally, we do not observe concentric interfaces separating
the different oxides and the iron at the pellet scale. The
kinetic parameters used are taken from our own experi-
ments (porosity measurements, specific surface) and from
model-experiment fitting for the rate constants [19]. From
a mathematical point of view, the model remains simple
because it is based on the law of additive characteristic
times [20]. This law, approximate but validated by several
studies, allows us an analytical calculation of the reaction
rate, even for mixed regimes. This single pellet model
adapted to the reduction of iron ore by H2 and/or CO is
described in more detail in [13,18,19,21]. Finally, when they compare the reduction kinetics by
CO and H2, the vast majority of authors find that the
reductionkineticsbyH2isfasterthanthatbyCO,e.g.[13–15]. In the case of CO–H2 mixtures, such as the reducing gas used
for the direct reduction furnaces of MIDREX [16] and HYL-
ENERGIRON [17] processes, the mixture gives results closer
to those with pure H2 than those with pure CO. Figure 7
illustrates these results. 4 Multiparticle model of the shaft furnace The modeling of reactors such as shaft furnaces requires a
predictive calculation of the rate of the reduction reactions
at any temperature and for any gas composition. For this
purpose, we have designed a specific model, the kinetic
model of a single pellet, which reproduces the observations
and results obtained in the laboratory and which can be
easily integrated into a multiparticle reactor model. Its
principle is based on a geometrical representation of a
spherical pellet made up of smaller grains (Fig. 8). Initially
the grains are dense hematite. The inter- and intra-
granular porosity evolves during the reduction. At the The shaft furnace for the reduction of iron ore with pure H2
is the core of the new process. In a conventional shaft
furnace, the iron ore, in the form of pellets or lump ore,
slowly descends by gravity into a vertical cylindrical shaft
and is reduced by a CO–H2 mixture flowing upwards
countercurrently. This bustle gas is introduced through the
sidewall at mid-height and goes out at the top. In the
MIDREX process (Fig. 9), the top gas is recycled to feed
the burners and the reformer, in combination with natural F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 6 Fig. 8. Representation of the pellet (top row) and grain (bottom row) evolution in the single pellet kinetic model [11]. esentation of the pellet (top row) and grain (bottom row) evolution in the single pellet kinetic model [ Fig. 9. Schematic diagram of the direct reduction process [21]. Fig. 9. Schematic diagram of the direct reduction process [21]. Fig. 9. Schematic diagram of the direct reduction process [21]. with or without intermediate and cooling zones), the
chemical reactions taken into account and the associated
kinetic models, the description of the gas and solid flows,
the numerical method of resolution. The most detailed
models [21–24] give access to very interesting character-
istics of the internal behavior of shaft furnaces. With the
exception of REDUCTOR, presented below, none of them
has been used so far to predict the operation of a shaft
furnace under pure H2. gas. In the HYL process, the gas loop is somewhat different,
the reformer may not exist. The bottom part of the shaft
furnace is a cone, in which the DRI is cooled by a looping
counterflow of natural gas. 4 Multiparticle model of the shaft furnace with or without intermediate and cooling zones), the
chemical reactions taken into account and the associated
kinetic models, the description of the gas and solid flows,
the numerical method of resolution. The most detailed
models [21–24] give access to very interesting character-
istics of the internal behavior of shaft furnaces. With the
exception of REDUCTOR, presented below, none of them
has been used so far to predict the operation of a shaft
furnace under pure H2. To predict whether a similar shaft furnace could be
operated under pure hydrogen, a powerful and economical
means of evaluation is mathematical modeling. Several
models of DR shaft furnaces have been published, which
differ according to the description of the reactor (1D or 2D, F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 7 Fig. 10. The four scales in the REDUCTOR model [21]. Fig. 11. Calculated solid mass fractions in a shaft furnace fed with H2 at 800 °C. The symmetry axis is on the left-hand side of each
map, and the wall is on the right-hand side. The main gas inlet is at the wall near the bottom of the reactor. F. Patisson et al.: Matériaux & Techniques 109, 303 (2021)
7 Fig. 10. The four scales in the REDUCTOR model [21]. Fig. 10. The four scales in the REDUCTOR model [21]. Fig. 11. Calculated solid mass fractions in a shaft furnace fed with H2 at 800 °C. The symmetry axis is on the left-hand side of each
map, and the wall is on the right-hand side. The main gas inlet is at the wall near the bottom of the reactor. Fig. 11. Calculated solid mass fractions in a shaft furnace fed with H2 at 800 °C. The symmetry axis is on the left-hand side of each
map, and the wall is on the right-hand side. The main gas inlet is at the wall near the bottom of the reactor. Fig. 11. Calculated solid mass fractions in a shaft furnace fed with H2 at 800 °C. The symmetry axis is on the left-hand side of each
map, and the wall is on the right-hand side. The main gas inlet is at the wall near the bottom of the reactor. The REDUCTOR model simulates the operation of a
MIDREX or HYL-ENERGIRON shaft furnace. A cylin-
drical upper part, a conical lower part and an intermediate
cylindrical part are considered. 4 Multiparticle model of the shaft furnace Calculated temperatures (K) in a shaft furnace fed with
H2 at 800 °C. Fig. 13. Calculated molar gas fractions in a shaft furnace fed
with H2 at 800 °C. The temperature map (Fig. 12) shows that gas and
solids are almost at the same temperature everywhere,
except at the very top where the solid goes very quickly
from 25 to 600 °C. Slight radial and especially axial thermal
gradients are noted in the upper half of the furnace due to
the endothermicity of the reduction. The map of H2 and
H2O molar fractions (Fig. 13) illustrates the progressive
consumption of H2 and the corresponding production of
H2O (from z = 3 m to z = 6 m) corresponding to the
reduction of oxides to iron. Not all H2 is used, far from
it, since 72% of H2 remains in the top gas. This is not due to
a poor utilization of H2 but to the fact that a flow rate much
higher (3.8 times) than the stoichiometric flow rate
necessary for the reduction must be circulated in order
to heat the furnace. maturity and very good performance in terms of emissions
reduction (more than 85% decrease). Hydrogen must be
green or produced through low-carbon processes, and the
solution that appears to be the most efficient to produce the
large quantities needed is the electrolysis of water with
decarbonized electricity (renewable or nuclear). Numerous
projects in line with this new route are starting, focusing
either on the development of large capacity electrolysers or,
more rarely until now, on the demonstration of the viability
of the direct reduction process with pure H2. The research carried out on this topic at the Institut
Jean Lamour, University of Lorraine, France, initiated
during the ULCOS program and continued since, has
provided a set of valuable results. The experimental study of the reduction of ore pellets
by H2 shows that the reaction is faster than with CO and
that a complete metallization is obtained in the laboratory,
even when a dense iron layer is formed around the wüstite
grains. The influence of the temperature is complex, with
the appearance of kinetic slowdowns at the end of the
reaction
at
700
and
950 °C. We
have
proposed
a
sophisticated single pellet kinetic model based on experi-
mental observations and capable of simulating mixed
kinetic regimes. 4 Multiparticle model of the shaft furnace The input data for the
calculation are the flow rates, compositions and temper-
atures of the incoming streams (solid at the top, reducing
gas at the side, and cooling gas at the bottom). The same
output quantities (DRI at the bottom and gas at the top)
are results of the calculation. Four scales are considered:
reactor, porous moving bed, pellets and grains (Fig. 10). The main equations solved are material balances on solid
species (Fe2O3, Fe3O4, FeO, Fe, C, gangue), gaseous
species (H2, CO, H2O, CO2, CH4, N2), heat balances on gas
and solid, momentum balances. The kinetic model of a
single pellet is included as a subroutine of the reactor code. The equations and the numerical method have already were simulated by REDUCTOR and the comparison of the
calculated results with measurements was used to validate
the model [18,21]. Below we present the results of the simulation of a
furnace that would operate under pure H2, without CO and
CH4, limited to the reduction zone. The height is 6 m, the
diameter 6.6 m. The solid charge consists of hematite
pellets, its flow rate is 52 kg s1. The gas (98%H2, 2%H2O)
enters at 800 °C with a flow rate of 3634 mol s1 from the
side and 100 mol s1 from the bottom. The map of the mass fractions of the solids (Fig. 11)
reveals that the reduction of hematite to magnetite is very
fast, followed by the reduction of magnetite to wüstite,
then the reduction of wüstite to iron is the slowest. It is
however entirely completed at 3 m below the solid inlet,
whereas the whole height is necessary for a metallization of
about 95% in a conventional MIDREX furnace operating
under CO–H2. The equations and the numerical method have already
been presented [11,13,21] and will not be repeated here. Two MIDREX direct reduction furnaces of different sizes 8 F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 8 Fig. 12. Calculated temperatures (K) in a shaft furnace fed with
H2 at 800 °C. 8
F. Patisson et al.: Matériaux Fig. 13. Calculated molar gas fractions in a shaft furnace fed
with H2 at 800 °C. Fig. 12. Calculated temperatures (K) in a shaft furnace fed with
H2 at 800 °C. Fig. 13. Calculated molar gas fractions in a shaft furnace fed
with H2 at 800 °C. Fig. 12. 4 Multiparticle model of the shaft furnace Figure 14 shows the effect of the reduction gas
temperature on the iron mass fraction. The case at
800 °C has already been presented. At higher temperatures,
a complete reduction is obtained faster. Between 900 and
950 °C, the difference is little because of the kinetic
slowdown mentioned. Moreover, above 900 °C, the gain
seems small compared to the need to spend more energy. At
700 °C, the temperature is not sufficient to heat the furnace
and iron appears only very partially, near the injection
point. Dealing
with
the
reactor
scale,
the
use
of
the
REDUCTOR model to simulate a shaft furnace operated
under pure H2 has provided significant first results. It seems
possible to obtain a complete metallization under H2 and to
use a squatter furnace than the current direct reduction
furnaces. Temperatures between 800 and 950 °C seems
adequate. However, these simulations should be extended
to the case of a complete furnace, including the lower
conical zone. The model can be used to guide towards
optimal dimensions and operating conditions. These simulations need to be complemented by other
ones, and confronted with experiments or other recent
modeling results [25,26]. They do not answer all the
questions related to the use of pure H2. However, they
clearly show that a complete reduction of iron ore pellets by
H2 in a shaft furnace is quite feasible. 5 Conclusion In the context of the fight against global warming, the need
to drastically reduce CO2 emissions in the industrial sectors
most concerned has recently led the steel industry to
seriously consider the hydrogen option. The most promis-
ing
hydrogen-based
steelmaking
route
is
the
direct
reduction of iron ore by pure H2 followed by electric
steelmaking. It indeed combines a certain technological The points that we believe deserve further investiga-
tions are: some very scientific questions on kinetic slow-
downs,
occurrence
of
sticking,
solid
flow,
more
technological questions such as the design and optimal
operating conditions of a H2 shaft furnace and its gas loop,
the consequences of using carbon-free DRI in the electric
furnace, the materials best suited to H2–H2O atmospheres, F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 9 Fig. 14. Effect of the bustle gas temperature (Tg,e: 700 to 1100 °C) on the iron mass fraction in a shaft furnace fed with H2. Fig. 14. Effect of the bustle gas temperature (Tg,e: 700 to 1100 °C) on the iron mass fraction in a bustle gas temperature (Tg,e: 700 to 1100 °C) on the iron mass fraction in a shaft furnace fed with H2. Fig. 14. Effect of the bustle gas temperature (Tg,e: 700 to 1100 °C) on the iron mass fraction in a shaft furnace fed with H2. more industrial questions such as the integration of a direct
reduction unit in a conventional integrated plant, with its
consequences on the valorization of the coke oven, BF and
BOF gases and on the best use of the DRI. 11. F. Patisson, O. Mirgaux, Hydrogen ironmaking: How it
works, Metals 10, 922 (2020) 12. Hybrit (Hydrogen Breakthrough Ironmaking Technology)
brochure, Available from https://ssabwebsitecdn.azureedge. net/-/media/hybrit/files/hybrit_brochure.pdf (accessed on
26/11/2020) 13. A. Ranzani da Costa, D. Wagner, F. Patisson, Modelling a
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of iron oxide pellets in a shaft furnace process, Renew. Energy
179, 1537–1547 (2021)
Cite this article as: Fabrice Patisson, Olivier Mirgaux, Jean-Pierre Birat, Hydrogen steelmaking. Part 1: Physical chemistry and
process metallurgy, Matériaux & Techniques 109, 303 (2021)
10
F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) F. Patisson et al.: Matériaux & Techniques 109, 303 (2021) 10
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English
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Alterations of gut microbiota are associated with blood pressure: a cross-sectional clinical trial in Northwestern China
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Journal of translational medicine
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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. Alterations of gut microbiota are associated
with blood pressure: a cross‑sectional clinical
trial in Northwestern China Jing Lv1†, Jihan Wang2†, Yan Yu1, Mengyao Zhao1, Wenjuan Yang1, Junye Liu1, Yan Zhao1, Yanjie Yang3,
Guodong Wang4, Lei Guo5* and Heping Zhao1* Jing Lv1†, Jihan Wang2†, Yan Yu1, Mengyao Zhao1, Wenjuan Yang1, Junye Liu1, Yan Zhao1, Yanjie Yang3,
Guodong Wang4, Lei Guo5* and Heping Zhao1* Abstract Background The human gut microbiota (GM) is involved in the pathogenesis of hypertension (HTN), and could be
affected by various factors, including sex and geography. However, available data directly linking GM to HTN based on
sex differences are limited. Methods This study investigated the GM characteristics in HTN subjects in Northwestern China, and evaluate the
associations of GM with blood pressure levels based on sex differences. A total of 87 HTN subjects and 45 controls
were recruited with demographic and clinical characteristics documented. Fecal samples were collected for 16S rRNA
gene sequencing and metagenomic sequencing. Results GM diversity was observed higher in females compared to males, and principal coordinate analysis showed
an obvious segregation of females and males. Four predominant phyla of fecal GM included Firmicutes, Bacteroi-
detes, Actinobacteria and Proteobacteria. LEfSe analysis indicated that phylum unidentified_Bacteria was enriched
in HTN females, while Leuconostocaceae, Weissella and Weissella_cibaria were enriched in control females (P < 0.05). Functionally, ROC analysis revealed that Cellular Processes (0.796, 95% CI 0.620 ~ 0.916), Human Diseases (0.773,
95% CI 0.595 ~ 0.900), Signal transduction (0.806, 95% CI 0.631 ~ 0.922) and Two-component system (0.806, 95% CI
0.631 ~ 0.922) could differentiate HTN females as effective functional classifiers, which were also positively correlated
with systolic blood pressure levels. Conclusions This work provides evidence of fecal GM characteristics in HTN females and males in a northwestern
Chinese population, further supporting the notion that GM dysbiosis may participate in the pathogenesis of HTN, and
the role of sex differences should be considered. Trial registration Chinese Clinical Trial Registry, ChiCTR1800019191. Registered 30 October 2018 – Retrospectively regis-
tered, http://www.chictr.org.cn/. †Jing Lv and Jihan Wang are co-first authors, and they have contributed
equally to this work. †Lei Guo and Heping Zhao are co-corresponding authors, and they have
contributed equally to this work. *Correspondence:
Lei Guo
guolei0711@163.com
Heping Zhao
redcrossjyk@163.com
Full list of author information is available at the end of the article *Correspondence:
Lei Guo
guolei0711@163.com
Heping Zhao
redcrossjyk@163.com
Full list of author informa *Correspondence:
Lei Guo
guolei0711@163.com
Heping Zhao
redcrossjyk@163.com
Full list of author information is available at the end of the article Lv et al. Journal of Translational Medicine (2023) 21:429
https://doi.org/10.1186/s12967-023-04176-6 Lv et al. Journal of Translational Medicine (2023) 21:429
https://doi.org/10.1186/s12967-023-04176-6 Journal of
Translational Medicine Open Access Background As a common and chronic medical condition, hyperten-
sion (HTN) has become a global health issue, account-
ing for approximately 10.8 million deaths worldwide [1]. HTN is a complex and modifiable risk factor for car-
diovascular diseases (CVDs) and stroke, while a diverse
range of endogenous and environmental factors contrib-
utes to both HTN onset and progression [2–4]. Although
an increased risk of HTN has been demonstrated to be
associated with approximately 900 genetic loci, only < 6%
of the variance in systolic blood pressure (BP) could be
explained by common genetic risk variants [5]. Given its
complexity and heterogeneity, the elucidation of HTN
pathogenesis remains challenging. Growing evidence has revealed the potential role of
the gut microbiota (GM) in host homeostasis and multi-
ple physiological processes [6], and suggests associations
between GM and various diseases including atheroscle-
rotic CVDs [7]. As the longest organ, the gastrointestinal
tract is mainly involved in the absorption of nutrients
and ions which greatly impacts BP [8], and a substantial
amount of work has supported the role of GM as a poten-
tial factor in BP regulation or even causal determinants
of HTN pathogenesis [2, 9–11]. There is evidence of gas-
trointestinal pathophysiology in animal HTN models, in
which fecal transplantation from HTN subjects increases
BP in germ-free mice [12]. In addition, GM dysregulation
is also associated with various metabolic diseases and
HTN-related risk factors, such as obesity, hyperlipidemia
and diabetes mellitus [13]. Overall, current data strongly
indicate that GM may play an important role in HTN
pathogenesis [12, 14–16]. This study was approved by the Ethics Committee
of Honghui Hospital, Xi’an Jiaotong University (Proto-
col Number: 201801022, approved January 8th, 2018),
and all subjects provided written informed consent
before enrollment. HTN was diagnosed if systolic BP
(SBP) ≥ 130 mmHg and/or diastolic BP (DBP) ≥ 80
mmHg; subjects with normal BP (SBP < 120 mmHg and
DBP < 80 mmHg) served as controls [26, 27]. The study
flow is shown in Fig. 1. Measurements of demographic and clinical parameters
On their first visit, all medical information was
recorded for the recruited subjects. Body weight and
height were measured without shoes and with light
clothing to the nearest 0.1 cm or to the nearest 0.1 kg,
respectively. Body mass index (BMI) was calculated as
weight in kilograms divided by the square of height in
meters. Study participants and study protocol From July 2018 to June 2020, we recruited 205 sub-
jects from our outpatient clinics at Honghui Hospital,
Xi’an Jiaotong University, China. All selected subjects
were aged between 18 and 80 years old, and were able
to provide written informed consent. Subjects with one
of the following conditions were excluded: ① pregnant
or lactating, or with chronic diseases including can-
cers, inflammation or surgical history in the alimentary
tract, or serious systematic dysfunctions; ② taking any
medications that may disrupt their original GM, such
as fiber supplements, probiotics or prebiotics within 6
weeks, or antimicrobial drugs within 6 months before
GM sampling [2, 23]; ③ taking any anti-inflammatory
agents, acid-suppressing agents, immunosuppressants,
or anti-HTN medication, which seem to modulate GM
[2, 24, 25]. © 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. Page 2 of 17 Lv et al. Journal of Translational Medicine (2023) 21:429 Lv et al. Journal of Translational Medicine (2023) 21:429 Keywords Blood pressure, Gut microbiota, Hypertension,
Metagenome, Northwestern China, Sex difference Background Waist circumference (WC) was measured mid-
way between the lower rib margin and the iliac crest in
the standing position with a non-expandable tape to the
nearest 0.1 cm. BP was assessed using a medical elec-
tronic sphygmomanometer (OMRON HEM-7130 pro-
fessional portable blood pressure monitor, OMRON,
Dalian, China) on the left arm positioned at heart level
in a seated position with palm face up. The subjects
were required to rest for at least 3 ~ 5 min before BP
measurement, and BP was measured in duplicate at a
1 ~ 2-min interval. The average of the 2 readings was
calculated. Mean arterial pressure (MAP) was calcu-
lated using the formula [(2 × diastolic BP) + systolic
BP]/3 [16]. Due to the novel concept of GM-influenced HTN
pathogenesis [2], it is critical to investigate GM altera-
tions in HTN subjects in different regions with different
genetic background and dietary habits [2, 16–19]. More-
over, obesity, hyperlipidemia and diabetes could result in
GM alterations [20], and these variations should also be
taken into consideration. Of note, few studies have inves-
tigated the associations between GM and HTN subjects
based on sex differences. Given that sex is important in
BP regulation [2, 21, 22], we investigated the GM charac-
teristics of female and male HTN subjects, respectively,
and the associations between GM characteristics and BP
levels were discussed. Peripheral venous blood samples were obtained in the
morning after an overnight (at least 8 h) fast. Levels of
fasting plasma glucose (FPG), serum triglyceride (TG), Page 3 of 17 Lv et al. Journal of Translational Medicine (2023) 21:429 Fig. 1 Study flow diagram. BMI body mass index; WC waist circumference; BP blood pressure; FBG fasting blood glucose; TG triglyceride; TC
total cholesterol; LDL-C low-density lipoprotein cholesterol; HDL-C high-density lipoprotein cholesterol; non-HDL-C non-high-density lipoprotein
cholesterol; GM gut microbiota Fig. 1 Study flow diagram. BMI body mass index; WC waist circumference; BP blood pressure; FBG fasting blood glucose; TG triglyceride; TC
total cholesterol; LDL-C low-density lipoprotein cholesterol; HDL-C high-density lipoprotein cholesterol; non-HDL-C non-high-density lipoprotein
cholesterol; GM gut microbiota 16S rRNA gene amplification, sequencing and analysish 16S rRNA gene amplification, sequencing and analysis
The V3 ~ V4 hypervariable regions of the 16S rRNA gene
were amplified using specific primers (338F: 5ʹ-ACT
CCTACGGGAGGCAGCAG-3ʹ;
806R:
5ʹ-GGACTA
CHVGGGTWTCTAAT-3ʹ) with barcodes, and all PCRs
were performed with Phusion® High Fidelity PCR Mas-
ter Mix (New England Biolabs, Ipswich, MA, USA). After mixing the PCR products, the mixture was purified
using a GeneJET™ Gel Extraction Kit (Thermo Scientific,
Waltham, MA, USA). Sequencing libraries were gener-
ated using the NEB Next® Ultra™ DNA Library Prep Kit
for Illumina (New England Biolabs, Ipswich, MA, USA)
following the manufacturer’s recommendations, and
library quality was assessed on the Qubit® 2.0 Fluorom-
eter (Thermo Scientific, Waltham, MA, USA). Finally, the
prepared libraries were sequenced on an Illumina HiSeq
platform (Illumina NovaSeq 6000, PE150, Illumina, San
Diego, CA, USA). Short Oligonucleotide Analysis Package 2 (SOAP2)
software
(v2.04,
http://soap.genomics.org.cn/soapd
enovo.html) and Bowtie2.2.4 software were used for
raw data processing, and the remaining Scaftigs were
used for subsequent analysis. Genes were predicted
on Scaftigs (≥ 500 bp) using MetaGeneMark (prokary-
otic GeneMark.hmm v2.10), and a non-redundant gene
catalog was constructed with CD-HIT (v4.5.8) software. For information on the abundance of genes, clean reads
were realigned to the gene catalog (Unigenes) using Bow-
tie2.2.4 software. Genes with more than 2 mapped reads
were deemed to be present in a sample [32]. The abun-
dance of genes was calculated by counting the number of
reads and normalizing based on gene length. Unigenes
were aligned to the KEGG database (Release 73.1, Ver-
sion: 2018.01, with animal and plant genes removed) for
gene functional annotation using DIAMOND software
(version 0.9.9), and GM functions were evaluated and
compared across groups in the present study. g
UPARSE software (Uparse v7.0.1001, http://drive5.
com/uparse/) [29] and Quantitative Insights Into Micro-
bial Ecology (QIIME) software (v1.7.0) [30] were intro-
duced for sequencing analysis. Acquired high-quality
clean reads with ≥ 97% similarity were de novo clustered
into the same operational taxonomic units (OTUs), and
the representative sequence for each OTU was screened
and used to annotate taxonomic information based on
the RDP classifier [31]. After OTUs with annotation
were produced, microbial diversity was assessed using
QIIME software (v1.7.0) [30]. Alpha-diversity analysis
was performed based on 4 indices, including Shannon,
Chao1, Simpson and abundance coverage-based estima-
tor (ACE). Statistical analysis y
Statistical analyses and figure constructions were con-
ducted using the SPSS PASW v23 (IBM SPSS Inc., Chi-
cago, IL), R platform v4.0.2 (R Foundation, Vienna,
Austria), GraphPad Prism v5.01 (GraphPad Software
Inc., San Diego, CA, USA) and MedCalc v19.0.4 (Med-
Calc Software Bvba, Ostend, Belgium) software. Quan-
titative variables are presented as mean ± standard
deviation (SD), and the normal distribution of quantita-
tive variables was assessed by the Shapiro-Wilk test. The
t-test was used for comparisons of the demographic and
clinical parameters between groups, depending on the
homogeneity of variance. The comparisons of GM diver-
sities between groups were assessed using the Wilcoxon
rank-sum test. The top 10 GM taxa in each level (phylum, 16S rRNA gene amplification, sequencing and analysish Beta-diversity of GM composition was esti-
mated using the unweighted UniFrac method to calculate
the distances between samples, and then visualized by
principal coordinates analysis (PCoA). Linear discrimi-
nant analysis (LDA) effect size (LEfSe) algorithm with an
LDA score threshold of 2 (on a log10 scale) was applied
to identify the enriched and significant bacteria in each
group, with a P value < 0.05. Fecal sample collection and DNA extraction total cholesterol (TC), low-density lipoprotein cho-
lesterol (LDL-C), high-density lipoprotein cholesterol
(HDL-C) and non-high-density lipoprotein cholesterol
(non-HDL-C) were measured using an automatic bio-
chemical analyzer (Cobas c701, Roche, Mannheim, Ger-
many). All instruments were calibrated regularly. We followed the standard protocols of fecal sample col-
lection and processing [28]. Briefly, fresh fecal samples
were collected from each participant at home, and tem-
porarily stored in foam boxes with frozen cold packs. After immediate transportation to the Clinical Labo-
ratory at Honghui Hospital, Xi’an Jiaotong University
within 6 h, fecal samples were stored at − 80 °C until fur-
ther processing. Page 4 of 17 Lv et al. Journal of Translational Medicine (2023) 21:429 Lv et al. Journal of Translational Medicine (2023) 21:429 Lv et al. Journal of Translational Medicine (2023) 21:429 Samples were sent in cold-chain with dry ice to Novo-
gene Co., Led. (Beijing, China) for subsequent proce-
dures. Genomic DNA was extracted from feces using
the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden,
Germany) according to the manufacturer’s instructions. After the evaluation of genomic DNA concentration
and quality, DNA samples, greater than 1 µg and with
an OD value between 1.8 ~ 2.0, were qualified for further
analysis. Sequencing libraries were generated using the NEBNext®
Ultra™ DNA Library Prep Kit (New England Biolabs,
Ipswich, MA, USA) following the manufacturer’s recom-
mendations. Briefly, the qualified DNA was fragmented
to a size of 350 bp by sonication, and the acquired DNA
fragments were end-polished, A-tailed, and ligated with
the full-length adaptor for Illumina sequencing. Then
PCR amplification was performed, and PCR products
were purified using the AMPure XP system. Then, librar-
ies were initially quantified using a Qubit® 2.0 Fluo-
rometer (Thermo Scientific, Waltham, MA, USA) and
diluted to 2 ng/µL. Finally, libraries were analyzed using
an Agilent2100 Bioanalyzer for size distribution, and
then quantified using real-time PCR to ensure the qual-
ity (effective concentration > 3 nM). After clustering, the
prepared libraries were sequenced on an Illumina HiSeq
platform (Illumina NovaSeq 6000, PE250, Illumina, San
Diego, CA, USA). Sequencing libraries were generated using the NEBNext®
Ultra™ DNA Library Prep Kit (New England Biolabs,
Ipswich, MA, USA) following the manufacturer’s recom-
mendations. Briefly, the qualified DNA was fragmented
to a size of 350 bp by sonication, and the acquired DNA
fragments were end-polished, A-tailed, and ligated with
the full-length adaptor for Illumina sequencing. Fecal sample collection and DNA extraction Then
PCR amplification was performed, and PCR products
were purified using the AMPure XP system. Then, librar-
ies were initially quantified using a Qubit® 2.0 Fluo-
rometer (Thermo Scientific, Waltham, MA, USA) and
diluted to 2 ng/µL. Finally, libraries were analyzed using
an Agilent2100 Bioanalyzer for size distribution, and
then quantified using real-time PCR to ensure the qual-
ity (effective concentration > 3 nM). After clustering, the
prepared libraries were sequenced on an Illumina HiSeq
platform (Illumina NovaSeq 6000, PE250, Illumina, San
Diego, CA, USA). Metagenomic sequencing and analysis Journal of Translational Medicine (2023) 21:429 Page 5 of 17 Table 1 Demographic and clinical characteristics of the enrolled subjects for GM 16S rRNA gene sequencing in this study
Data are presented as mean ± standard deviation (SD)
BMI body mass index, WC waist circumference, SBP systolic blood pressure, DBP diastolic blood pressure, MAP mean arterial pressure, FBG fasting plasma glucose,
TG triglyceride, TC total cholesterol, LDL-C low-density lipoprotein cholesterol, HDL-C high-density lipoprotein cholesterol, non-HDL-C non-high-density lipoprotein
cholesterol
P values are from t-test depending on the homogeneity of variance, *P < 0.05
Variables
Female
P value
Male
P value
HTN
control
HTN
control
Number
45
25
–
42
20
–
Age (year)
55 ± 7
51 ± 11
0.064
54 ± 11
48 ± 12
0.059
Height (cm)
153.2 ± 6.7
152.7 ± 7.7
0.800
165.7 ± 6.4
164.6 ± 8.5
0.564
Weight (kg)
56.9 ± 9.8
55.7 ± 9.8
0.626
64.3 ± 9.6
61.5 ± 8.4
0.262
BMI (kg/m2)
24.2 ± 3.6
23.8 ± 3.3
0.646
23.4 ± 3.2
22.7 ± 2.9
0.411
WC (cm)
85.9 ± 9.2
84.6 ± 8.5
0.554
86.0 ± 8.4
83.3 ± 7.2
0.213
SBP (mmHg)
145 ± 15
110 ± 7
0.000*
142 ± 17
112 ± 7
0.000*
DBP (mmHg)
82 ± 9
68 ± 6
0.000*
86 ± 8
69 ± 5
0.000*
MAP (mmHg)
103 ± 9
82 ± 6
0.000*
105 ± 9
83 ± 5
0.000*
FPG (mmol/L)
5.06 ± 0.94
5.09 ± 0.78
0.889
4.82 ± 1.00
4.81 ± 0.65
0.948
TG (mmol/L)
1.61 ± 0.55
1.51 ± 0.59
0.460
1.40 ± 0.70
1.27 ± 0.77
0.510
TC (mmol/L)
4.40 ± 1.07
4.36 ± 1.62
0.909
3.75 ± 0.70
3.53 ± 1.14
0.428
LDL-C (mmol/L)
2.49 ± 0.78
2.60 ± 1.31
0.653
2.07 ± 0.56
1.99 ± 0.72
0.630
HDL-C (mmol/L)
1.37 ± 0.44
1.20 ± 0.40
0.115
1.22 ± 0.40
1.07 ± 0.47
0.179
non-HDL-C (mmol/L)
3.03 ± 0.84
3.16 ± 1.56
0.646
2.53 ± 0.59
2.46 ± 0.91
0.774 Table 1 Demographic and clinical characteristics of the enrolled subjects for GM 16S rRNA gene sequencing in this study cal characteristics of the enrolled subjects for GM 16S rRNA gene sequencing in this study Data are presented as mean ± standard deviation (SD) waist circumference, SBP systolic blood pressure, DBP diastolic blood pressure, MAP mean arterial pressure, FBG fasting plasma glucose,
olesterol, LDL-C low-density lipoprotein cholesterol, HDL-C high-density lipoprotein cholesterol, non-HDL-C non-high-density lipoprotein BMI body mass index, WC waist circumference, SBP systolic blood pressure, DBP diastolic blood pressure, MAP mean arterial pressure, FBG fasting plasma glucose,
TG triglyceride, TC total cholesterol, LDL-C low-density lipoprotein cholesterol, HDL-C high-density lipoprotein cholesterol, non-HDL-C non-high-density lipoprotein
cholesterol
P values are from t test depending on the homogeneity of variance *P<0 05 P values are from t-test depending on the homogeneity of variance, *P < 0.05 shows the correlations between any two characteristics of
the enrolled females and males. Metagenomic sequencing and analysis class, order, family, genus and species) sorted by higher
relative abundances were identified, and the significant
differences between groups were assessed using the Wil-
coxon rank-sum test. The “PerformanceAnalytics” pack-
age in R was utilized for Spearman’s correlation analysis
between any two demographic or clinical parameters of
the subjects. In addition, Spearman’s correlation test
was used to analyze the correlations between GM com-
position/KEGG functions and BP levels. A P value < 0.05
was considered statistically significant, whereas a P
value between 0.05 and 0.1 was considered a tendency. Receiver operator characteristic (ROC) curve analysis
was performed, and GM functions with an area under
the curve (AUC) ≥ 0.700 were selected for presentation. 16S rRNA gene sequencing analysis of gut microbiota
in the study subjects After 16S rRNA gene sequencing, the species accumula-
tion curve of GM in Fig. 3A and the rarefaction curves in
Fig. 3B showed a plateau of species richness, indicating
that the sample numbers and sequencing depth herein
covered enough information for the following analysis. Finally, we acquired a total of 1653 unique OTUs (rang-
ing from 208 to 416 per sample), and please refer to
Fig. 3C ~ 3E for details. g
Initially, we evaluated the α-diversities of GM in
females and males, which were significantly lower in
males compared with females (Fig. 4A). Moreover, the
PCoA model for β-diversity analysis revealed a segrega-
tion between females and males (Fig. 4B). Therefore, the
study subjects should be divided into female and male
groups in the following analysis. Then, we conducted the
α- and β-diversity analyses between the HTN and con-
trol subjects in females and males, respectively. However,
the differences of GM diversities were not significant
between either HTN vs. control females (Fig. 4C, D), or
HTN vs. control males (Fig. 4E, F). Data are presented as mean ± standard deviation (SD) Metagenomic sequencing and analysis After DNA extraction and quality control, a total of
1 µg DNA per sample was used for library preparation. Lv et al. Demographic and clinical characteristics of the study
subjects As described above, 87 HTN subjects and 45 controls
were finally recruited in this study. Table 1 shows the
demographic and clinical characteristics of the study
subjects grouped by sex and BP levels. With the excep-
tion of SBP, DBP and MAP (P < 0.05), the other param-
eters showed no significances between HTN and control
females, as well as HTN and control males. Figure 2 Lv et al. Journal of Translational Medicine (2023) 21:429 Page 6 of 17 on analysis between demographic and clinical characteristics of the study subjects. Correlation analysis betw
ristics of the enrolled females (A) and males (B), respectively. Bar plots present the distribution of each param
istribution for each two parameters. The numbers in this figure indicate the correlation coefficients of each
od in R, and the red dots and asterisks indicate the degrees of statistical significances, •P < 0.1, *P < 0.05, **P < 0 Fig. 2 Correlation analysis between demographic and clinical characteristics of the study subjects. Correlation analysis between demographic and
clinical characteristics of the enrolled females (A) and males (B), respectively. Bar plots present the distribution of each parameter, and the scatter
plots show the distribution for each two parameters. The numbers in this figure indicate the correlation coefficients of each two parameters by
Spearman method in R, and the red dots and asterisks indicate the degrees of statistical significances, •P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001 Fig. 2 Correlation analysis between demographic and clinical characteristics of the study subjects. Correlation analysis between demographic and
clinical characteristics of the enrolled females (A) and males (B), respectively. Bar plots present the distribution of each parameter, and the scatter
plots show the distribution for each two parameters. The numbers in this figure indicate the correlation coefficients of each two parameters by
Spearman method in R, and the red dots and asterisks indicate the degrees of statistical significances, •P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001 Lv et al. Journal of Translational Medicine (2023) 21:429 Page 7 of 17 Fig. 3 The basic information of the 16S rRNA gene sequencing. The species accumulation curve of gut microbiota detected in HTN and control
subjects in this study (A). The line indicates the averaged accumulated increase of detected OTUs vs. number of samples. The box-plots show
the 25th, 50th and 75th percentile at each sample size. Demographic and clinical characteristics of the study
subjects The rarefaction curve of the number of sequence reads and their corresponding number
of OTUs in females and males in this study (B). Venn diagrams of observed OTU numbers in different comparison groups: females vs. males (C),
HTN group vs. control group in females (D) and males (E), respectively. Every circle depicts the number of unique OTUs observed in one group. Overlapping OTUs shared by two groups are represented in the areas of intersection among corresponding circles Fig. 3 The basic information of the 16S rRNA gene sequencing. The species accumulation curve of gut microbiota detected in HTN and control
subjects in this study (A). The line indicates the averaged accumulated increase of detected OTUs vs. number of samples. The box-plots show
the 25th, 50th and 75th percentile at each sample size. The rarefaction curve of the number of sequence reads and their corresponding number
of OTUs in females and males in this study (B). Venn diagrams of observed OTU numbers in different comparison groups: females vs. males (C),
HTN group vs. control group in females (D) and males (E), respectively. Every circle depicts the number of unique OTUs observed in one group. Overlapping OTUs shared by two groups are represented in the areas of intersection among corresponding circles The differences of GM composition between HTN
and control subjects were taxonomically evaluated at
six different levels, including phylum, class, order, fam-
ily, genus and species, and the top 10 GM taxa with
higher relative abundances were summarized in each
level (Fig. 5). Consistent with previous results, the
GM taxa were mostly included in four predominant
phyla, which were Firmicutes, Bacteroidetes, Actino-
bacteria and Proteobacteria. The relative abundances
of Firmicutes, Bacteroidetes, Clostridia, Bacteroidia,
Clostridiales, Bacteroidales, Ruminococcaceae, Lach-
nospiraceae and Faecalibacterium were greater than
0.100 at respective taxonomic levels in HTN and con-
trol females. Of note, the unidentified_Bacteria abun-
dance was greater in HTN females, whereas the relative abundances of Bacteroidia, Bacteroidales, Leuconosto-
caceae, Weissella and Weissella_cibaria were lower in
HTN females compared with control females (Fig. 5A). LEfSe analysis indicated that unidentified_Bacteria was
enriched in HTN females; while, Leuconostocaceae,
Weissella and Weissella_cibaria were enriched in con-
trol females (Fig. 6A). In contrast, no significant differ-
ences of GM composition were noted between HTN
and control males except for Erysipelotrichia, which
was enriched in HTN males (Figs. 5B and 6B). Demographic and clinical characteristics of the study
subjects Further-
more, the correlations between GM taxa and SBP/DBP/
MAP levels were investigated in females and males,
respectively, and data were showed in Additional file 1:
Tables S1 and S2 in the Additional files. Lv et al. Journal of Translational Medicine (2023) 21:429 Page 8 of 17 Fig. 4 The diversity analysis of gut microbiota in the study subjects. Violin plots of α-diversity analysis in different comparison groups: females vs. males (A), HTN group vs. control group in females (C) and males (E), respectively. Each plot represents one index of the α-diversity distribution,
including Shannon, Chao1, Simpson and ACE indices, for each comparison groups. Wilcoxon rank-sum test was used for the analysis of significant
differences between different comparison groups. #P < 0.1, *P < 0.05, **P < 0.01. Plots of principal coordinate analysis (PCoA) based on the OTU
level in different comparison groups: females vs. males (B), HTN group vs. control group in females (D) and males (F), respectively. Each square/
circle indicates one sample. The distance between samples represents the GM similarity or differences in the samples, and the PCoA analysis was
conducted with unweighted UniFrac method Fig. 4 The diversity analysis of gut microbiota in the study subjects. Violin plots of α-diversity analysis in different comparison groups: females vs. males (A), HTN group vs. control group in females (C) and males (E), respectively. Each plot represents one index of the α-diversity distribution,
including Shannon, Chao1, Simpson and ACE indices, for each comparison groups. Wilcoxon rank-sum test was used for the analysis of significant
differences between different comparison groups. #P < 0.1, *P < 0.05, **P < 0.01. Plots of principal coordinate analysis (PCoA) based on the OTU
level in different comparison groups: females vs. males (B), HTN group vs. control group in females (D) and males (F), respectively. Each square/
circle indicates one sample. The distance between samples represents the GM similarity or differences in the samples, and the PCoA analysis was
conducted with unweighted UniFrac method Lv et al. Journal of Translational Medicine (2023) 21:429 Page 9 of 17 Fig. 5 Relative abundances and comparative analysis of the taxonomic composition of gut microbiota in the study subjects. Relative abundances
and comparative analysis of the taxonomic composition of gut microbiota in the enrolled females (A) and males (B), respectively. Demographic and clinical characteristics of the study
subjects Bar plots show the
relative abundances of the top 10 taxa at respective levels, including phyla, class, orders, family, genera and species, in HTN and control females and
males. Each component of the cumulative bar chart indicates a phylum, a class, an order, a family, a genus or a species, respectively. The taxa with
significant difference between groups are presented using Wilcoxon rank-sum test. #P < 0.1, *P < 0.05, **P < 0.01 Fig. 5 Relative abundances and comparative analysis of the taxonomic composition of gut microbiota in the study subjects. Relative abundances
and comparative analysis of the taxonomic composition of gut microbiota in the enrolled females (A) and males (B), respectively. Bar plots show the
relative abundances of the top 10 taxa at respective levels, including phyla, class, orders, family, genera and species, in HTN and control females and
males. Each component of the cumulative bar chart indicates a phylum, a class, an order, a family, a genus or a species, respectively. The taxa with
significant difference between groups are presented using Wilcoxon rank-sum test. #P < 0.1, *P < 0.05, **P < 0.01 Metagenomic sequencing analysis of gut microbiota After GM gene and functional annotation aligned to
the KEGG database, the functional changes in the micro-
bial community were evaluated. GM functions showed
different patterns between HTN and control subjects
in females and males, as presented in Fig. 7. Our results
revealed that Cellular Processes and Human Diseases
represented the enrichment in Level 1 KEGG functions
in HTN females (P < 0.05), as well as Metabolism, Envi-
ronmental Information Processing and Organismal Sys-
tem (P < 0.1, Fig. 7A). In addition, the relative abundance g
q
g
y
g
After the preliminary analysis based on the 16S rRNA
gene sequencing, we conducted further analysis of GM
functions by performing matagenomic sequencing. A total of 36 HTN subjects (24 females and 12 males)
and 18 controls (9 females and 9 males) were randomly
selected for metagenomic analysis. Their demographic
and clinical characteristics were demonstrated in Table 2
grouped by sex and BP levels. Page 10 of 17 Lv et al. Journal of Translational Medicine (2023) 21:429 of Signal transduction in Level 2 and Two-componen
system in Level 3 were increased in HTN females com
Fig. 6 Linear discriminant analysis (LDA) effect size (LEfSe) analysis
of gut microbiota taxa in the study subjects. LDA scores indicate
differentially represented taxa in HTN and control groups in females
(A) and males (B), respectively. The logarithmic threshold for
discriminative features was set to 2.0 (P < 0.05); please refer to Additional file 1: Figure S1 in the
additional files for details. These data further supported
our previous strategy of separation of female and male
subjects in GM analysis. j
y
To explore the implications of altered GM functions
in HTN pathogenesis, Spearman’s correlation analy-
sis was introduced to evaluate the correlations between
GM functions and BP levels. Significantly positive cor-
relations of Cellular Processes, Human Diseases, Signal
transduction and Two-component system with SBP were
found in females (P < 0.05, Fig. 8A). Besides, the cor-
relation of Human Diseases with MAP was also noted
(P < 0.05, Fig. 8B). To further explore the altered GM
functions that could facilitate the identification of HTN
subjects, ROC curve analysis was introduced herein
(Fig. 8C, D). Metagenomic sequencing analysis of gut microbiota The AUCs of GM functions in Level 1 that
could differentiate HTN females from controls included
Cellular Processes (0.796, 95% CI 0.620 ~ 0.916), Human
Diseases (0.773, 95% CI 0.595 ~ 0.900), Environmental
Information Processing (0.718, 95% CI 0.534 ~ 0.860),
Organismal Systems (0.718, 95% CI 0.534 ~ 0.860) and
Metabolism (0.704, 95% CI 0.520 ~ 0.849). Besides, Signal
transduction (0.806, 95% CI 0.631 ~ 0.922), Two-compo-
nent system (0.806, 95% CI 0.631 ~ 0.922), Carbohydrate
metabolism (0.722, 95% CI 0.539 ~ 0.863), Metabolism of
cofactors and vitamins (0.718, 95% CI 0.534 ~ 0.860), and
Amino acid metabolism (0.708, 95% CI 0.525 ~ 0.853) in
Level 2 and 3 could effectively distinguish HTN females
from controls. Discussion Hypertension (HTN) is a multifactorial and complicated
condition [3, 4], and growing evidence suggests a novel
role of GM in HTN onset and progression [16, 33, 34]. The adult GM consists trillions of microorganisms domi-
nated by phyla Firmicutes, Bacteroidetes, Actinobacteria
and Proteobacteria, and maintains the gut immunity and
whole-body homeostasis [35]; dysbiosis or imbalance
in the GM community may have detrimental effects on
health [36]. Several demographic factors, such as age,
BMI, sex, ethnicity and culture, geographic location and
socioeconomic environment, could influence both GM
and BP levels [2, 15, 21, 24, 37–39], and it is important to
consider these confounding factors in GM-related stud-
ies on HTN. Although biological sex could shape the host
GM [40–42], available data directly linking GM to HTN
based on sex differences are limited [21]. Therefore, resi-
dents with similar dietary habits living in Shaanxi Prov-
ince, China, were recruited to minimize the regional
differences of GM composition in this study; and the dif-
ferences of GM characteristics between female and male
subjects were analyzed. Of note, an obvious segregation
of GM diversity between females and males was found, Fig. 6 Linear discriminant analysis (LDA) effect size (LEfSe) analysis
of gut microbiota taxa in the study subjects. LDA scores indicate
differentially represented taxa in HTN and control groups in females
(A) and males (B), respectively. The logarithmic threshold for
discriminative features was set to 2.0 of Signal transduction in Level 2 and Two-component
system in Level 3 were increased in HTN females com-
pared with controls (P < 0.05, Fig. 7B, C). Please refer to
Fig. 7D ~ 7 F for detailed information about male sub-
jects. Notably, the relative abundances of Metabolism,
Environmental Information Processing, Cellular Pro-
cesses, Human Diseases, Carbohydrate metabolism,
Amino acid metabolism, Membrane transport, Cellular
community – prokaryotes, ABC transporters and Quo-
rum Sensing were enriched in females compared to males Lv et al. Discussion Journal of Translational Medicine (2023) 21:429 Page 11 of 17 Table 2 Demographic and clinical characteristics of the enrolled subjects for GM metagenomic sequencing in this study
Data are presented as mean ± standard deviation (SD)
BMI body mass index, WC waist circumference, SBP systolic blood pressure, DBP diastolic blood pressure, MAP mean arterial pressure, FBG fasting plasma glucose,
TG triglyceride, TC total cholesterol, LDL-C low-density lipoprotein cholesterol, HDL-C high-density lipoprotein cholesterol, non-HDL-C non-high-density lipoprotein
cholesterol
P values are from t-test depending on the homogeneity of variance, *P < 0.05
Variables
Female
P value
Male
P value
HTN
control
HTN
control
Number
24
9
–
12
9
–
Age (year)
54 ± 7
54 ± 7
0.925
57 ± 6
52 ± 4
0.040*
Height (cm)
153.1 ± 6.1
153.7 ± 7.4
0.822
167.2 ± 6.5
160.9 ± 7.3
0.052
Weight (kg)
58.7 ± 10.1
55.2 ± 11.0
0.395
66.3 ± 9.8
58.9 ± 11.2
0.126
BMI (kg/m2)
25.0 ± 3.7
23.1 ± 2.9
0.190
23.7 ± 2.9
22.7 ± 3.2
0.453
WC (cm)
88.5 ± 9.8
85.1 ± 11.4
0.405
89.2 ± 9.5
82.4 ± 9.3
0.118
SBP (mmHg)
145 ± 17
105 ± 8
0.000*
136 ± 12
112 ± 6
0.000*
DBP (mmHg)
83 ± 9
65 ± 7
0.000*
83 ± 6
69 ± 6
0.000*
MAP (mmHg)
103 ± 9
78 ± 5
0.000*
100 ± 5
83 ± 5
0.000*
FPG (mmol/L)
5.24 ± 1.15
5.38 ± 0.74
0.740
4.58 ± 0.67
4.84 ± 0.88
0.447
TG (mmol/L)
1.71 ± 0.62
1.68 ± 0.83
0.898
1.40 ± 0.78
1.05 ± 0.55
0.255
TC (mmol/L)
4.35 ± 1.14
5.39 ± 1.99 ±
0.068
3.51 ± 0.95
3.62 ± 0.86
0.780
LDL-C (mmol/L)
2.45 ± 0.82
3.36 ± 1.70
0.155
1.88 ± 0.78
2.01 ± 0.63
0.702
HDL-C (mmol/L)
1.26 ± 0.42
1.34 ± 0.46
0.662
1.11 ± 0.44
1.22 ± 0.48
0.559
non-HDL-C (mmol/L)
3.09 ± 0.90
4.05 ± 2.16
0.226
2.40 ± 0.69
2.40 ± 0.77
0.987 cal characteristics of the enrolled subjects for GM metagenomic sequencing in this study Data are presented as mean ± standard deviation (SD) BMI body mass index, WC waist circumference, SBP systolic blood pressure, DBP diastolic blood pressure, MAP mean arterial pressure, FBG fasting plasma glucose,
TG triglyceride, TC total cholesterol, LDL-C low-density lipoprotein cholesterol, HDL-C high-density lipoprotein cholesterol, non-HDL-C non-high-density lipoprotein
cholesterol P values are from t-test depending on the homogeneity of variance, *P < 0.05 levels have been observed in obese pregnant women
[59], and fiber and acetate supplementation could
improve GM dysbiosis and increase the relative abun-
dances of certain bacteria that may play a protective
role in HTN [49]. Data are presented as mean ± standard deviation (SD) Discussion In summary, SCFAs and a range of
SCFA-producing taxa could play potent roles in main-
taining GM homeostasis and BP levels [33, 60–62]. It is
known that Bacteroidetes phylum members could pro-
duce high levels of acetate and propionate, whereas cer-
tain species in Firmicutes may produce high amounts
of butyrate [63–65]. Consistent with these data, our
results showed that the relative abundances of Bac-
teroidia and Bacteroidales were greater than 0.10 at
respective taxonomic levels, and were lower in HTN
females compared with controls. Besides, Leuconos-
tocaceae, Weissella and Weissella_cibaria were also
enriched in control females. As Firmicutes phylum
members, Leuconostocaceae, Weissella and Weissella_
cibaria are producers of SCFAs, and Weissella cibaria
may have antihypertensive and antioxidant effects in
spontaneously hypertensive rats models [66, 67], indi-
cating their potential for HTN prevention and further
supporting our results herein. supporting a respective analysis for female and male sub-
jects in the subsequent GM analysis. GM has been implicated in the pathogenesis of HTN
by means of influencing sodium intake, production of
certain metabolites, low-grade inflammation, etc. [37,
43]. Epidemiological data have linked salt and fiber
intake with changes in BP levels, suggesting a connec-
tion between gut and HTN [3, 4, 44–47]. High sodium
intake could reduce the relative abundances of certain
beneficial taxa such as Lactobacillus spp, and GM-pro-
duced metabolites may also influence sodium absorp-
tion [48]. Besides, an increased consumption of dietary
fiber, which could modulate GM as well as be fer-
mented by GM, has been indicated to decrease BP lev-
els [45, 46, 48, 49]. The potent mechanism may involve
the production of short-chain fatty acids (SCFAs), such
as acetate, butyrate and propionate, which are mainly
derived from the fermentation of indigestible carbohy-
drates and consumption of protein or peptide [15, 49–
52]. GM-produced SCFAs are rapidly absorbed in the
colon and taken up by the liver or enter the circulation,
and serve as precursors or substrates in various physi-
ological processes [53–55]. In rodent models, SCFAs
were reported to bind to G-protein-coupled recep-
tors involved in the regulation of vasoreactivity and
BP levels [56–58]. The negative correlations between
the abundances of butyrate-producing bacteria and BP Ruminococcaceae is involved in intestinal epithe-
lium maintenance as it is inversely correlated with
intestinal permeability [68–70], and its abundance was
found diminished in elderly HTN patients [71]. Lower Lv et al. Discussion Journal of Translational Medicine (2023) 21:429 Page 12 of 17 Fig. 7 Relative abundances and comparative analysis of the annotated KEGG functions of gut microbiota in the study subjects. Relative
abundances and comparative analysis of the annotated KEGG functions of GM at three levels in HTN and control groups in females (A–C) and males
(D–F), respectively. The KEGG functions with significant differences between groups are presented with Wilcoxon rank-sum test. #P < 0.1, *P < 0.05,
**P < 0.01 Fig. 7 Relative abundances and comparative analysis of the annotated KEGG functions of gut microbiota in the study subjects. Relative
abundances and comparative analysis of the annotated KEGG functions of GM at three levels in HTN and control groups in females (A–C) and males
(D–F), respectively. The KEGG functions with significant differences between groups are presented with Wilcoxon rank-sum test. #P < 0.1, *P < 0.05,
**P < 0.01 Fig. 7 Relative abundances and comparative analysis of the annotated KEGG functions of gut microbiota in the study subjects. Relative
abundances and comparative analysis of the annotated KEGG functions of GM at three levels in HTN and control groups in females (A–C) and males
(D–F), respectively. The KEGG functions with significant differences between groups are presented with Wilcoxon rank-sum test. #P < 0.1, *P < 0.05,
**P < 0.01 abundance of Ruminococcus may associate with HTN
[72–74], and a particular reduction in Ruminococcus was
also found in SD rats with minocycline-induced pro-
grammed HTN [75]. However, Kim SR et al. reported
a higher abundance of Ruminococcus torques in HTN,
which was significantly associated with SBP [15]; while,
Dan et al. showed increased Ruminococcaceae and
decreased Ruminococcus in HTN subjects [76]. In this
study, Ruminococcus_bromii, as a member of Firmicutes
and Ruminococcus, was positively correlate with DBP. In
other words, certain inconsistent results exist, which may be due to variations in genetics, sex, diet and lifestyle,
geographical differences or other unknown factors [37]. Moreover, these data implicated a more important role of
individual GM taxon than phyla in BP regulation [21]. be due to variations in genetics, sex, diet and lifestyle,
geographical differences or other unknown factors [37]. Moreover, these data implicated a more important role of
individual GM taxon than phyla in BP regulation [21]. be due to variations in genetics, sex, diet and lifestyle,
geographical differences or other unknown factors [37].
Moreover, these data implicated a more important role of
individual GM taxon than phyla in BP regulation [21].
GM-related studies indicate that GM could exert
potential influences on various diseases [35, 77–80],
and certain GM characteristics could be utilized as non-
invasive biomarkers for early diagnosis in clinical prac-
tice [81, 82]. As one of the most prevalent CVDs and
a leading risk factor of other CVDs, HTN is certainly
accompanied by GM alterations [83–85]. Exploration Discussion GM-related studies indicate that GM could exert
potential influences on various diseases [35, 77–80],
and certain GM characteristics could be utilized as non-
invasive biomarkers for early diagnosis in clinical prac-
tice [81, 82]. As one of the most prevalent CVDs and
a leading risk factor of other CVDs, HTN is certainly
accompanied by GM alterations [83–85]. Exploration GM-related studies indicate that GM could exert
potential influences on various diseases [35, 77–80],
and certain GM characteristics could be utilized as non-
invasive biomarkers for early diagnosis in clinical prac-
tice [81, 82]. As one of the most prevalent CVDs and
a leading risk factor of other CVDs, HTN is certainly
accompanied by GM alterations [83–85]. Exploration Lv et al. Journal of Translational Medicine (2023) 21:429 Page 13 of 17 Fig. 8 Exploration of the altered KEGG functions of gut microbiota in the enrolled HTN females. Correlations of altered KEGG functions of gut
microbiota with SBP (A) and MBP (B) levels in the enrolled females. The horizontal axis represents the relative abundances of KEGG functions, and
the vertical axis represents different BP levels. Data was analyzed and plotted with Spearman method in R. Receiver operator characteristic (ROC)
curve analysis of identification of HTN females from controls based on KEGG functions of gut microbiota (C, D) Fig. 8 Exploration of the altered KEGG functions of gut microbiota in the enrolled HTN females. Correlations of altered KEGG functions of gut
microbiota with SBP (A) and MBP (B) levels in the enrolled females. The horizontal axis represents the relative abundances of KEGG functions, and
the vertical axis represents different BP levels. Data was analyzed and plotted with Spearman method in R. Receiver operator characteristic (ROC)
curve analysis of identification of HTN females from controls based on KEGG functions of gut microbiota (C, D) of the altered KEGG functions of GM in HTN may help
illustrate its functional roles in HTN pathogenesis, and
might provide a new perspective on the interpretation
of HTN and additional auxiliary diagnosis in the future
[86]. In this study, we randomly selected HTN and con-
trol subjects from the recruited females and males, who
also lacked significant differences in the majority of these
demographic and clinical characteristics, for metagen-
omic sequencing analysis. Discussion The relative abundances of
Human Diseases, Signal transduction and Two-compo-
nent system were increased in HTN females compared
with controls, positively correlated with increased SBP and MAP levels, and contributed to the effective identi-
fication of HTN females from controls. Besides, our data
indicated that the altered GM functions differed between
HTN females and males compared to respective controls,
further supporting the notion that females and males
should be separately analyzed in GM-related analysis. In terms of sex differences in this study, we found higher
α-diversities of GM in females, as well as an obvious seg-
regation in β-diversity between females and males, and
the GM composition and functions were also different
between female and male subjects, which could be con-
tributed by genetic and epigenetic factors, sex steroid Lv et al. Journal of Translational Medicine (2023) 21:429 Lv et al. Journal of Translational Medicine (2023) 21:429 Page 14 of 17 Page 14 of 17 milieu, gonadal status, dietary and lifestyle-related fac-
tors, etc. [40, 87–89]. Specifically, previous data indicated
that the primary sex steroid hormones, such as estrogen,
progesterone and testosterone, may participate in the
regulation of GM diversity, composition and function
[38, 40, 89]. For example, it was reported that estrogen
could promote the growth of certain beneficial bacteria
such as Lactobacillus and Bifidobacterium, which were
also considered to improve the cardiovascular health
[18, 89–91]. Moreover, sex differences also exist in life-
style-related factors, such as dietary preferences, sex*diet
interactions and physical activity levels, which may influ-
ence the GM characteristics and HTN onset and pro-
gression differently in females and males [18, 19, 42]. The
present study was conducted in northwestern China, and
regional variations in diet, lifestyle and environmental
factors may potentially contribute to the observed differ-
ences in GM characteristics between females and males. It is crucial to note that the specific mechanisms under-
lying the observed differences herein are multifactorial
and complicated, and may not be fully elucidated. Fur-
ther researches are needed to better reveal the potential
causes for the observed sex-based differences in GM, to
elucidate the underlying mechanisms, and to facilitate
understanding of the implications of GM for health and
diseases, including HTN. Author contributions L-J and G-L searched and reviewed published articles, interpreted data, con-
structed tables, drafted the article, and made substantial contributions to the
conception and design of this study. W-JH, Z-HP, Y-Y, Z-MY, Y-WJ, L-JY and Z-Y
conducted the experiments, performed sequencing analysis and constructed
the figures and tables. W-JH and W-GD performed statistical analysis. Y-YJ
contributed to the conception of the study. All authors read and approved the
final manuscript. Supplementary Information Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s12967-023-04176-6. Additional file 1: Table S1 Correlations between gut microbiota taxa and
blood pressure levels in female subjects. Table S2 Correlations between
gut microbiota taxa and blood pressure levels in male subjects. Fig. S1
Relative abundances and comparative analysis of the annotated KEGG
functions of gut microbiota in female and male subjects. The KEGG
functions with significant differences between groups are presented by
Wilcoxon rank-sum test. #P < 0.1, *P < 0.05, **P < 0.01. Additional file 1: Table S1 Correlations between gut microbiota taxa and
blood pressure levels in female subjects. Table S2 Correlations between
gut microbiota taxa and blood pressure levels in male subjects. Fig. S1
Relative abundances and comparative analysis of the annotated KEGG
functions of gut microbiota in female and male subjects. The KEGG
functions with significant differences between groups are presented by
Wilcoxon rank-sum test. #P < 0.1, *P < 0.05, **P < 0.01. Conclusions In conclusion, the pathogenesis of HTN is multifactorial
and complicated, and evidence suggests that GM may
play a novel role in HTN onset and progression. This
study provide the first evidence of GM characteristics
and alterations in HTN females and males, respectively,
in northwestern China, further supporting the theory
that GM dysbiosis underlies HTN pathogenesis. Future
studies are needed to elucidate the underlying mecha-
nisms and potential therapeutic interventions targeting
GM for HTN prevention and management [14]. Abbreviations
CVDs
Cardiovascular diseases
HTN
Hypertension
BP
Blood pressure
GM
Gut microbiota
SBP
Systolic blood pressure
DBP
Diastolic blood pressure
BMI
Body mass index
WC
Waist circumference
MAP
Mean arterial pressure
FPG
Fasting plasma glucose
TG
Triglyceride
TC
Total cholesterol
LDL-C
Low-density lipoprotein cholesterol
HDL-C
High-density lipoprotein cholesterol
non-HDL-C Non-high-density lipoprotein cholesterol
OTUs
Operational taxonomic units
ACE
Abundance coverage-based estimator
PCoA
Principal coordinates analysis
LDA
Linear discriminant analysis
LEfSe
Linear discriminant analysis effect size
SD
Standard deviation
ROC
Receiver operator characteristic
AUC
Area under the curve Although our data revealed certain GM changes in
HTN subjects, it has several limitations. First, this inves-
tigation was conducted with a limited sample size due
to the practical limitations of data availability. Second,
the subjects were recruited in a single hospital and were
grouped based on BP levels alone. Third, BP measure-
ment in clinic is used for HTN diagnosis [92] rather than
office BP monitoring [9]. Nevertheless, certain confound-
ing factors were taken into consideration herein, such
as genetics, geography, sex and treatment-naive recruit-
ment, thus our data were reliable despite some other or
unknown factors. Furthermore, evidence directly linking
GM and sex differences in BP regulation is rather lim-
ited, and the role of GM in sex-dependent HTN is only
hypothesized [2, 21, 22, 42]. Our data could provide
evidence of fecal GM characteristics in HTN females
and males, respectively, which might fill in the gap to a
certain degree. However, this is only a cross-sectional
clinical study in China, and it is infeasible to draw any
definite conclusions about the causal relationships
between GM and HTN based on sex differences. Issues
regarding the differences in GM profiles of HTN females
and males remain to be addressed in future studies [72],
and detailed evaluations are needed in larger numbers of
treatment-naive HTN patients. Consent for publication
Not applicable. 17. He Y, Wu W, Zheng H-M, Li P, McDonald D, Sheng H-F, Chen M-X, Chen
Z-H, Ji G-Y, Zheng Z-D-X, et al. Regional variation limits applications of
healthy gut microbiome reference ranges and disease models. Nat Med. 2018;24:1532–5. Funding
Thi
k u d
g
This work was supported by the Youth Program of Xi’an Municipal Health
Commission of China, No. 2022qn07; the General Program of Xi’an Municipal Page 15 of 17 Lv et al. Journal of Translational Medicine (2023) 21:429 Health Commission of China, No. 2023ms11 and No. 2020ms14; the National
Natural Science Foundation of China, No. 81702067. 12. Li J, Zhao FQ, Wang YD, Chen JR, Tao JE, Tian G, Wu SL, Liu WB, Cui QH,
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https://openalex.org/W4206374466
|
https://spektra.unsiq.ac.id/index.php/spek/article/download/217/pdf
|
Indonesian
| null |
Bagaimana Menjadi Guru Sains di Era 4.0 bagi Generasi Y dan Z?
|
Spektra : Jurnal Kajian Pendidikan Sains/Spektra
| 2,021
|
cc-by-sa
| 6,388
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Abstract The learning process in the 4.0 era for generations Y and Z requires special attention, considering that these two
generations have relatively different characteristics from the previous generation. These two generations have a life
that is very familiar with technology but lacks in terms of socializing. Learning in the 4.0 era should not only
emphasize the knowledge aspect but also have life skills in the 21st century. This is like science itself which includes
products, processes, and approaches to values, attitudes, and skills. Several things need to be done to become a
science teacher in Era 4.0 for generation Y and generation Z, including (a) applying science learning with a scientific
approach, (b) seeking meaningful learning through a Contextual Teaching and Learning (CTL) approach, (c)
developing learning with multiple intelligences approach, (d) collaborating learning proses with information
technology, (e) integrating character building in every learning process. In addition, era 4.0 teachers must also have
the following competencies: (a) educational competence, (b) technology commercialization competence, (c)
competence in globalization, (d) future strategy competence, and (e) counsellor competence. The application of
learning and mastery of these competencies is expected to have a positive impact on the development of generations
Y and Z. Keywords: era 4.0, science teacher, teacher competence, science learning Abstrak Proses pembelajaran sains di era 4.0 bagi generasi Y dan Z membutuhkan perhatian khusus mengingat kedua
generasi ini memiliki karakteristik yang relatif berbeda dengan generasi sebelumnya. Kedua generasi ini memiliki
kehidupan yang sangat akrab dengan teknologi namun kurang dalam hal bersosialisasi. Pembelajaran sains di era
4.0 tidak boleh hanya menekankan pada aspek pengetahuan, tetapi juga memiliki keterampilan hidup di abad 21. Hal ini sebagaimana hakikat sains itu sendiri yang meliputi produk, proses, dan pendekatan nilai, sikap, serta
keterampilan. Beberapa hal yang perlu dilakukan untuk menjadi guru sains di Era 4.0 bagi generasi Y dan Z di
antaranya: (a) menerapkan pembelajaran sains berpendekatan saintifik, (b) mengupayakan pembelajaran dengan
pendekatan Contextual Teaching and Learning (CTL), (c) mengembangkan pembelajaran berbasis teori multiple
intelligences, (d) mengkolaborasikan pembelajaran dengan teknologi informasi, dan (e) mengintegrasikan
pembentukan karakter dalam proses pembelajatan. Selain itu, guru sains era 4.0 juga harus memiliki kompetensi
berikut, di antaranya: (a) educational competence, (b) competence for technological commercialization, (c)
competence in globalization, (d) competence in future strategies, dan (e) conselor competence, Penerapan
pembelajaran dan penguasaan kompetensi tersebut diharapkan dapat memberikan dampak positif bagi
perkembangan generasi Y dan Z. p
g
g
Kata Kunci: era 4.0, guru sains, kompetensi guru, pembelajaran sains Diterima: 07/12/2021. Dipublikasikan: 13/12/2021. Dikirimkan: 02/11/2021. P-ISSN : 2442-9910
E-ISSN : 2548-642X
S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021)
DOI: http://dx.doi.org/10.32699/spektra.v7i2.217 P-ISSN : 2442-9910
E-ISSN : 2548-642X
S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021)
DOI: http://dx.doi.org/10.32699/spektra.v7i2.217 P-ISSN : 2442-9910
E-ISSN : 2548-642X
S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021)
DOI: http://dx.doi.org/10.32699/spektra.v7i2.217 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) Bagaimana Menjadi Guru Sains di Era 4.0 bagi Generasi Y dan Z? Panggih Priyambodo1)*, Wulandari Saputri2)
1Program Studi Ilmu Pendidikan, Pascasarjana, Universitas Negeri Yogyakarta, Yogyakarta
2Program Studi Pendidikan Biologi, Pascasarjana, Universitas Muhammadiyah Palembang, Palembang Panggih Priyambodo1)*, Wulandari Saputri2)
1Program Studi Ilmu Pendidikan, Pascasarjana, Universitas Negeri Yogyakarta, Yogyakarta
2Program Studi Pendidikan Biologi, Pascasarjana, Universitas Muhammadiyah Palembang, Palembang *email: panggihpriyambodo.2019@student.uny.ac.id S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) Oleh karenanya, proses pendidikan tidak bisa
diterapkan dengan cara-cara yang selalu sama
melainkan harus mampu menjawab setiap
kebutuhan dari generasi tersebut. Paradigma ini
harus benar-benar disadari oleh para pendidik dan
tidak hanya sekadar menjadi bahan ulasan
ataupun wacana keilmuan tanpa disertai dengan
tindakan nyata di lapangan. Pengembangan soft
skills maupun hard skills pada generasi terkini
tidak
bisa
ditawar
lagi
mengingat
laju
perkembangan IPTEK dan juga kebutuhan di
dunia kerja sudah begitu mendesak untuk segera
mendapatkan respons yang tepat. Pembelajaran sains itu sendiri meliputi tiga
focus utama, yaitu sains sebagai produk, sains
sebagai proses, dan sains sebagai pendekatan. Istilah sains sebagai produk mengacu pada
pengetahuan yang diperoleh peserta didik dari
proses pembelajaran (hard skill). Kemudian, sains
sebagai
proses
berkenaan
dengan
metode
pemecahan
masalah
yang
menjadi
sarana
mengembangkan kompetensi peserta didik (hard
skill) dan (soft skill). Terakhir, sains sebagai
pendekatan sikap dan nilai ilmiah mengacu pada
nilai-nilai yang diperoleh setelah mempelajari
sains [3]. Geliat kehadiran generasi terkini telah
memberikan warna baru bagi dunia dengan segala
bentuk tingkah laku dan karakternya yang
berbeda dengan generasi-generasi sebelumnya. Kenyataan di lapangan menunjukkan bahwa telah
terjadi pergeseran paradigma yang menyertai
kemunculan ataupun kehadiran dari setiap
generasi terbaru. Pembeda generasi milenial atau
generasi Y yang lahir antara tahun 1980-2000
dengan generasi sebelumnya salah satunya adalah
pola hidup ataupun perilaku yang tidak lepas dari
internet dan entertaiment sebagai gaya hidup [1]. Generasi milenial dan generasi Z kini telah
mendominasi lingkungan kerja, di mana sebe-
lumnya telah didahului oleh generasi X dan baby
boomers [2]. Sedangkan terkait kondisi masa
depan, Setiawan menjelaskan berdasarkan riset
yang dilakukan oleh Beano Studios jika generasi
alpha
(generasi
yang
terlahir
dari
tahun
2010/penerus
generasi
Z)
akan
mencapai
puncaknya pada tahun 2025 mendatang, di mana
pada saat itu akan ada 2 miliar generasi alpha di
seluruh
dunia. Data
dan
fakta
di
atas
menunjukkan bahwa sebentar lagi Indonesia juga
akan didominasi oleh orang-orang baru dengan
segala pandangan dan gaya hidupnya yang
berbeda dengan yang terjadi saat ini ataupun di
masa lalu. Berkaitan dengan hal yang telah dipaparkan,
penyelenggaraan
pendidikan
membutuhkan
perencanaan yang konkret dan inovatif dengan
mengarah pada orientasi masa depan. Hal ini
bukan lagi hanya sebatas rencana ataupun wacana
semata namun harus benar-benar terealisasi di
lapangan. Peran guru tidak hanya sebatas pada
penggalian wacana keilmuwan namun juga
membutuhkan tindakan nyata di kelas-kelas
maupun di berbagai kegiatan penelitian dan
pengembangan. PENDAHULUAN /pengelola pendidikan untuk berbenah agar selalu
adaptif terhadap setiap dinamika yang ada. Setiap
era juga melahirkan generasi yang kian ber-
kembang. Karakteristik, pola hidup, dan panda-
ngan yang melekat pada setiap generasi tersebut
membutuhkan pola-pola pengajaran yang sesuai. Perubahan tata kelola sistem dunia yang
berkembang begitu cepat membawa dampak
serius di berbagai bidang kehidupan termasuk
pendidikan. Hal ini memaksa setiap pelaku- 154 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) Beragam konsep, teori serta
keterampilan yang telah dikuasai guru baik yang
diperoleh dari proses perkuliahan maupun dari
beragam kegiatan akademis lainnya membutuh-
kan ekspresi dalam bentuk tindakan nyata secara
terprogram, terencana dan berkelanjutan. Guru,
khususnya guru sains, juga tidak bisa hanya
bertahan pada gaya ataupun pola-pola pengajaran
lama yang dirasa mudah, simpel, dan terkesan
tidak merepotkan. Meskipun metode mengajar
konvensional (seperti ceramah) tidak selalu salah
dan tidak pula harus serta merta ditinggalkan
namun kiranya perlu dikombinasikan dengan
metode pembelajaran lain guna menghasilkan
pembelajaran yang bermakna [4]. 155 HASIL DAN PEMBAHASAN Guru menurut Undang-Undang RI Nomor
14 Tahun 2005 adalah profesi dengan tugas utama
pendidik
profesional
dengan
tugas
utama
mendidik, mengajar, membimbing, mengarahkan,
melatih, menilai, dan mengevaluasi peserta didik
pada pendidikan anak usia dini melalui jalur
formal
pendidikan
dasar
dan
pendidikan
menengah. Berkenaan
dengan
tugasnya,
berdasarkan
Undang-Undang
tersebut
pula
disebutkan
bahwa
guru
harus
memiliki
kompetensi pedagogik, kompetensi kepribadian,
kompetensi sosial, dan kompetensi profesional
[5]. Standar ini harus benar-benar menjadi acuan
dalam pelaksanaan tugas di lapangan dan
sekaligus harus dihayati oleh para guru untuk
mendapatkan pemahaman yang utuh tentang diri
mereka sendiri dengan segala kelebihan ataupun
kekurangan-kekurangan yang harus dibenahi. Ruang lingkup maupun aspek-aspek dari tiap-tiap
kompetensi guru tersebut dijabarkan dalam tabel
berikut. Penelitian
ini
menggunakan
metode
kepustakaan atau literatur review. Studi literatur
merupakan
tindakan
yang
dipakai
untuk
menghimpun data atau sumber - sumber yang
berhubungan dengan topik yang diangkat dalam
suatu penulisan. Studi literatur didapatkan dari
berbagai sumber baik jurnal, buku, dokumentasi,
dan pustaka. Metode studi literatur ini merupakan
rangkaian kegiatan berkenaan dengan metode
pengumpulan
data
pustaka,
membaca
dan
mencatat, serta mengelola bahan penulisan. Dalam
hal ini digunakan untuk menganalisis dan
mengetahui Guru Sains di Era 4.0 bagi Generasi
Y dan Z. Teknik analisis data yang digunakan dalam
penelitian ini adalah teknik analisis data deskriptif
yaitu
teknik
analisis
yang
dipakai
untuk
menganalisis data dengan mendeskripsikan atau
menggambarkan
data-data
yang
sudah
dikumpulkan tanpa membuat generalisasi dari
hasil penelitian. Tabel 1. Kompetensi Guru dan Aspek-Aspeknya
Kompetensi
Definisi
Aspek-Aspek
Kompetensi
Pedagogik
Kemampuan yang berkenaan
dengan karakteristik peserta
didik dari berbagai aspek
seperti moral, emosional,
dan intelektual yang
kemudian berimplikasi pada
tuntutan bahwa guru harus
menguasai teori belajar dan
prinsip-prinsip belajar. Penguasaan terhadap karakteristik peserta didik dari aspek
fisik, moral, sosial, kultural, emosional dan intelektual. Penguasaan terhadap teori belajar dan prinsip-prinsip
pembelajaran yang mendidik. Mampu mengembangkan kurikulum yang terkait dengan
bidang pengembangan yang diampu. Menyelenggarakan
kegiatan
pengembangan
yang
mendidik. Memanfaatkan teknologi informasi dan komunikasi untuk
kepentingan penyelenggaraan kegiatan pengembangan
yang mendidik. Memfasilitasi pengembangan potensi peserta didik untuk
mengaktualisasikan berbagai potensi yang dimiliki. Berkomunikasi secara efektif, empatik, dan santun dengan
peserta didik. Melakukan penilaian dan evaluasi proses dan hasil belajar,
memanfaatkan hasil penilaian dan evaluasi untuk
kepentingan pembelajaran. Melakukan tindakan reflektif untuk peningkatan kualitas
pembelajaran. Kompetensi
Kepribadian
Kemampuan kepribadian
yang mantap, berakhlak
mulia, arif, dan berwibawa
Bertindak sesuai dengan norma agama, hukum, sosial, dan
kebudayaan nasional Indonesia. Tabel 1. HASIL DAN PEMBAHASAN Kompetensi Guru dan Aspek-Aspekny
Kompetensi
Definisi
Aspek-Aspek
Kompetensi
Pedagogik
Kemampuan yang berkenaan
dengan karakteristik peserta
didik dari berbagai aspek
seperti moral, emosional,
dan intelektual yang
kemudian berimplikasi pada
tuntutan bahwa guru harus
menguasai teori belajar dan
prinsip-prinsip belajar. Penguasaan terhadap karakte
fisik, moral, sosial, kultural,
Penguasaan terhadap teori
pembelajaran yang mendidik
Mampu mengembangkan ku
bidang pengembangan yang
Menyelenggarakan
kegiat
mendidik. Memanfaatkan teknologi inf
kepentingan penyelenggara
yang mendidik. Memfasilitasi pengembanga
mengaktualisasikan berbaga
Berkomunikasi secara efekti
peserta didik. Melakukan penilaian dan eva
memanfaatkan hasil peni
kepentingan pembelajaran. Melakukan tindakan reflekti
pembelajaran. Kompetensi
Kepribadian
Kemampuan kepribadian
yang mantap, berakhlak
mulia, arif, dan berwibawa
Bertindak sesuai dengan norm
kebudayaan nasional Indone Tabel 1. Kompetensi Guru dan Aspek-Aspeknya
Definisi
Aspek-Aspek Menampilkan diri sebagai pribadi yang jujur, berak
mulia, dan teladan bagi peserta didik dan masyarakat. Menampilkan diri sebagai pribadi yang mantap, st
dewasa, arif, dan berwibawa. Menunjukan etos kerja, tanggung jawab yang tinggi,
bangga menjadi guru, dan rasa percaya diri. Menjunjung tinggi kode etik profesi guru. Kompetensi
Sosial
Kemampuan guru untuk
berkomunikasi dan
berinteraksi secara efektif
dan efisien dengan peserta
didik, sesama guru,
orangtua/wali peserta didik,
dan masyarakat sekitar. Bertindak objektif serta tidak diskriminatif karena
pertimbangan jenis kelamin, agama, ras, kondisi fisik
latar belakang keluarga, dan status sosial ekonomi. Berkomunikasi secara efektif, empatik, dan santun
dengan sesama pendidik, tenaga kependidikan, orang
dan masyarakat. Beradaptasi di tempat bertugas di seluruh wilayah
Republik Indonesia yang memiliki keragaman sosial
budaya. Berkomunikasi dengan komunitas profesi sendiri dan
profesi lain secara lisan dan tulisan atau bentuk lain. Kompetensi
Profesional
Kemampuan penguasaan
materi pelajaran secara luas
dan mendalam. Menguasai materi, struktur, konsep, dan pola pikir
keilmuan yang mendukung mata pelajaran yang diam
Menguasai standar kompetensi dan kompetensi dasar
mata pelajaran/bidang pengembangan yang diampu. Mengembangkan materi pembelajaran yang diampu
secara kreatif. Mengembangkan keprofesionalan secara berkelanjuta
dengan melakukan tindakan reflektif. Memanfaatkan teknologi informasi dan komunikasi
untuk berkomunikasi dan mengembangkan diri. Sumber: [5] Menampilkan diri sebagai pribadi yang jujur, berakhlak
mulia, dan teladan bagi peserta didik dan masyarakat. Menampilkan diri sebagai pribadi yang mantap, stabil,
dewasa, arif, dan berwibawa. Menunjukan etos kerja, tanggung jawab yang tinggi, rasa
bangga menjadi guru, dan rasa percaya diri. Menjunjung tinggi kode etik profesi guru. Bertindak objektif serta tidak diskriminatif karena
pertimbangan jenis kelamin, agama, ras, kondisi fisik,
latar belakang keluarga, dan status sosial ekonomi. Berkomunikasi secara efektif, empatik, dan santun
dengan sesama pendidik, tenaga kependidikan, orang tua,
dan masyarakat. Beradaptasi di tempat bertugas di seluruh wilayah
Republik Indonesia yang memiliki keragaman sosial
budaya. Berkomunikasi dengan komunitas profesi sendiri dan
profesi lain secara lisan dan tulisan atau bentuk lain. Menguasai materi, struktur, konsep, dan pola pikir
keilmuan yang mendukung mata pelajaran yang diampu. Menguasai standar kompetensi dan kompetensi dasar
mata pelajaran/bidang pengembangan yang diampu. Mengembangkan materi pembelajaran yang diampu
secara kreatif. Mengembangkan keprofesionalan secara berkelanjutan
dengan melakukan tindakan reflektif. Memanfaatkan teknologi informasi dan komunikasi
untuk berkomunikasi dan mengembangkan diri. Tabel 1. Kompetensi Guru dan Aspek-Aspeknya
Definisi
Aspek-Aspek Penguasaan terhadap karakteristik peserta didik dari aspek
fisik, moral, sosial, kultural, emosional dan intelektual. Penguasaan terhadap karakteristik peserta didik dari aspek
fisik, moral, sosial, kultural, emosional dan intelektual. Penguasaan terhadap teori belajar dan prinsip-prinsip
pembelajaran yang mendidik. Mampu mengembangkan kurikulum yang terkait dengan
bidang pengembangan yang diampu. Menyelenggarakan
kegiatan
pengembangan
yang
mendidik. Memanfaatkan teknologi informasi dan komunikasi untuk
kepentingan penyelenggaraan kegiatan pengembangan
yang mendidik. Memfasilitasi pengembangan potensi peserta didik untuk
mengaktualisasikan berbagai potensi yang dimiliki. Berkomunikasi secara efektif, empatik, dan santun dengan
peserta didik. Melakukan penilaian dan evaluasi proses dan hasil belajar,
memanfaatkan hasil penilaian dan evaluasi untuk
kepentingan pembelajaran. Melakukan tindakan reflektif untuk peningkatan kualitas
pembelajaran. Bertindak sesuai dengan norma agama, hukum, sosial, dan
kebudayaan nasional Indonesia. 156 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) Kompetensi
Definisi
Aspek-Aspek
serta menjadi teladan bagi
peserta didik. Menampilkan diri sebagai pribadi yang jujur, berakhlak
mulia, dan teladan bagi peserta didik dan masyarakat. Menampilkan diri sebagai pribadi yang mantap, stabil,
dewasa, arif, dan berwibawa. Menunjukan etos kerja, tanggung jawab yang tinggi, rasa
bangga menjadi guru, dan rasa percaya diri. Menjunjung tinggi kode etik profesi guru. Kompetensi
Sosial
Kemampuan guru untuk
berkomunikasi dan
berinteraksi secara efektif
dan efisien dengan peserta
didik, sesama guru,
orangtua/wali peserta didik,
dan masyarakat sekitar. Bertindak objektif serta tidak diskriminatif karena
pertimbangan jenis kelamin, agama, ras, kondisi fisik,
latar belakang keluarga, dan status sosial ekonomi. Berkomunikasi secara efektif, empatik, dan santun
dengan sesama pendidik, tenaga kependidikan, orang tua,
dan masyarakat. Beradaptasi di tempat bertugas di seluruh wilayah
Republik Indonesia yang memiliki keragaman sosial
budaya. Berkomunikasi dengan komunitas profesi sendiri dan
profesi lain secara lisan dan tulisan atau bentuk lain. Kompetensi
Profesional
Kemampuan penguasaan
materi pelajaran secara luas
dan mendalam. Menguasai materi, struktur, konsep, dan pola pikir
keilmuan yang mendukung mata pelajaran yang diampu. Menguasai standar kompetensi dan kompetensi dasar
mata pelajaran/bidang pengembangan yang diampu. Mengembangkan materi pembelajaran yang diampu
secara kreatif. Mengembangkan keprofesionalan secara berkelanjutan
dengan melakukan tindakan reflektif. Memanfaatkan teknologi informasi dan komunikasi
untuk berkomunikasi dan mengembangkan diri. Sumber: [5] Kompetensi
Definisi
Aspek-Aspek
serta menjadi teladan bagi
peserta didik. Sumber: [5] Globalisasi di era 4.0 membawa tantangan
baru bagi para guru. Era 4.0 membutuhkan
penerapan pembelajaran yang mampu menum-
buhkembangkan kemampuan peserta didik secara
menyeluruh. Peserta didik generasi X dan Y
membutuhkan pengembangan baik pada aspek
keterampilan (psikomotorik) maupun aspek
pengetahuan (kognitif) aktual. Selain itu, peserta
didik juga membutuhkan kesempatan untuk
mengembangkan diri dari segi ranah sikap
(afektif) sebagaimana yang menyangkut penana-
man dan juga internalisasi nilai, norma, dan juga
karakter. Fisk menyatakan bahwa terdapat
sembilan
tren
atau
kecenderungan
terkait
pendidikan 4.0, yaitu: (a) belajar dapat dilakukan
pada waktu dan tempat berbeda, di mana e-
learning memfasilitasi kesempatan untuk pem-
belajaran jarak jauh dan mandiri; (b) pem-
belajaran individual, di mana peserta didik akan
belajar dengan peralatan belajar yang adaptif dengan kemampuannya dengan memberikan
tugas dan tantangan yang lebih sulit ketika peserta
didik telah mencapai derajat kompetensi tertentu
sedangkan bagi peserta didik yang mengalami
kesulitan belajar diberikan kesempatan yang lebih
banyak untuk belajar (memperdalam dan meng-
ulang materi); (c) peserta didik diberikan kesem-
patan untuk belajar sesuai dengan gaya belajarnya
masing-masing sekaligus dengan peralatan mau-
pun sarana dan prasarana yang diperlukannya; (d)
pembelajaran berbasis proyek, di mana peserta
didik diarahkan untuk mencoba menerapkan
keterampilan mereka dalam jangka pendek ke
berbagai situasi nyata terutama untuk peserta
didik di level sekolah menengah; (e) peningkatan
kesempatan peserta didik untuk memperoleh
pengalaman lapangan ataupun keterampilan dunia
nyata yang mewakili pekerjaan mereka baik
dengan program magang, proyek dengan bim-
bingan ataupun proyek kolaborasi; (f) penekanan 157 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) kemampuan peserta didik dalam hal interpretasi
data, di mana peserta didik dituntut untuk
memiliki kecakapan dalam menerapkan penge-
tahuan teoritis ke dalam angka-angka dan
menggunakan
keterampilan
mereka
untuk
membuat kesimpulan berdasarkan logika dan tren
data; (g) penerapan sistem penilaian yang
beragam dan menyeluruh baik untuk mengukur
kemampuan
faktual
peserta
didik
selama
pembelajaran maupun kemampuan peserta didik
dalam menerapkan pengetahuan saat melakukan
pekerjaan proyek di lapangan; (h) peningkatan
keterlibatan peserta didik dalam perancangan
kurikulum, di mana peserta didik dilibatkan
dalam menentukan materi pembelajaran untuk
menghasilkan kurikulum kontemporer yang
mutakhir dan bernilai guna; serta (i) kegiatan
mentoring, yaitu kegiatan pendampingan atau
pemberian bimbingan kepada peserta didik dalam
rangka membangun kemandirian dalam belajar
[6]. diselenggarakan dalam bentuk kegiatan belajar
yang normatif dan teoritis, di mana peserta didik
semata-mata berkewajiban untuk mendengarkan,
menghafal materi, berlatih hitung-hitungan dan
rumus-rumus, ataupun berlatih untuk menjawab
soal ujian hingga mendapatkan nilai yang bagus. Sumber: [5] Lebih dari itu, proses pembelajaran di sekolah
harus benar-benar terhubung dengan masyarakat,
dunia kerja, tantangan-tantangan maupun isu-isu
popular di sekitar lingkungan peserta didik. Keterhubungan ini akan menjadikan peserta didik
memiliki kesiapan untuk berekspresi, berkreasi,
maupun
memberikan
kontribusinya
dalam
kehidupan nyata setelah meninggalkan/lulus dari
lembaga pendidikan formal. Beberapa hal yang hendaknya dimiliki oleh
guru sains era 4.0 agar dapat membelajarkan
generasi Y dan Z di antaranya diuraikan sebagai
berikut. Mampu
Menyelenggarakan
Pembelajaran
Berbasis Pendekatan Saintifik Guru sains hendaknya menerapkan pem-
belajaran berbasis ilmiah atau lebih dikenal
dengan istilah pendekatan saintifik. Pemerintah
Indonesia melalui Permendikbud No. 81A Tahun
2013 mengamanatkan agar guru dalam proses
pembelajaran menerapkan pendekatan saintifik,
yang meliputi, mengamati, menanya, mengum-
pulkan informasi (mencoba), mengasosiasikan
dan mengkomunikasikan (5M) [8]. Adapun
beberapa kriteria yang menjadikan suatu proses
pembelajaran dapat dikatakan ilmiah meliputi: (a)
substansi
atau
materi
pembelajaran
yang
diajarkan berdasarkan fakta atau fenomena yang
benar terjadi dan dapat dijelaskan dengan logika
atau penalaran tertentu, bukan sebatas perkiraan,
khayalan, legenda, atau dongeng semata; (b)
penjelasan guru, respons peserta didik, dan
interaksi edukatif antara guru dengan peserta
didik harus terbebas dari prasangka, pemikiran
subyektif, atau penalaran yang menyimpang dari
alur piker yang logis; (c) mendorong dan Berkaitan dengan hal tersebut guru sains
sudah saatnya menyadari akan pentingnya
perubahan dalam penyelenggaraan pembelajaran
dalam rangka memenuhi kebutuhan peserta didik
generasi di era revolusi industri 4.0. Penye-
lenggaraan pembelajaran yang kurang sesuai
dengan minat maupun karakteristik pembelajar
berpotensi menjadikan tujuan pembelajaran tidak
tercapai secara optimal. Generasi X dan Y dido-
minasi oleh individu-individu yang cenderung
kritis sehingga mereka membutuhkan kegiatan
pembelajaran yang bermakna. National Council
for the Sosial Studies (NCSS) tahun 1994
menyatakan bahwa pembelajaran bermakna
adalah pembelajaran yang memungkinkan peserta
didik mampu menghubungkan pengetahuan,
keterampilan, dan sikap yang dipelajari di sekolah
dan luar sekolah, di mana penyampaian bahan ajar
ditujukan pada pemahaman, apresiasi dan
aplikasinya dalam kehidupan [7]. Dengan demi-
kian, proses pembelajaran di era 4.0 tidak sekadar 158 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) lajar dengan menghafal pengetahuan; (d) belajar
juga menyangkut latihan dalam mempraktikkan
pengetahuan serta pengalaman yang telah
diperoleh dari aktivitas belajar dalam kehidupan
nyata (applying knowledge) dalam rangka mem-
bangun perilaku yang lebih baik; dan (e) mela-
kukan refleksi (reflecting knowledge) terhadap
strategi pengembangan pengetahuan sebagai
umpan balik dalam menyempurnakan/memper-
baiki proses pembelajaran [10]. Beberapa pen-
dekatan CTL yang dapat diterapkan oleh guru di
antaranya: (a) Problem-Based Learning, (b)
Authentic
Instruction,
(c)
Inquiry-Based
Learning, (d) Project-Based Learning, (e) Work-
Based Learning, (f) Service Learning, dan (g)
Cooperative Learning [11]. Mampu
Menyelenggarakan
Pembelajaran
Berbasis Pendekatan Saintifik menginspirasi peserta didik untuk berpikir secara
kritis, analitis, dan tepat dalam mengidentifikasi
suatu hal, memahami, memecahkan masalah, dan
mengaplikasikan substansi atau materi pembe-
lajaran tertentu; (d) mengarahkan dan mengin-
spirasi peserta didik untuk mampu berpikir
hipotetik
(membuat
dugaan)
saat
melihat
perbedaan, kesamaan, ataupun kaitan satu materi
pembelajaran
dengan
materi
lainnya;
(e)
mengarahkan dan menginspirasi peserta didik
untuk mampu memahami, menerapkan, dan
mengembangkan pola pikir rasional dan obyektif
dalam menanggapi materi pembelajaran; (f)
kegiatan belajar harus berbasis pada konsep, teori,
serta fakta empiris yang dapat dipertanggung-
jawabkan; dan (g) tujuan pembelajaran harus
dirumuskan secara jelas, sederhana, dan disajikan
dengan suatu sistem yang menarik [9]. Mampu
Menyelenggarakan
Pembelajaran
Berbasis Teori Multiple Intellegences Mampu
Menyelenggarakan
Pembelajaran
Berbasis Teori Multiple Intellegences Guru sains juga hendaknya mampu menye-
lenggarakan pembelajaran yang berbasis ber-
pendekatan multiple intelligences. Hal ini karena
kebutuhan di era 4.0 mencakup berbagai bidang
kehidupan yang begitu variatif dan kompleks. Ragam kebutuhan di tiap-tiap bidang ini mem-
butuhkan perhatian serius dari lembaga pen-
didikan untuk membekali peserta didik dengan
kemampuan yang beragam pula sekaligus
aplikatif. Setiap orang tidak mengerti dengan
pasti terkait bagaimana bentuk maupun kondisi
masa depan yang akan menyambutnya nanti. Berbagai bentuk kecakapan pada peserta didik
perlu disipakan sedemikian rupa sesuai dengan
bakat, minat, dan juga kemampuannya. Fenomena
disrupsi yang telah terjadi pada era ini
membutuhkan penyiapan individu-individu masa
depan yang siap berkompetisi di berbagai bidang
kehidupan. Oleh karena itu, diperlukan program
pendidikan yang mampu menstimulus bahkan
mengembangkan kecakapan-kecakapan spesifik
pada peserta didik hingga mampu menjadi sebuah
kompetensi yang layak diperhitungkan. Potensi
diri harus mampu berkembang menjadi kom-
petensi diri. Ada sembilan jenis kecerdasan yang Mampu
M
Kontekstual Guru sains perlu menyelenggarakan pembe-
lajaran yang kontekstual berbasis masalah
kehidupan nyata. Pembelajaran
kontekstual
merupakan kegiatan pembelajaran yang menge-
depankan pada pemaknaan, di mana peserta didik
benar-benar diarahkan dan dibimbing untuk
memahami sekaligus mampu menerapkan penge-
tahuan yang diperoleh di sekolah dalam meng-
atasi persoalan nyata dalam kehidupan sehari-
hari. Fahmi menjelaskan tentang karakteristik dari
CTL (Contextual Teaching Learning) yaitu: (a)
membangun pengetahuan baru di atas pe-
ngetahuan yang telah ada sebelumnya hingga ter-
bentuk keterkaitan antar komponen pengetahuan
menjadi sebuah pengetahuan baru yang utuh; (b)
membangun pengetahuan baru (acquiring know-
ledge) secara deduksi, di mana pembelajaran
dimulai dari keseluruhan untuk kemudian mem-
perhatikan detailnya; (c) belajar adalah dengan
memahami dan meyakini pengetahuan (under-
standing knowledge) berdasarkan studi literatur
ataupun tanggapan dari orang lain untuk ke-
mudian mengembangkannya, bukan sekadar be- 159 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) Strategi Penokohan, yaitu strategi pembelajaran
yang mengkaitkan antara konsep pembelajaran
dengan sosok terkenal untuk membantu proses-
proses menghafal maupun pemahaman konsep
pada peserta didik; (h) Strategi Flash-Card, yaitu
strategi pembelajaran yang mengaitkan konsep
pembelajaran dengan gambar dalam kartu baik
dengan pola kwartet, urutan, dan yang lainnya; (i)
Strategi Gambar Visual, yaitu strategi pembe-
lajaran yang mengaitkan konsep pembelajaran
dengan gambar, lambang, atau simbol tertentu; (j)
Strategi Wayang, yaitu strategi pembelajaran
yang mengaitkan konsep pembelajaran dengan
dialog tokoh-tokoh yang ada hubungannya
dengan konsep atau materi; (k) Strategi Applied
Learning, yaitu strategi pembelajaran yang me-
ngaitkan konsep pembelajaran dengan manfa-
atnya untuk kebutuhan sehari-hari; (l) Strategi
Movie Learning, yaitu strategi pembelajaran yang
mengaitkan konsep pembelajaran dengan ta-
yangan film yang mengandung muatan materi
yang hendak dibelajarkan; (m) Strategi Envi-
ronment Learning, yaitu strategi pembelajaran
dengan mengunjungi suatu tempat tertentu yang
punya
manajemen
tertentu
dalam
rangka
menggali informasi dan pengetahuan; serta (n)
Strategi Service Learning, yaitu strategi pembe-
lajaran dengan mengunjungi suatu tempat (fasi-
litas publik) atau lingkungan tertentu dengan
melakukan pelayanan informasi kepada tempat
tersebut sebagaimana pengetahuan dan ketera-
mpilan yang telah dipelajari di kelas [14]. Mengenai bentuk-bentuk strategi ini, guru dapat
menggali lebih lanjut terkait strategi mana yang
menjadi ketertarikannya dalam menumbuhkan
jenis kecerdasan tertentu pada peserta didik. S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) logical Content Knowledge), TPK (Technological
Pedagogical Knowledge), dan TPACK (Techno-
logy, Pedagogy, and Content Knowledge) [16]. dapat menyiapkan poin-poin indikator terkait
kecerdasan tersebut meliputi: (a) menyukai ko-
munikasi secara efektif; (b) senang dan terampil
dalam mengarang suatu cerita; (c) senang
berdiskusi dan mengikuti debat; (d) senang dan
terampil dalam berlatih bahasa asing; (e) senang
bermain game bahasa; (f) senang membaca dan
mampu memahami suatu bacaan dengan baik; (g)
mudah mengingat kutipan dari para ahli ataupun
pakar; (h) tidak mudah salah tulis atau salah eja
dan lain sebagainya [15]. Proses pembelajaran
dengan pendekatan multiple intelligences mampu
membekali peserta didik dengan beragam jenis
kecerdasan yang kemudian dapat dikembangkan
secara mandiri dalam kehidupan sehari-hari. Peningkatan mutu dan kualitas pembelajaran
baik di level sekolah dasar, menengah, hingga ke
perguruan tinggi harus terus-menerus diupa-
yakan. Menteri Riset, Teknologi, dan Pendidikan
Tinggi periode 2014-2019, Mohammad Nasir
menyatakan bahwa disruption technology atau
gangguan teknologi akan berdampak pada
munculnya profesi atau bidang pekerjaan baru
yang berbasis pada kombinasi teknologi antara
lain: (a) Internet of Things, (b) Artificial Intel-
ligence, (c) New Materials, (d) Big Data, (e)
Robotics, (f) Augmented Reality, (g) Cloud Com-
puting, (h) Additive Manufacturing 3D Printing,
(i) Nanotech & Biotech, (j) Genetic Editing, serta
(k) E-Learning [17]. Beberapa media yang dapat
digunakan dalam pembelajaran berasis Teknologi
Informasi dan Komunikasi (ICT) adalah: Mampu
M
Kontekstual Satu hal yang tidak boleh dilupakan guru
dalam menerapkan pendekatan multiple intel- penting dikembangkan menurut teori multiple
intelligences, di antaranya: (a) kecerdasan verbal-
/linguistik, (b) kecerdasan logis/matematis, (c)
kecerdasan visual/spasial, (d) kecerdasan kines-
tatik/jasmani, (e) kecerdasan musikal, (f) kecer-
dasan interpersonal, (7) kecerdasan intrapersonal,
(g) kecerdasan naturalis, dan (h) kecerdasan ek-
sistensial) [12], [13]. Penerapan pendekatan multiple intelligences
menjadikan proses pembelajaran akan menjadi
berwarna dan tidak membosankan. Beberapa
strategi pembelajaran berbasis multiple intel-
ligences yang dapat digunakan sebagai rujukan
bagi para pendidik sebagaimana yang dirumuskan
Munif Chatif adalah sebagai berikut: (a) Strategi
Diskusi, yaitu aktivitas pembelajaran dengan
komunikasi dan interaksi di antara dua orang atau
lebih dengan mengacu pada sebuah topik
permasalahan yang akan dipecahkan; (b) Strategi
Action Research, yaitu proses pembelajaran yang
dilakukan dengan meminta peserta didik untuk
merumuskan suatu hipotesis atas suatu materi
tertentu lalu kemudian dibuktikan melalui proses-
proses pengumpulan data, analisis data, hingga
penarikan kesimpulan; (c) Strategi Klasifikasi,
yaitu aktifitas belajar dengan cara melakukan
pengelompokan banyak data ke dalam (minimal)
dua area atau premis yang berbeda berdasarkan
kriteria, ciri-ciri dan indikator tertentu; (d)
Strategi Analogi, yaitu proses pembelajaran yang
berorientasi pada pemahaman konsep dengan cara
membuat persamaan suatu bentuk dengan bentuk
lainnya yang mengakibatkan adanya hubungan
kesamaan di antara kedua hal/bentuk tersebut
untuk memecahkan masalah yang dihadapi beri-
kutnya; (e) Strategi Identifikasi, yaitu pema-
haman konsep dengan cara melakukan iden-
tifikasi guna mencari beberapa ciri yang melekat
pada suatu obyek; (f) Strategi Sosiodrama, yaitu
pembelajaran dengan mendramakan situasi sosial
tertentu terutama untuk pengembangan kecer-
dasan linguistik, kinestetik, dan interpersonal; (g) Satu hal yang tidak boleh dilupakan guru
dalam menerapkan pendekatan multiple intel-
ligences adalah penyiapan indikator-indikator
yang merepresentasikan ketercapaian setiap jenis
kecerdasan yang dibelajarkan. Seperti contohnya
untuk indikator kecerdasan verbal-linguistik, guru 160 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) Mampu
Menyelenggarakan
dengan
Memanfaatkan Teknologi Informasi Guru sains di era 4.0 dituntut kreatif dan
inovatif
dalam
mengkolaborasikan
proses
pembelajaran dengan teknologi. Sebagaimana
yang telah dibahas di awal bahwa, teknologi
sudah bukan merupakan hal asing bagi generasi Y
dan generasi Z. Bahkan, kehidupan mereka
sehari-hari sudah tidak bisa lagi dipisahkan
dengan perangkat berbasis jaringan internet. Oleh
karena itu, pelibatan unsur teknologi dalam
pembelajaran menjadi sesuatu yang sangat
penting dengan beberapa alasan, seperti untuk
membangkitkan
ketertarikan
peserta
didik
terhadap aktivitas belajar, menyajikan kegiatan
pembelajaran yang sesuai dengan gaya hidup dan
pola pikir peserta didik sehari-hari, memperkaya
sumber belajar, meningkatkan efektivitas kegia-
tan pembelajaran dan lain sebagainya. Dalam hal
penerapan teknologi pembelajaran, guru dapat
mengacu pada prinsip The TPACK Framework
yang secara umum meliputi tiga komponen yaitu
content, pedagogy, dan technology. Komponen-
komponen ataupun badan pengetahuan tersebut
kemudian saling berinteraksi sehingga mem-
bentuk irisan-irisan dalam area PCK (Peda-
gogical Content Knowledge), TCK (Techno- a. Internet, merupakan jaringan komputer
global sebagai media utama/media yang
sesungguhnya dalam pendidikan berbasis TI
karena mendasari terciptanya beberapa
model pembelajaran seperti e-learning,
distance learning, web based learning, dan
lain-lain. Keberadaan internet menjadikan
akses dan distribusi informasi maupun
pengetahuan yang berkaitan dengan materi
pelajaran menjadi lebih mudah dan lebih
cepat. Pemanfaatan dalam kegiatan pem-
belajaran dapat berupa tiga bentuk, yaitu: 1) Web Course, yaitu penggunaan internet
dalam pembelajaran tanpa memerlukan
tatap muka. Keseluruhan bahan belajar,
diskusi, konsultasi, penugasan, latihan
dan ujian disampaikan/didisposisikan
melalui internet baik melalui e-mail,
chat room, bulletin board maupun
online conference. Bentuk pembela-
jaran seperti ini umumnya digunakan
untuk pembelajaran jarak jauh (distance 161 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) apabila koneksi jaringan internet ataupun
intranet belum tersedia [18]. education). Aplikasi bentuk ini antara
lain Virtual Campus/University. 2) 2) Web Centric Course, di mana sebagian
bahan
belajar,
diskusi,
konsultasi,
penugasan, dan latihan disampaikan
melalui internet, sedangkan ujian dan
sebagian konsultasi, diskusi maupun
latihan dilakukan secara tatap muka. Presentasi tatap muka lebih sedikit
dibandingkan
penggunaan
internet. Bentuk ini banyak diterapkan di
perguruan tinggi yang menerapkan
sistem belajar off campus. 2) Web Centric Course, di mana sebagian
bahan
belajar,
diskusi,
konsultasi,
penugasan, dan latihan disampaikan
melalui internet, sedangkan ujian dan
sebagian konsultasi, diskusi maupun
latihan dilakukan secara tatap muka. Presentasi tatap muka lebih sedikit
dibandingkan
penggunaan
internet. Bentuk ini banyak diterapkan di
perguruan tinggi yang menerapkan
sistem belajar off campus. Mampu Memfasilitasi Pengembangan Moral p
g
g
Guru sains juga harus mampu memfasilitasi
penumbuhkembangan moral serta karakter pada
peserta didik sebagai penyeimbang arus budaya
maupun ilmu pengetahuan dan teknologi yang
berkembang semakin dinamis. Permasalahan
karakter menjadi permasalahan sendiri bagi
generasi X dan Y. Salah satunya adalah terkait
generasi muda yang mudah menghujat dan
berbicara kotor di media sosial. Roberts dan
Gilbert menyatakan bahwa pola jejaring sosial
yang negatif dapat saja terjadi, misalnya terlalu
kritis ataupun saling menuntut satu sama lain,
atau memperkuat atau mendorong perilaku yang
membahayakan atau antisosial, di mana komu-
nikasi-komunikasi semacam itu tak jarang hingga
menimbulkan problematika yang berujung pada
meja hijau [19]. 3) Web Enhanced Course atau dikenal
dengan
Web
Lite
Course,
yaitu
penggunaan
internet
yang
lebih
berposisi sebagai penunjang kegiatan
pembelajaran tatap muka. Dalam hal ini,
pembelajaran
tatap
muka
lebih
dominan. Penerapan
sistem
ini
umumnya digunakan sebagai langkah
awal dalam penerapan pembelajaran
berbasis internet. 3) Web Enhanced Course atau dikenal
dengan
Web
Lite
Course,
yaitu
penggunaan
internet
yang
lebih
berposisi sebagai penunjang kegiatan
pembelajaran tatap muka. Dalam hal ini,
pembelajaran
tatap
muka
lebih
dominan. Penerapan
sistem
ini
umumnya digunakan sebagai langkah
awal dalam penerapan pembelajaran
berbasis internet. Penanaman karakter peserta didik dapat
dilakukan terintegrasi dengan materi yang
disampaikan guru. Misalnya, dengan mengganti
peran peserta didik dari sekedar ‘menerima’
pengetahuan menjadi ‘pengkonstruksi’ penge-
tahuan. Penggantian peran tersebut dapat men-
dorong peserta didik untuk aktif berpartisipasi
dalam proses pembelajaran di kelas. Dengan
demikian, peserta didik secara tidak langsung
dapat
terfasilitasi
dalam
mengembangkan
keterampilan berpikir kritis dan logis, rasa ingin
tahu, toleransi, dan tanggung jawab [20]. Selain
itu, guru juga dapat menanamkan nilai-nilai
islam dengan mengintegrasikan ayat-ayat al-
qur’an dalam pembelajaran sains (biologi) [21]. Ataupun juga dengan memanfaatkan jejaring
sosial untuk menanamkan karakter religious pada
diri peserta didik [22]. b. Intranet, yaitu sistem pembelajaran dengan
karakteristik sebagaimana dengan basis
internet namun dengan area yang lebih
sempit/bersifat lokal (misal dalam suatu
kelas, sekolah, gedung, atau antar gedung). Umumnya penggunaan intranet dilakukan
apabila terjadi hambatan terkait penggunaan
infrastruktur internet. b. Intranet, yaitu sistem pembelajaran dengan
karakteristik sebagaimana dengan basis
internet namun dengan area yang lebih
sempit/bersifat lokal (misal dalam suatu
kelas, sekolah, gedung, atau antar gedung). Umumnya penggunaan intranet dilakukan
apabila terjadi hambatan terkait penggunaan
infrastruktur internet. c. Mobile Phone (Smart Phone), yaitu proses
pembelajaran yang dilakukan dengan me-
manfaatkan teknologi telepon selular yang
kian maju dan berkembang. Teknologi te-
lepon selular umumnya dapat digunakan
untuk mengakses materi pelajaran. Mampu Memfasilitasi Pengembangan Moral Pembe-
lajaran seperti ini biasa disebut dengan M-
Learning (Mobile Learning). Selain beberapa rekomendasi yang sudah
dijabarkan,
berikut
dirinci
pula
beberapa
kompetensi yang harus diperhatikan guru dalam
rangka meningkatkan kemampuan profesiona- d. CD-ROM/Flash Disk. Umumnya, CD ROM
dan flash disk digunakan untuk menyimpan
materi pelajaran dan cenderung digunakan 162 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) lismenya di era revolusi industri 4.0, yakni
meliputi: (1) educational competence, yaitu ke-
mampuan
guru
yang
berkaitan
dengan
kompetensi mendidik atau menyelenggarakan
pembelajaran berbasis Internet of Things (IoT)
sebagai basic skill; (2) competence for techno-
logical commercialization, yaitu kompetensi
guru dalam membawa ataupun mengarahkan
peserta didik untuk memiliki sikap entre-
preneurship (kewirausahaan) berbasis teknologi
dan hasil karya inovasi peserta didik; (3)
competence in globalization, yaitu kemampuan
hybrid dan kemampuan dalam memecahkan
masalah secara unggul (problem solver com-
petence) sehingga tidak gagap terhadap berbagai
budaya dan sekaligus mampu memberdayakan
lifeskill pada peserta didik; (4) competence in
future strategies, yaitu kemampuan dalam
memprediksi kondisi, peluang-peluang, maupun
tantangan yang dapat terjadi di masa depan; serta
(5) conselor competence, yaitu kemampuan guru
dalam menjalankan tugas sebagai seorang
konselor/psikolog guna menangani masalah-
masalah psikologis peserta didik seperti terkait
stres akibat tekanan dari keadaan yang semakin
kompleks [6]. yang dapat diperoleh dari pergaulan maupun
dunia maya dan lain sebagainya. Dalam hal ini,
koordinasi antara guru dan para orang tua sangat
dibutuhkan untuk menghadirkan pola pendidikan
maupun pola pengasuhan yang menyeluruh dan
sekaligus dengan pengawasan yang lebih intens
namun tetap proporsional. Kondisi ini tidak bisa
dipandang remeh mengingat beragam jenis
tayangan maupun konten di media sosial sangat
beragam, di mana hampir setiap orang dapat
membuat akun pribadi yang cenderung bebas
mengunggah konten video apapun tanpa adanya
standar kebaikan yang jelas untuk bisa menjadi
contoh bagi para generasi penerus DAFTAR PUSTAKA [1]
F. Y. Apsari, M. D. Rama, and E. Prasetyo,
“Pemanfaatan Teknologi Bagi Generasi
Millenial :
Konseling
Berbasis
Teks
Menggunakan Riliv-Aplikasi Android,” J. Exp., vol. 6, no. 1, pp. 45–49, 2018. [12] [12]
H. Gardner and T. Hatch, “Multiple
Intelligences Go to School: Educational
Implications of the Theory of Multiple
Intelligences,” Educ. Res., vol. 18, no. 8,
pp. 4–10,
1989,
doi:
10.3102/0013189X018008004. [2]
S. R. D. Setiawan, “Setelah Millenial dan
Gen Z, Muncul Generasi Alpha?,” 2019. [13]
M. Zhou and D. Brown, Educational
Learning Theories, 2nd ed. 2017. [3] [3]
B. Sumintono, “Pembelajaran Sains,
pengembangan ketrampilan sains dan
sikap
ilmiah
dalam
meningkatkan
kompetensi guru,” Al Bidayah J. Pendidik. Dasar Islam, vol. 2, no. 1, pp. 63–85,
2010,
[Online]. Available:
http://eprints.utm.my/11660/. [14]
S. Andriyani, “Implementasi Strategi
Multiple Intelligences pada Pembelajaran
Bahasa Inggris (Studi Kasus di SD Inklusi
Semai Jepara),” At-Tarbawi J. Kaji. Kependidikan Islam, vol. 2, no. 2, p. 121,
2017, doi: 10.22515/attarbawi.v2i2.829. [4] [4]
A. Khoiri, “Analisis Kritis Pendidikan
Sains Di Indonesia: (Problematika, Solusi
dan Model Keterpaduan Sains Dasar),”
SPEKTRA J. Kaji. Pendidik. Sains, vol. 6,
no. 1,
p. 19,
2020,
doi:
10.32699/spektra.v6i1.132. [15]
T. Musfiroh, Pengembangan Kecerdasan
Majemuk. Jakarta: Universitas Terbuka,
2010. [16]
M. J. Koehler, P. Mishra, and W. Cain,
“What is Technological Pedagogical
Content Knowledge (TPACK)?,” J. Educ.,
vol. 193, no. 3, pp. 13–19, 2013, doi:
10.1177/002205741319300303. [5] [5]
Undang-Undang
Republik
Indonesia
Nomor 14 Tahun 2005 Tentang Guru dan
Dosen. 2005. [17]
D. Yunanto, S. Herlina, S. Boediono, A. Pravita, and M. S. Fajri, “Kreatif dan
Inovatif di Era Revolusi Industri 4.0,”
Kemenristekdikti, vol. 8, no. I, Jakarta, pp. 1–56, 2018. [6] [6]
D. Lase, “Pendidikan di Era Revolusi
Industri 4.0,” Sundermann J. Ilm. Teol. Pendidikan, Sains, Hum. dan Kebud., vol. 12, no. 2, pp. 28–43, Nov. 2019, doi:
10.36588/sundermann.v1i1.18. [18]
T. Andriani,
“Sistem
Pembelajaran
Berbasis
Teknologi
Informasi
dan
Komunikasi,” Sos. Budaya Media Komun. Ilmu-Ilmu Sos. dan Budaya, vol. 12, no. 1,
2015. [7] [7]
R. Faslah, “Pemanfaatan Internet Dalam
Pengembangan
Konsep
IPS
dan
Implikasinya
terhadap
Pembelajaran
Bermakna,” Econosains J. Online Ekon. dan Pendidik., vol. 9, no. 2, pp. 161–170,
2011, doi: 10.21009/econosains.0092.07. [19]
M. M. Pandie and I. T. J. Weismann,
“Pengaruh Cyberbullying Di Media Sosial
Terhadap Perilaku Reaktif Sebagai Pelaku
Maupun Sebagai Korban Cyberbullying
Pada Siswa Kristen SMP Nasional
Makassar,” J. Jaffray, vol. 14, no. 1, pp. 43–62,
2016,
doi:
10.25278/jj.v14i1.188.43-62. [8] [8]
M. PENUTUP Pembelajaran
di
era
4.0
juga
membutuhkan guru dengan kemampuan yang
sangat baik. Guru dituntut untuk lebih memahami
dan
juga
menghayati
rumusan
standar
kompetensi utama yang meliputi kompetensi
pedagogik, personal, sosial, dan profesional. Guru sains harus memahami hakikat sains itu
pula, yang meliputi produk, proses, dan nilai serta
sikap. Pemahaman
terhadap
hal
tersebut
diharapkan
dapat
berimplikasi
kepada
kompetensi guru sains, di mana hendaknya
mampu membelajarkan sains dengan pendekatan
saintifik,
menerapkan
pembe-lajaran
yang
kontekstual, menyelenggarakan pembelajaran
berbasis teori multiple intelligences dan mampu
menyelenggarakan pembe-lajaran berbasis ICT
dengan berbagai bentuk desain yang sesuai dan
juga
meningkatkan
kepekaannya
terhadap
kondisi sosial peserta didik dalam kehidupan
sehari-hari. Hal ini sangat penting dalam rangka
menentukan
program
pendam-pingan
yang
terbaik
hingga
mampu
mendukung
penumbuhkembangan kepribadian dan juga
keterampilan
peserta
didik. Kegiatan
pembelajaran
yang
berhasil
akan
mampu Tugas guru sains di era 4.0 mencakup area
yang luas. Selain fokus dalam menjalankan tugas
profesionalnya di sekolah, guru seharusnya juga
meningkatkan kepekaan terhadap kondisi realitas
peserta didik di luar sekolah. Kondisi peserta
didik di luar sekolah seringkali luput dari
perhatian baik para guru maupun orang tua. Seakan peserta didik dalam keadaan baik dan
normal seperti hari-hari biasa, namun para guru
dan orang tua terkadang tidak mengetahui secara
detail tentang bagaimana bentuk-bentuk maupun
kondisi pergaulannya, apa yang sedang mereka
pikirkan, apa saja jenis tayangan di media sosial
seperti youtube yang sering ditonton, apa tren
pergaulan yang sedang banyak digandrungi,
nilai-nilai apa atau bahkan potensi keburukan apa 163 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) Keterampilan Beripikir Tingkat Tinggi
Melalui Pembelajaran IPA,” 2017, no. September 2016, pp. 121–128. memberikan kontribusi yang signifikan bagi
perubahan sikap dan perilaku peserta didik
sekaligus berimplikasi positif terhadap pengem-
bangan pengetahuan dan keterampilannya. [11] [11]
A. Kadir,
“Konsep
Pembelajaran
Kontekstual Di Sekolah,” Din. Ilmu, vol. 13, no. 1, pp. 17–38, 2013, [Online]. Available:
http://journal.iain-
samarinda.ac.id/index.php/dinamika_ilmu
/article/view/20. DAFTAR PUSTAKA Abduh, “Interaksi pada pendekatan
saintifik (kajian teori scaffolding),” in
Seminar Nasional Pendidikan PGSD UMS
& HDPGSDI Wilayah Jawa, 2017, pp. 307–318. [9]
Musfiqon and Nurdyansyah, Pendekatan
Pembelajaran
Sainstifik. Sidoarjo:
Nizamia Learning Center, 2015. [20] [20]
H. Sulistyaningrum, A. Winata, and S. Cacik, “Analisis Kemampuan Awal 21st
Century Skills Mahasiswa Calon Guru
SD,” J. Pendidik. Dasar Nusant., vol. 5,
no. 1,
p. 142,
2019,
doi:
10.29407/jpdn.v5i1.13068. [10]
Fahmi, “Strategi Pembelajaran Contextual
Teaching and Learning Untuk,” in
Conference:
Seminar
Nasional
Pendidikan
IPA
“Mengembangkan 164 S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) S P E K T R A: Jurnal Kajian Pendidikan Sains 7 (2) (2021) [21]
A. Yaqin, “Integrasi Ayat-Ayat Al-Qur’an
Dalam Pembelajaran Sains (Biologi)
Berdasarkan Pemikiran Ian G. Barbour,”
SPEKTRA J. Kaji. Pendidik. Sains, vol. 6,
no. 1,
p. 78,
2020,
doi:
10.32699/spektra.v6i1.119. [22]
A. R. Anis Rahmawati, “Integrasi Sains
Islam Penggunaan Jejaring Sosial Dalam
Pembentukan Karakter Siswa,” SPEKTRA
J. Kaji. Pendidik. Sains, vol. 4, no. 1, p. 49,
2018, doi: 10.32699/spektra.v4i1.45. 165
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References Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor,
R. (1987). J. Chem. Soc. Perkin Trans. 2 pp. S1–19. Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor,
R. (1987). J. Chem. Soc. Perkin Trans. 2 pp. S1–19. Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555-1573. Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl. 34, 1555-1573. Experimental
Crystal data
C10H15NO
Mr = 165.23
Monoclinic, P21=n
a = 5.9816 (8) A˚
b = 23.8962 (19) A˚
c = 7.4653 (8) A˚
= 111.119 (7)
V = 995.40 (19) A˚ 3
Z = 4
Cu K radiation Bruker (1996). XSCANS. Bruker AXS Inc., Madison, Wisconsin, USA. Sh
i
(1
)
A
Ch
S
1
16 6 16 Bruker (1996). XSCANS. Bruker AXS Inc., Madison, Wisconsin, USA. DeShong, P. & Leginus, J. M. (1983). J. Am. Chem. Soc. 105, 1686-1688. DeShong, P. & Leginus, J. M. (1983). J. Am. Chem. Soc. 105, 1686-1688. Hennebo¨hle, M., Le Roy, P.-Y., Hein, M., Ehrler, R. & Ja¨ger, V. (2004). Z. Naturforsch. Teil B, 59, 451– 467. Ibrahim, M. M. (2009). Dissertation, Universita¨t Stuttgart, Germany. Ja¨ger, V. & Buss, V. (1980). Liebigs Ann. Chem. pp. 101–121. Ja¨ger, V. & Colinas, P. (2002). Synthetic Applications of 1,3-Dipolar
Cycloaddition Chemistry Toward Heterocycles and Natural Products, The
Chemistry of Heterocyclic Compounds, edited by A. Padwa & W. H. Pearson, pp. 361–472. New York: Wiley. Experimental Experimental
Crystal data
C10H15NO
Mr = 165.23
Monoclinic, P21=n
a = 5.9816 (8) A˚
b = 23.8962 (19) A˚
c = 7.4653 (8) A˚
= 111.119 (7)
V = 995.40 (19) A˚ 3
Z = 4
Cu K radiation Crystal data
C10H15NO
Mr = 165.23
Monoclinic, P21=n
a = 5.9816 (8) A˚
b = 23.8962 (19) A˚ Ja¨ger, V., Frey, W., Bathich, Y., Shiva, S., Ibrahim, M., Hennebo¨hle, M., LeRoy,
P. Y. & Imerhasan, M. (2010). Z. Naturforsch. Teil B, 65b, 821–832. P. Y. & Imerhasan, M. (2010). Z. Naturforsch. Teil B, 65b, Ja¨ger, V., Mu¨ller, I. & Paulus, E. F. (1985). Tetrahedron Lett. 26, 2997-3000. Jager, V., Muller, I. & Paulus, E. F. (1985). Tetrahedron Lett. 26, 2997-3000. Lubell, W. D., Kitamura, M. & Noyori, R. (1991). Tetrahedron Asymmetry, 2,
543-554. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Frey et al. Table 1 Received 17 June 2012; accepted 27 August 2012 Table 1
Hydrogen-bond geometry (A˚ , ). D—H A
D—H
H A
D A
D—H A
N1—H1B O1i
1.02 (4)
2.06 (3)
3.023 (4)
157 (2)
O1—H1A N1ii
1.12 (4)
1.70 (4)
2.815 (3)
176 (3)
Symmetry codes: (i) x þ 1; y; z; (ii) x þ 2; y; z þ 1. Key indicators: single-crystal X-ray study; T = 293 K; mean (C–C) = 0.005 A˚;
R factor = 0.071; wR factor = 0.201; data-to-parameter ratio = 14.3. The title compound, C10H15NO, is an amino alcohol with the
hydroxy group residing on the terminal C atom. Apart from
the hydroxy group and the phenyl ring, all non-H atoms are
almost coplanar. In the crystal, classical O—H N and N—
H O hydrogen bonds connect the molecules into centro-
symmetric dimers [R2
2(12) descriptor] and tetrameric units
[R4
4(8) descriptor] as ring motifs, consolidating a three-
dimensional network. Data collection:
XSCANS (Bruker, 1996); cell refinement:
XSCANS; data reduction: XSCANS; program(s) used to solve
structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine
structure: SHELXL97 (Sheldrick, 2008); molecular graphics: XP in
SHELXTL-Plus (Sheldrick, 2008); software used to prepare material
for publication: XP in SHELXTL-Plus. We thank
the German Academic
Exchange
Service
(DAAD) for PhD scholarship to MMI. Related literature For the syntheses of amino alcohols from isoxazolidines,
isoxazolines and isoxazolinium salts, see: DeShong & Leginus,
(1983); Hennebo¨hle et al. (2004); Ibrahim (2009); Ja¨ger &
Buss, (1980); Ja¨ger et al. (1985, 2010); Ja¨ger & Colinas (2002);
Lubell et al. (1991). For hydrogen-bond motifs see: Bernstein
et al. (1995). For standard bond lengths, see: Allen et al. (1987). Supplementary data and figures for this paper are available from the
IUCr electronic archives (Reference: IM2389). organic compounds = 0.56 mm1
T = 293 K Acta Crystallographica Section E Acta Crystallographica Section E 0.80 0.50 0.20 mm Acta Crystallographica Section E
Structure Reports
Online
ISSN 1600-5368 Data collection
Siemens P4 diffractometer
3535 measured reflections
1704 independent reflections
896 reflections with I > 2(I)
Rint = 0.087
3 standard reflections every 100
reflections
intensity decay: 3%
Refinement
R[F 2 > 2(F 2)] = 0.071
wR(F 2) = 0.201
S = 1.04
1704 reflections
119 parameters
H atoms treated by a mixture of
independent and constrained
refinement
max = 0.22 e A˚ 3
min = 0.22 e A˚ 3 Data collection
Siemens P4 diffractometer
3535 measured reflections
1704 independent reflections
896 reflections with I > 2(I) ISSN 1600-5368 Rint = 0.087
3 standard reflections every 100
reflections
intensity decay: 3% Wolfgang Frey,a Mohammad M. Ibrahim,b Basem F. Alib
and Volker Ja¨gera* H atoms treated by a mixture of
independent and constrained
refinement
max = 0.22 e A˚ 3
min = 0.22 e A˚ 3 aUniversita¨t Stuttgart, Institut fu¨r Organische Chemie, Pfaffenwaldring 55, D-70569
Stuttgart, Germany, and bDepartment of Chemistry, Al al-Bayt University, Mafraq
25113, Jordan Correspondence e-mail: jager.ioc@oc.uni-stuttgart.de Table 1
Hydrogen-bond geometry (A˚ , ). Comment Isoxazolidines, isoxazolines, and isoxazolinium salts are useful intermediates for syntheses of 1,3-amino alcohols by
reduction with cleavage of the N–O bond (DeShong & Leginus, 1983; Jäger & Buss, 1980; Jäger et al., 1985; Jäger &
Colinas, 2002; Henneböhle et al., 2004; Jäger et al., 2010). The structures and conformations of previously synthesized
amino alcohols were all assigned on the basis of analytical as well as IR, 13C and 1H NMR data. When the 2-methyl-3-
phenylisoxazolidine-3-carbonitrile was heated to reflux with lithium aluminium hydride in ether (abs.), the title
compound I was formed in good yield. The starting isoxazolidine had been obtained from the corresponding N-methyl-
isoxazolinium salt by cyanide addition (Henneböhle et al., 2004; Ibrahim, 2009; Jäger et al. 2010). The formation of the
amino alcohol I was rationalized elsewhere (Ibrahim, 2009). The title compound I is already known from other routes
(Lubell et al., 1991), yet, the crystal structure of I has not been published so far. We herein report the synthesis and the
crystal structure of I, along with the supramolecular motifs present in the crystal lattice. The asymmetric unit of I consists of one amino alcohol molecule with bond distances and angles in the normal range
(Allen et al., 1987). The molecule, a primary alcohol and a secondary amine, adopts a planar zigzag-chain conformation
(C1/C2/C3/N1/C4 almost coplanar), with both the hydroxy and the phenyl group being out-of-plane. The hydroxy and the
phenyl group enclose dihedral angles of -60.3 (4)° and -63.0 (3)°, respectively, with the atoms of the carbon-chain
(hydroxyl-O1/phenyl-C5-C1-C2-C3), see Fig. 1. In the crystal structure, molecules are hydrogen-bonded through the hy-
droxy groups as well as the amino groups (Table 1) giving rise to a three-dimensional network. The cooperative hydrogen
bonds (alternating between hydroxy and amino groups) connect the molecules into chains down the crystallographic a
axis (Fig. 2). These chains consist of alternating centrosymmetric dimers, with each dimer further interacting through the
hydroxyl and amino groups with the adjacent one to form tetrameric units (Fig. 2). In terms of graph-set description, the
hydrogen-bonded molecules might be described as forming two types of rings (Bernstein et al., 1995), the
centrosymmetric dimers being R22(12) while R44(8) represents the descriptor for the tetramer units. These interactions
consolidate a three-dimensional network. 3-Methylamino-3-phenylpropan-1-ol Wolfgang Frey, Mohammad M. Ibrahim, Basem F. Ali and Volker Jäger Comment This amino alcohol conformation in the crystal found here is in contrast to the conformations elucidated in solution on
the basis of IR dilution experiments and extensive collections of 13C and 1H NMR data, notably coupling constants and
substituent increments - there intramolecular hydrogen bonds O—H···N prevail to form monomers with chair-like
arrangements (Jäger & Buss, 1980). Acta Cryst. (2012). E68, o2857 [doi:10.1107/S160053681203694X] Acta Cryst. (2012). E68, o2857 [doi:10.1107/S160053681203694X] References o2857 Acta Cryst. (2012). E68, o2857 doi:10.1107/S160053681203694X supplementary materials Refinement Hydrogen atoms were located from the difference fourier map, but refined with fixed individual displacement parameters,
using a riding model with d(C—H) ranging from 0.93 to 0.98 Å and Uiso(H) = 1.2 Ueq(C) or Uiso(H) = 1.5 Ueq(Cmethyl). In
addition, the methyl group is allowed to rotate but not to tip. Hydrogen atoms attached to the hydroxy function and to the
amino moiety are refined freely because of their relevance in hydrogen bonding. supplementary materials H2O (1.5 ml) were added sequentially. The reaction mixture was extracted with ether (4 x 15 ml) and CH2Cl2 (2 x 10 ml). The organic layers were dried over Na2SO4. The solvent was evaporated in vacuo (5 mbar, 40°C) to afford the amino
alcohol I in analytically and spectroscopically pure form as a colorless solid [100 mg, 84%, m.p. 56–57°C; lit. 56–57°C
(Lubell et al., 1991)]. Crystallization of the solid from ether afforded colorless crystals suitable for crystal structure
determination. Analysis for C10H15NO, Calc.: C 72.69, H 9.15, N 8.48; Found: C 72.39, H 9.11, N 8.07. Computing details Data collection: XSCANS (Bruker, 1996); cell refinement: XSCANS (Bruker, 1996); data reduction: XSCANS (Bruker,
1996); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97
(Sheldrick, 2008); molecular graphics: XP in SHELXTL-Plus (Sheldrick, 2008); software used to prepare material for
publication: XP in SHELXTL-Plus (Sheldrick, 2008). publication: XP in SHELXTL-Plus (Sheldrick, 2008). Figure 1
The molecular structure of I, with thermal ellipsoids drawn at the 30% probability level. Experimental A solution of 2-methyl-3-phenylisoxazolidine-3-carbonitrile (150 mg, 0.80 mmol) in anhydrous diethylether (5 ml) at 0°C
was added to a suspension of LiAlH4 (61.0 mg, 1.6 mmol, 2 eq) in anhydrous diethylether (15 ml) and stirred for 30 min. The mixture was allowed to warm up to room temperature and stirred for 1 h. The reaction mixture was then heated to
reflux for 12 h. The solution was cooled to r.t., then at 0°C with vigorous stirring. H2O (0.5 ml), 5% NaOH (0.5 ml), and Acta Cryst. (2012). E68, o2857 sup-1 supplementary materials Figure 2 g
A view down c axis showing chains of hydrogen bonding molecules along a axis. The chains consist of alternating
centrosymmetric dimers, with each dimer further interacting through the hydroxyl and amino groups with the adjacent
one to form tetrameric units. A view down c axis showing chains of hydrogen bonding molecules along a axis. The chains consist of alternating
centrosymmetric dimers, with each dimer further interacting through the hydroxyl and amino groups with the adjacent
one to form tetrameric units. Figure 1 gu e
The molecular structure of I, with thermal ellipsoids drawn at the 30% probability level. g
The molecular structure of I, with thermal ellipsoids drawn at the 30% probability level. The molecular structure of I, with thermal ellipsoids drawn at the 30% probability level. sup-2 Acta Cryst. (2012). E68, o2857 supplementary materials Figure 2
A view down c axis showing chains of hydrogen bonding molecules along a axis. The chains consist of alternating
centrosymmetric dimers with each dimer further interacting through the hydroxyl and amino groups with the adjacent Figure 2
A view down c axis showing chains of hydrogen bonding molecules along a axis. The chains consist of alternating 3-Methylamino-3-phenylpropan-1-ol sup-3
Acta Cryst. (2012). E68, o2857
Crystal data
C10H15NO
Mr = 165.23
Monoclinic, P21/n
Hall symbol: -P 2yn
a = 5.9816 (8) Å
b = 23.8962 (19) Å
c = 7.4653 (8) Å
β = 111.119 (7)°
V = 995.40 (19) Å3
Z = 4
F(000) = 360
Dx = 1.103 Mg m−3
Cu Kα radiation, λ = 1.54178 Å
Cell parameters from 30 reflections Crystal data
C10H15NO
Mr = 165.23
Monoclinic, P21/n
Hall symbol: -P 2yn
a = 5.9816 (8) Å
b = 23.8962 (19) Å
c = 7.4653 (8) Å sup-3 Acta Cryst. (2012). E68, o2857 supplementary materials θ = 21.0–22.5°
µ = 0.56 mm−1
T = 293 K
Data collection
Siemens P4
diffractometer
Radiation source: fine-focus sealed tube
Graphite monochromator
ω scans
3535 measured reflections
1704 independent reflections
896 reflections with I > 2σ(I)
Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.071
wR(F2) = 0.201
S = 1.04
1704 reflections
119 parameters
0 restraints
Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map θ = 21.0–22.5°
µ = 0.56 mm−1
T = 293 K
Data collection
Siemens P4
diffractometer
Radiation source: fine-focus sealed tube
Graphite monochromator
ω scans
3535 measured reflections
1704 independent reflections
896 reflections with I > 2σ(I)
Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.071
wR(F2) = 0.201
S = 1.04
1704 reflections
119 parameters
0 restraints
Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map Block, colourless
0.80 × 0.50 × 0.20 mm Rint = 0.087
θmax = 67.5°, θmin = 3.7°
h = −7→7
k = −28→28
l = −8→8 3 standard reflections every 100 reflections
intensity decay: 3% 3 standard reflections every 100 reflections
intensity decay: 3% Hydrogen site location: inferred from
neighbouring sites
H atoms treated by a mixture of independent
and constrained refinement
w = 1/[σ2(Fo2) + (0.0232P)2 + 0.9564P]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max < 0.001
Δρmax = 0.22 e Å−3
Δρmin = −0.22 e Å−3
Extinction correction: SHELXL97 (Sheldrick,
2008), Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
Extinction coefficient: 0.026 (2) Extinction correction: SHELXL97 (Sheldrick,
2008), Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
Extinction coefficient: 0.026 (2) Special details Experimental. 1H NMR (500.2 MHz, MeOD): d = 1.82 (dddd, 3J1a,2a = 7.9 Hz, 3J1b,2a = 5.6 Hz, 2J2a,2b = 14.0 Hz, 3J2a,3 = 5.2
Hz, 1 H, 2-Ha), 2.06 (dddd, 3J1a,2b = 6.2 Hz, 3J1b,2b = 7.5 Hz, 2J2a,2b = 14.2 Hz, 3J2b,3 = 5.8 Hz, 1 H, 2-Hb), 2.18 (s, 3 H,
NCH3), 3.42 (ddd, 2J1a,1b = 10.8 Hz, 3J1a,2a = 8.1 Hz, 3J1a,2b = 6.1 Hz, 1 H, 1-Ha), 3.49 (ddd, 2J1a,1b = 8.0 Hz, 3J1b,2a = 5.0 Hz,
3J1b,2b = 6.8 Hz, 1 H, 1-Hb), 3.65 (dd, 3J2a,3 = 8.3 Hz, 3J2b,3 = 6.0 Hz, 1 H, 3-H), 7.23-7.35 (m, 5 H, C6H5); 13C NMR (125.8
MHz, MeOD): d = 34.0 (q, NCH3), 40.7 (t, C-2), 60.4 (t, C-1), 63.7 (d, C-3), 128.2, 128.4, 129.6 (3 d, o-, m-, p-C of
C6H5), 143.8 (s, i-C of C6H5). Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full
covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and
torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2,
conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used
only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2
are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. sup-4
Acta Cryst. (2012). Special details E68, o2857
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq
O1
0.6223 (4)
0.02730 (9)
0.2754 (3)
0.0633 (7)
H1A
0.622 (7)
−0.0130 (18)
0.351 (6)
0.111 (14)*
N1
1.3595 (5)
0.07300 (10)
0.5221 (4)
0.0610 (8)
H1B
1.458 (6)
0.0688 (12)
0.436 (5)
0.063 (9)*
C1
0.7636 (6)
0.02462 (14)
0.1583 (5)
0.0639 (9)
H1C
0.7208
0.0555
0.0681
0.077*
H1D
0.7277
−0.0099
0.0849
0.077* onal atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq sup-4 Acta Cryst. (2012). Acta Cryst. (2012). E68, o2857 Special details E68, o2857 supplementary materials C2
1.0300 (5)
0.02701 (12)
0.2739 (4)
0.0564 (8)
H2A
1.1168
0.0238
0.1868
0.068*
H2B
1.0732
−0.0049
0.3599
0.068*
C3
1.1102 (5)
0.08083 (12)
0.3929 (4)
0.0565 (8)
H3
1.0121
0.0848
0.4727
0.068*
C4
1.4610 (8)
0.11951 (15)
0.6516 (6)
0.0922 (14)
H4A
1.4654
0.1522
0.5782
0.138*
H4B
1.6207
0.1102
0.7351
0.138*
H4C
1.3637
0.1268
0.7270
0.138*
C5
1.0643 (6)
0.13176 (12)
0.2623 (5)
0.0603 (9)
C6
0.8826 (7)
0.16881 (14)
0.2513 (6)
0.0790 (11)
H6
0.7948
0.1640
0.3307
0.095*
C7
0.8291 (8)
0.21376 (16)
0.1211 (7)
0.0936 (14)
H7
0.7072
0.2387
0.1152
0.112*
C8
0.9553 (9)
0.22067 (16)
0.0047 (7)
0.0925 (14)
H8
0.9167
0.2496
−0.0844
0.111*
C9
1.1390 (8)
0.18531 (15)
0.0176 (6)
0.0851 (12)
H9
1.2292
0.1911
−0.0595
0.102*
C10
1.1927 (7)
0.14055 (14)
0.1451 (5)
0.0737 (10)
H10
1.3169
0.1163
0.1511
0.088*
Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
O1
0.0526 (13)
0.0690 (14)
0.0665 (15)
0.0047 (10)
0.0194 (11)
0.0030 (11)
N1
0.0552 (15)
0.0620 (16)
0.0583 (17)
−0.0054 (12)
0.0114 (14)
−0.0037 (12)
C1
0.0563 (18)
0.075 (2)
0.0562 (19)
−0.0061 (15)
0.0158 (16)
−0.0016 (15)
C2
0.0557 (17)
0.0585 (17)
0.0554 (18)
−0.0043 (13)
0.0206 (15)
−0.0044 (13)
C3
0.0547 (17)
0.0579 (17)
0.0563 (19)
0.0004 (13)
0.0193 (16)
−0.0015 (13)
C4
0.101 (3)
0.071 (2)
0.079 (3)
−0.015 (2)
0.001 (2)
−0.0142 (19)
C5
0.0563 (18)
0.0562 (17)
0.063 (2)
0.0016 (14)
0.0145 (16)
0.0007 (14)
C6
0.078 (2)
0.066 (2)
0.092 (3)
0.0102 (17)
0.030 (2)
−0.0017 (19)
C7
0.090 (3)
0.065 (2)
0.109 (4)
0.021 (2)
0.016 (3)
0.005 (2)
C8
0.114 (3)
0.067 (2)
0.082 (3)
0.009 (2)
0.019 (3)
0.011 (2)
C9
0.106 (3)
0.071 (2)
0.084 (3)
−0.001 (2)
0.040 (3)
0.0100 (19)
C10
0.082 (2)
0.068 (2)
0.073 (2)
0.0038 (17)
0.031 (2)
0.0077 (17)
Geometric parameters (Å, º)
O1—C1
1.420 (4)
C4—H4B
0.9600
O1—H1A
1.12 (4)
C4—H4C
0.9600
N1—C4
1.454 (4)
C5—C10
1.373 (5)
N1—C3
1.468 (4)
C5—C6
1.381 (4)
N1—H1B
1.02 (4)
C6—C7
1.406 (5)
C1—C2
1.516 (4)
C6—H6
0.9300
C1—H1C
0.9700
C7—C8
1.351 (6)
C1—H1D
0.9700
C7—H7
0.9300
C2—C3
1.538 (4)
C8—C9
1.362 (6) C2
1.0300 (5)
0.02701 (12)
0.2739 (4)
0.0564 (8)
H2A
1.1168
0.0238
0.1868
0.068*
H2B
1.0732
−0.0049
0.3599
0.068*
C3
1.1102 (5)
0.08083 (12)
0.3929 (4)
0.0565 (8)
H3
1.0121
0.0848
0.4727
0.068*
C4
1.4610 (8)
0.11951 (15)
0.6516 (6)
0.0922 (14)
H4A
1.4654
0.1522
0.5782
0.138*
H4B
1.6207
0.1102
0.7351
0.138*
H4C
1.3637
0.1268
0.7270
0.138*
C5
1.0643 (6)
0.13176 (12)
0.2623 (5)
0.0603 (9)
C6
0.8826 (7)
0.16881 (14)
0.2513 (6)
0.0790 (11)
H6
0.7948
0.1640
0.3307
0.095*
C7
0.8291 (8)
0.21376 (16)
0.1211 (7)
0.0936 (14)
H7
0.7072
0.2387
0.1152
0.112*
C8
0.9553 (9)
0.22067 (16)
0.0047 (7)
0.0925 (14)
H8
0.9167
0.2496
−0.0844
0.111*
C9
1.1390 (8)
0.18531 (15)
0.0176 (6)
0.0851 (12)
H9
1.2292
0.1911
−0.0595
0.102*
C10
1.1927 (7)
0.14055 (14)
0.1451 (5)
0.0737 (10)
H10
1.3169
0.1163
0.1511
0.088* Atomic displacement parameters (Å2) sup-5 Acta Cryst. Symmetry codes: (i) x+1, y, z; (ii) −x+2, −y, −z+1. Special details (2012). E68, o2857 supplementary materials C3—C5
1.521 (4)
C9—H9
0.9300
C3—H3
0.9800
C10—H10
0.9300
C4—H4A
0.9600
C1—O1—H1A
112 (2)
N1—C4—H4B
109.5
C4—N1—C3
114.9 (3)
H4A—C4—H4B
109.5
C4—N1—H1B
107.1 (17)
N1—C4—H4C
109.5
C3—N1—H1B
106.3 (18)
H4A—C4—H4C
109.5
O1—C1—C2
112.6 (3)
H4B—C4—H4C
109.5
O1—C1—H1C
109.1
C10—C5—C6
118.2 (3)
C2—C1—H1C
109.1
C10—C5—C3
121.2 (3)
O1—C1—H1D
109.1
C6—C5—C3
120.5 (3)
C2—C1—H1D
109.1
C5—C6—C7
120.6 (4)
H1C—C1—H1D
107.8
C5—C6—H6
119.7
C1—C2—C3
113.9 (3)
C7—C6—H6
119.7
C1—C2—H2A
108.8
C8—C7—C6
119.8 (4)
C3—C2—H2A
108.8
C8—C7—H7
120.1
C1—C2—H2B
108.8
C6—C7—H7
120.1
C3—C2—H2B
108.8
C7—C8—C9
120.2 (4)
H2A—C2—H2B
107.7
C7—C8—H8
119.9
N1—C3—C5
115.4 (2)
C9—C8—H8
119.9
N1—C3—C2
107.7 (2)
C8—C9—C10
120.5 (4)
C5—C3—C2
110.6 (2)
C8—C9—H9
119.8
N1—C3—H3
107.6
C10—C9—H9
119.8
C5—C3—H3
107.6
C5—C10—C9
120.7 (4)
C2—C3—H3
107.6
C5—C10—H10
119.7
N1—C4—H4A
109.5
C9—C10—H10
119.7
O1—C1—C2—C3
−60.3 (4)
C10—C5—C6—C7
0.9 (5)
C4—N1—C3—C5
58.3 (4)
C3—C5—C6—C7
−175.6 (3)
C4—N1—C3—C2
−177.6 (3)
C5—C6—C7—C8
0.4 (6)
C1—C2—C3—N1
169.9 (3)
C6—C7—C8—C9
−2.1 (7)
C1—C2—C3—C5
−63.0 (3)
C7—C8—C9—C10
2.5 (7)
N1—C3—C5—C10
54.1 (4)
C6—C5—C10—C9
−0.6 (5)
C2—C3—C5—C10
−68.5 (4)
C3—C5—C10—C9
175.9 (3)
N1—C3—C5—C6
−129.5 (3)
C8—C9—C10—C5
−1.1 (6)
C2—C3—C5—C6
107.9 (3)
Hydrogen-bond geometry (Å, º)
D—H···A
D—H
H···A
D···A
D—H···A
N1—H1B···O1i
1.02 (4)
2.06 (3)
3.023 (4)
157 (2)
O1—H1A···N1ii
1.12 (4)
1.70 (4)
2.815 (3)
176 (3)
Symmetry codes: (i) x+1, y, z; (ii) −x+2, −y, −z+1. sup-6 Acta Cryst. (2012). E68, o2857
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https://openalex.org/W4362378636
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https://figshare.com/articles/journal_contribution/Figure_S4_from_Long_Noncoding_RNA_DRAIC_Inhibits_Prostate_Cancer_Progression_by_Interacting_with_IKK_to_Inhibit_NF-_B_Activation/22424473/1/files/39870730.pdf
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Greek, Modern
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Figure S2 from Long Noncoding RNA DRAIC Inhibits Prostate Cancer Progression by Interacting with IKK to Inhibit NF-κB Activation
| null | 2,023
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cc-by
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KO1+FL
KO1+E1-3
KO1+E4-5
701-1705
900-1705
1100-1705
701-1105
701-905
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DRAIC KO
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NF-κB ReporterActivity
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2
3
4
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6
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E4-5
EV
Ca-IKKβ
(S177E/S181E)
-
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* F Figure S4 Figure S4
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Directed content analysis: A life course approach to understanding the impacts of the COVID-19 pandemic with implications for public health and social service policy
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PLOS ONE RESEARCH ARTICLE Directed content analysis: A life course
approach to understanding the impacts of the
COVID-19 pandemic with implications for
public health and social service policy Eva PurkeyID1,2*, Imaan Bayoumi1,2, Colleen M. DavisonID2, Autumn Watson1,3 1 Queen’s University Department of Family Medicine, Kingston, Ontario, Canada, 2 Queen’s University
Public Health Sciences, Kingston, Ontario, Canada, 3 Indigenous Diabetes Health Circle, Curve Lake First
Nation, Ontario, Canada a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * eva.purkey@queensu.ca * eva.purkey@queensu.ca * eva.purkey@queensu.ca OPEN ACCESS The COVID-19 pandemic has had broad impacts on individuals, families and communities
which will continue to require multidimensional responses from service providers, program
developers, and policy makers. Citation: Purkey E, Bayoumi I, Davison CM,
Watson A (2022) Directed content analysis: A life
course approach to understanding the impacts of
the COVID-19 pandemic with implications for
public health and social service policy. PLoS ONE
17(12): e0278240. https://doi.org/10.1371/journal. pone.0278240 Objectives The purpose of this study was to use Life Course theory to understand and imagine public
health and policy responses to the multiple and varied impacts of the COVID-19 pandemic
on different groups. Editor: Charlotte Lennox, The University of
Manchester Division of Psychology and Mental
Health, UNITED KINGDOM Editor: Charlotte Lennox, The University of
Manchester Division of Psychology and Mental
Health, UNITED KINGDOM
Received: June 14, 2022
Accepted: November 14, 2022
Published: December 16, 2022 Editor: Charlotte Lennox, The University of
Manchester Division of Psychology and Mental
Health, UNITED KINGDOM Methods “The Cost of COVID-19” was a research study carried out in Kingston, Frontenac, Lennox
and Addington counties in South Eastern Ontario, Canada, between June and December
2020. Data included 210 micronarrative stories collected from community members, and 31
in-depth interviews with health and social service providers. Data were analyzed using
directed content analysis to explore the fit between data and the constructs of Life Course
theory. Copyright: © 2022 Purkey 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. PLOS ONE PLOS ONE Background The COVID-19 pandemic has affected individuals, families, communities and countries. Rela-
tionships within and between countries have been altered as illustrated by restrictions on inter-
national travel, changes to foreign aid, and varying degrees of international collaboration on
disease surveillance and vaccine development and distribution. Public and private institutions
(health and social service agencies, the education system, among others) have had to change
and adapt dramatically. Communities have had to reconfigure how members interact, how
they support each other, and the care they provide to the most vulnerable. Families and com-
munities have been divided by geographic distance and in some cases by ideology. Individuals
have seen fundamental changes to their ability to socialize, to work, to attend school, and to
feel safe in their environment [1–4]. While some Canadian political and public health messag-
ing has emphasized that we are “all in this together”, this has in fact not been the case, with
very different impacts being felt by different groups, varying by socioeconomic status, gender,
race, geography, ability, political inclination or age among others [3]. Differences in the experi-
ence of the pandemic include disproportionate infection rates as well as mortality in neighbor-
hoods with high proportions of racialized people, as well as those with material deprivation
and low income [5,6]. Other differences include the disproportionate impact of the restriction
in services on people living with intellectual and developmental disabilities [7]. The breadth of the impact of the COVID-19 pandemic is unlike anything seen in a genera-
tion. The disruption it has caused to all aspects of individual, family, community and social life
and development echo the impacts of Spanish Influenza in 1918, including the politicization of
a public health response, disproportionate impact on minority groups, and scapegoating of
certain groups with respect to origin and spread of the disease [8], however important differ-
ences between these pandemics include the speed of global communication, and the scientific
response with respect to vaccination and supportive medical care. Many academics and policy
makers have spoken about the impacts of secondary pandemics in the context of the COVID-
19 pandemic, for instance pandemics of child abuse and neglect, of mental health disorders, of
loneliness and isolation, of widening gaps in education and others [1,2,4,9]. Recovery from the
ongoing acute, infectious pandemic, requires imagining a way forward through the pervasive
impacts of COVID-19, while avoiding simplistic or reductionist thinking and policy making. Data Availability Statement: All data files are
available in Queen’s dataverse: https://doi.org/10.
5683/SP3/YQS0PR. Data Availability Statement: All data files are
available in Queen’s dataverse: https://doi.org/10. 5683/SP3/YQS0PR. Social pathways were significantly disrupted by changes to education and employment, as
well as changes to roles which further altered anticipated pathways. Transitions were by
and large missed, creating a sense of loss. While some respondents articulated positive
turning points, most of the turning points reported were negative, including fundamental
changes to relationships, family structure, education, and employment with lifelong implica-
tions. Participants’ trajectories varied based on principles including when they occurred in
their lifespan, the amount of agency they felt or did not feel over circumstances, where they Funding: This study was funded through two
sources: PSI Health Research Grant, as well as an
internal grant through Queen’s University SARS-
Co-V2 Rapid Research Response Grant. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of 1 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 PLOS ONE Life course approach to the COVID-19 pandemic lived (rural versus urban), what else was going on in their lives at the time the pandemic
struck, how their lives were connected with others, as well as how the pandemic impacted
the lives of those dear to them. An additional principle, that of Culture, was felt to be missing
from the Life Course theory as currently outlined. lived (rural versus urban), what else was going on in their lives at the time the pandemic
struck, how their lives were connected with others, as well as how the pandemic impacted
the lives of those dear to them. An additional principle, that of Culture, was felt to be missing
from the Life Course theory as currently outlined. the manuscript. Funds from PSI Foundation were
used to pay for the salary of Autumn Watson,
research assistant and co-author. No other authors
received salary support from either of these grants. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. Conclusions A Life Course analysis may improve our understanding of the multidimensional long-term
impacts of the COVID-19 pandemic and associated public health countermeasures. This
analysis could help us to anticipate services that will require development, training, and
funding to support the recovery of those who have been particularly affected. Resources
needed will include education, mental health and job creation supports, as well as programs
that support the development of individual and community agency. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Life course theory Life course theory is a theoretical orientation pioneered by Glen H Elder Jr that situates people
within a historical moment in time and place, in the context of specific social institutions,
bound by certain normative social and cultural pathways [11]. While it takes into account age,
it goes beyond a lifespan approach to contextualize age cohorts in a specific historical and
social time and place. This theory has been used to understand large social changes such as the impacts of the
Great Depression [12] or the Chinese Cultural Revolution [13], as well as seemingly less sweep-
ingly transformative and more individual social phenomena such as criminality and teen preg-
nancy [14,15]. Because Life Course theory seeks to explore the individual, family, social and
cultural impacts of experiences from a complex and multifaceted perspective, this theory may
be helpful in understanding both the immediate, short term consequences of the pandemic, as
well as the long-term impacts that are likely to be experienced throughout the entire lives of
individuals and societies affected. Life Course theory is not familiar to many public health
practitioners, andoutlining the effects of the pandemic in this way can provide a novel and use-
ful paradigm with which to move forward towards pandemic recovery. The constructs of Life Course theory can be divided into three groups: Constructs related to
timing (cohort or period effects), those related to trajectories (social pathways, transitions, and
turning points), and then broader principles through which any impactful event can be exam-
ined (life span development, agency, time and place, timing, and linked lives). These constructs
are described below [11]. Timing constructs include the cohort effect and the period effect. The cohort effect is the
phenomenon by which certain historical events differentiate the lives of successive birth
cohorts (for example, children born during the great depression, or young men coming of age
during the Vietnam war), while a period effect arises when a particular event occurs over a lon-
ger time, producing relatively similar impact on a number of successive birth cohorts. This
paper considers the cohort effect as the primary construct underlying the experience of
COVID-19. The three constructs in the trajectories domain include social pathways, referring to
expected sequence of life stages and events. Background There is an opportunity to consider the interconnections, the width and breadth of the human PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 2 / 20 PLOS ONE Life course approach to the COVID-19 pandemic experience as societies creatively imagine what recovery will be. We believe that Life Course
theory can be useful when understanding the impacts of social upheaval, such as the COVID-
19 pandemic, particularly when these upheavals pervasively affect all or many components of
individual, family, social and cultural life [10]. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Life course theory These will vary depending on cultural influences,
and over time and place, but can be altered by historical forces that are outside the control of
the individual (recessions, differential funding for higher education, civil or political unrest). Transitions refer to changes in role that make up trajectories. Graduating from school, moving
out of the family home, starting a job, having a first child, and retirement, would all be transi-
tions, which happen at different chronological ages, and the timing of which will affect one’s
life course and trajectory. Finally, turning points are major events that produce a substantial
change in the direction of one’s life. All of these constructs can be viewed through the lens of five principles. (1) Life span devel-
opment is the principle by which we view individuals in a constant state of development. Development does not only happen to young children but is occurring at every point in the
life cycle. Any major historical event occurring at any point in the life of an individual, will
have an impact on their ongoing development including how they assess and build physical,
social, spiritual, and social abilities and goals (2). The principle of agency [16] articulates the PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 3 / 20 PLOS ONE Life course approach to the COVID-19 pandemic ways in which people are active in making choices that impact their life and life course. Agency
is a complex principle that includes concrete constructs such as self-efficacy, but also more
philosophical notions of freedom and free will. Fundamentally, it proposes that people con-
struct their own life course by making choices in the context of opportunities and constraints
presented to them [11]. (3) The principle of time and place proposes that life courses are
impacted by geographic and historical events. The same historical event will have different
impact in different geographic locations. (4) The principle of timing implies that an event will
have differential impact based on how it is timed in a person’s life. Family poverty and food
deprivation will be different when experienced by a toddler than when experienced by a
responsible adult caregiver. Methods
Setting The data used for this analysis is data from “The Cost of COVID-19” research study carried
out in Kingston, Frontenac, Lennox and Addington counties (KFL&A) in South Eastern
Ontario. The data for this analysis was collected between June and December 2020 and
includes two data sources described below. KFL&A is an area with a population of roughly
210,000 people [17] that was relatively spared during the first year of the pandemic, with only
758 cases of COVID-19 and a single death [18,19]. The region had a strong public health
response, with strong leadership and excellent communication strategies, and high levels of
compliance with public health guidelines. As elsewhere in Ontario, primary and secondary
schools were closed to in person learning from March 2020 to September 2020, and reopened
for masked, in person learning until the Christmas break in December 2020, at which point
there was a second period of school closures [20]. Following a strict restriction of gathering
size at the onset of the pandemic, people were allowed to gather in larger groups from June to
September, when gatherings were again restricted following a rise in cases and remained
tightly restricted into early 2021 [20]. An initial period of extensive closures, including of
indoor and outdoor play spaces, parks, and recreational activities, was followed by a gradual
and selective reopening of services by the summer of 2020 [20] but people were still being
encourage to avoid common spaces and to physically distance as much as possible. With
respect to economic support, between March and September 2020, the Federal government
implemented the Canada Emergency Response Benefit (CERB)–a benefit providing people
with 500$CDN per week for lost income caused by the pandemic [21]. This benefit required
people to have had a minimum income in 2019 to be eligible. Life course theory (5) Finally, the principle of linked lives refers to the fact that we do
not live our lives in isolation, and that our life courses are profoundly shaped by our social net-
works, our culture, and by the impact of events on the lives of the people around us. In using Life Course theory to explore the impact of the COVID-19 pandemic and public
health countermeasures, this paper seeks to illustrate the complexities of the impact of the pan-
demic on many aspects of people’s lives, as well as the implications these complex impacts will
have for health, social and economic policy and recovery. Spryng.io micronarratives The first data source was participant-offered short stories, or micronarratives, collected
between June 30th and November 3rd 2020 using the Spryng.io platform [19], a technology that
allows participants to type or upload an audio story using a laptop, tablet or phone in response
to one of three pre-programmed prompts (ex: “Please tell a story about the worst OR best
impact of COVID-19 on you and/or your household”). Participants then interpreted their own
story by plotting responses to a series of pre-defined questions, and finally to respond to cer-
tain demographic questions anonymously. These micronarratives and accompanying
responses were uploaded to a secure server and transcripts downloaded. In this study stories
ranged in length from an average of approximately 150 to 800 words, with most in the 250–
500 word range. Respondents to this portion of the study were adults 16 years of age or older. Sampling was in the first instance opportunistic: the online data collection tool was distributed
through individual networks, social media, and partner organizations. Second, intentional
sampling was performed with certain groups, namely urban Indigenous People, people living
in poverty, and people experiencing homelessness. The purpose of this intentional sampling
was to ensure representation of segments of our population who are often underrepresented in
such studies due to inability to access the study platforms (eg lack of technology or technologi-
cal literacy) and/or lack of trust in research processes, among others [23,24]. Recruitment con-
tinued until 200 participants had been involved. This number was chosen due to sampling
recommendations for quantitative analyses in Spryng.io work, which are not applicable to the
qualitative analysis completed here. Two research assistants actively recruited respondents out-
side an integrated care hub established in Kingston as a response to homelessness during the
COVID-19 crisis, as well as participants attending cultural events held by the local Indigenous
community. The research assistants also attended two local food programs serving people
experiencing food insecurity. Ethics This study was reviewed and approved by Queen’s University Health Sciences and Affiliated
Teaching Hospitals Research Ethics Board. All participants provided informed written or
recorded consent to participate in this study. All methods were performed in accordance with
the relevant guidelines and regulations. In addition to this ethics board, “The Cost of COVID-
19” study was planned and conducted with support of an oversight committee from the Indig-
enous Health Council to ensure that the design, data management, and dissemination of 4 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 PLOS ONE Life course approach to the COVID-19 pandemic findings were consistent with the principles of OCAP (Ownership, Control, Access and Pos-
session) [22]. AW (author) was hired as an Indigenous Research Assistant to support commu-
nication with the oversight committee and outreach to the Indigenous Community. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Key informant interviews The second data source used for this analysis was in-depth interviews with key informants
recruited from organizations providing healthcare and/or a variety of social services, including
child welfare services, school support, sexual assault and domestic violence services, and men-
tal health services among others. These interviews served to triangulate the responses from
individual first-person micronarratives by asking interview participants to reflect more
broadly on the experiences of individuals in the groups they served as well as within their own
organization. Participants from organizations providing services to Indigenous People and
other marginalized groups often excluded from research were intentionally recruited for the
reasons mentioned above. Sampling of key informants was intentional. Participants were
recruited by phone or email. Questions included populations served and services provided, as
well as questions in five main themes: (1) impact of the pandemic on mental health; (2) impact
of the pandemic on family and partner conflict; (3) impact on access to services; (4) service
adaptations; and (5) ideas for future adaptations. Participants were provided space and time
for open ended answers, and to add anything they felt was important and not covered by the
questionnaire. Interviews were completed by trained Research Assistants via Zoom to adhere
to public health guidelines, audio recorded, and transcribed verbatim. Interviews were com-
pleted between October and December 2020 and participants were recruited until saturation PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 5 / 20 PLOS ONE Life course approach to the COVID-19 pandemic of themes was reached. Interviews ranged from approximately 45 minutes to 90 minutes in
duration. While these interviews concluded slightly later than the micronarrative collection,
the local pandemic situation (infection rates, public health countermeasures) was essentially
the same throughout that time. Data analysis Data was analyzed using directed content analysis. In directed content analysis, a framework
with key concepts or constructs is identified to create initial coding categories. These must be
clearly defined (as has been outlined above for Life Course constructs). Transcripts are read
and coded to these existing codes, with text that cannot be categorized into the existing coding
framework is given new codes, if appropriate, or assigned to sub-categories of existing codes. In this way, previous theoretical frameworks can be tested against new data, and possibly
expanded and strengthened. Likewise, new data can be understood through the lens of existing
theoretical frameworks, which can help expand the use and actionability of findings [25,26]. For the purposes of this analysis, transcripts were reviewed and coded to the core Life Course
theory constructs [11,25,26]. Both sets of transcripts were reviewed in their entirety by four researchers on the research
team (EP, CD, AW, IB) to identify emerging themes that warranted further investigation and
analysis. EP is a qualitative researcher and clinician whose teaching, clinical care and research
focus on equity deserving groups and the impact of trauma and adversity on health. CD is a
public health scientist and mixed methods researcher whose research interests include chil-
dren and youth. AW is a community based Indigenous researcher. IB is a predominantly
quantitative researcher and clinician whose research focuses on child health. Emerging themes
included complex experiences of COVID-19 and public health countermeasures that appeared
to far outweigh the impact of the virus. Life Course theory was identified as a theory with suffi-
cient complexity to illustrate many of the impacts of the COVID-19 pandemic and associated
countermeasures. One researcher (EP) used Directed Content Analysis [26,27] to code the
entire data set using the eight Life Course constructs outlined above as primary categories or
codes. Data that could not fit well into existing Life Course constructs was coded separately. Following the initial coding by EP, the analysis was brought back to the three other researchers
(IB, CD, AW) who, having already read the transcripts in detail, reviewed the coding process
and themes for reliability and consistency. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Demographics Table 1 provides information on key characteristics of the respondents for the Spryng.io
micronarratives. While the majority of respondents were female, there was a broad age range
and income distribution. Participants were given the opportunity to self-identify as Indige-
nous. Other specific ethnic backgrounds were not identified. Thirty one service providers or volunteers participated in the in-depth interviews (see
Table 2), including fifteen who identified their primary target population as Indigenous Peo-
ples. Sixteen of the organizations had a more general mandate. The organizations provided a
broad variety of services, including primary care, mental health for children and adults, sexual
assault and domestic violence services, shelter services, child and family services, school sup-
ports, and supports for people living with disabilities, among others. Respondents were evenly
split between service providers and administrators and ranged in experience from 6 months to
20 years with their organization. Some interview participants had held many roles within their
organization and community. Organizations provided a variety of types of service delivery PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 6 / 20 PLOS ONE Life course approach to the COVID-19 pandemic Table 1. Description of the sample providing Spryng.io micronarratives and sociodemographic information. Variable
Category
n = 210 (%)
Gender
Female:
Male:
Other/prefer not to answer:
158 (75)
47 (22)
5 (2)
Age
16–21:
22–34:
35–49:
50–64:
65–79:
Older/prefer not to answer:
12 (6)
57 (27)
55 (26)
59 (28)
23 (10)
4 (2)
Income
10,000–39,000:
40,000–79,999:
80,000–124,999:
>125,000
Prefer not to say:
62 (30)
41 (20)
47 (22)
32 (15)
28 (13)
Ethnicity/Race
Indigenous
Visible Minority (Other)
28 (13)
3 (1.4)
Where do you live
KLF&A:
Ontario (other):
Outside of Ontario:
176 (84)
28 (13)
6 (3)
https://doi.org/10.1371/journal.pone.0278240.t001 https://doi.org/10.1371/journal.pone.0278240.t001 (phone, drop in, in person appointments, groups, home visits, etc) and significant adaptations
had been made to all services since the start of the pandemic. Due to the qualitative nature of this analysis, inter-group differences were not specifically
quantified or compared, however the themes that emerged were consistent between the first-
person micronarratives and the in-depth service provider interviews. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Trajectories Social pathways. Respondents illustrated ways in which their social pathways have been
disrupted since the onset of the pandemic. People’s trajectories were actively disrupted by the
public health measures put in place in response to the pandemic, including disruptions to edu-
cation or employment, such as delaying entry to university, disengaging from an academic
program partway, or to being unable to find work. I am on [Ontario Disability Support Program]. I want to get a job but so many businesses
have closed down and they’re not willing to start businesses due to worry about COVID. Been
on [Ontario Disability Support Program] since August. I’d love to go back to work but I can’t. (Spryng 5654) My summer job was cancelled, and despite applying at several other places, I am unemployed. I have had a job since grade 10 of high school, working all summer every summer. Being home
has made me feel very lonely. (Spryng 4051) I also have several [First Nations, Inuit, Metis] students who have moved from their home ter-
ritories thinking that they are going to be doing school in-person. So I have folks from like
northern communities um and from like not Ontario. And they came here, thinking that they
would be in a cohort. And now they’re sitting by themselves, in an apartment, in the south,
and they don’t know anyone. (6A) There were also significant disruptions to roles, such as students having to become caregiv-
ers, and parents who felt they were no longer able to effectively provide for their children due
to new poverty or food insecurity. And uh so what became really evident is how many children in the middle class of the demo-
graphic were accessing that fresh fruit and vegetables through the Food Sharing Program and
through [program]. So now we have a whole section of our community that has not had to access Food Banks, has
not had to access support from Salvation Army, has not had to go to churches, didn’t know
the network to connect themselves to bridge that gap that they were experiencing in their own
reality of what this pandemic looked like. (9A) That’s how people felt, is like, I’m failing as parent. [. . .] People were afraid they were going to
lose their children to Children’s Aid because they didn’t have food to feed [them]. Directed content analysis: Life course theoretical framework The majority of data fit meaningfully into the constructs of the Life Course Framework. Sec-
ondary codes under each construct included whether the experience reported by the partici-
pant with positive or negative with respect to this construct. Most of the data reported negative
impacts of the pandemic on the constructs in question, however these variances are discussed
below. The only theme in our data that did not meaningfully fit into the constructs of Life Table 2. Description of the sample of service providers who participated in the in-depth interviews. Variable
Category
Number N = 31 (%)
Target population
Urban (exclusively)
Rural (exclusively)
Both
8 (26)
0 (0)
23 (74)
Service sector
Health care services
Housing/Shelter
Education
Social services/other
9 (29)
2 (6)
3 (10)
17 (55)
Role in organizations
Admin/management:
Direct service provision:
Volunteer:
12 (39)
14 (45)
5 (16)
Length of time in organization
<2 yrs
2–5 yrs
5–10 yrs
>10 yrs
4 (13)
12 (39)
6 (19)
9 (29)
note that there is overlap between sectors; organizations have been categorized based on primary mandate. https://doi.org/10.1371/journal.pone.0278240.t002 Table 2. Description of the sample of service providers who participated in the in-depth interviews. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 7 / 20 PLOS ONE Life course approach to the COVID-19 pandemic Course Framework was related to the impact of culture on Life Course. This was particularly
salient in our findings due to our sampling of Indigenous people. While many participant
quotes related to culture could fit under other Life Course constructs such as transitions, there
seemed to be an overall theme of importance of culture that was inadequately addressed by
existing constructs and such an additional theme was developed. Pregnancy can be very lonely even if you are surrounded by the most supportive husband and
family—and being pregnant during COVID was extra lonely (Spryng 5876) After competing my course work in late April, I was now a university graduate, but I do not
feel like it. I feel that [university name] has made VERY little effort to celebrate their gradu-
ates, especially compared to other universities and high schools. My parents made no effort to
celebrate my accomplishment, and my mental health was affected as I felt my past four years
of hard work has gone uncelebrated. (Spryng 4051) I realize that there was no way around the circumstances caused by the pandemic but the
combination of quarantine, self-isolation and grieving is brutal. (Spryng 3962) People also described more subtle but equally important role transitions such as youth tran-
sitioning out of family and children’s services and being unable to find meaningful work (or
any work at all) during their transition to adulthood. So our youth that have aged out [of the child welfare system] and are not in school. They have
no supports. We do work with some of those youths that have aged out of [care] and are no
longer sort of under their control. And yeah this this whole pandemic and not being able to
access the services and the places that they would normally go to, connect to WIFI or to, you
know, to go in and get out of the heat or out of the cold, whatever the case may be, it’s all been
taken away, right. Um so seeing a lot of increased drug use, increased risky drug use um and
unfortunately with a few deaths in town, directly related to that. [. . .] I mean they’re just
starting out kind of their adult life, so to speak. And then everything shuts down and they
have nowhere to go and no contacts, no social life. And I think that’s been the hardest for
them. (4A) Indigenous students who had enrolled in Indigenous-specific higher education programs
and support services to explore their Indigenous identity and to transition to the role of
teacher lost out on mentorship opportunities to help them with these transitions. For some of our students who now don’t step foot in the college, you know, [..] the reality is
that we could have students that get a certificate or a diploma without ever stepping foot on
our campus. Trajectories And that is
going to carry with them for a long time [and] we’re not ever going to see that. (9A) Some of the impacts of the pandemic on social pathways were unique based on cultural
groups, with Indigenous People and newcomers more likely to be identified as having new
obligations to family members due to the disruptions of the pandemic [28]. Some participants
did report positive change, such as improvements in income through access to government
pandemic relief programs, delayed evictions, and reprioritization of personal and family time. While the initial disruptions were viewed as a crisis and responded to as such, as the pandemic 8 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 PLOS ONE Life course approach to the COVID-19 pandemic lingered, social pathways became more and more disrupted with implications for people on
identity, sense of agency (see below), and choices people made about employment and educa-
tion, among others. lingered, social pathways became more and more disrupted with implications for people on
identity, sense of agency (see below), and choices people made about employment and educa-
tion, among others. Transitions The impact of the COVID-19 pandemic on transitions was very clear from our respondents
and many kinds of transitions were profoundly affected. People described the isolation of
being a first-time parent, without access to friends or family members to mark special or diffi-
cult times. Others spoke of missing key symbolic moments which are recognized as meaning-
ful across cultures and throughout time: graduation ceremonies, birthdays, funerals, and other
rites of passage. These ceremonies anchor us as social beings within our communities, and the
long-term impact of missing them should not be underestimated. Pregnancy can be very lonely even if you are surrounded by the most supportive husband and
family—and being pregnant during COVID was extra lonely (Spryng 5876) PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Pregnancy can be very lonely even if you are surrounded by the most supportive husband and
family—and being pregnant during COVID was extra lonely (Spryng 5876) And if that’s the case, they kind of missed that opportunity to reconnect with
their culture [to] explore some of that and then continue to educate their children, other peo-
ple in the community, their families. And so I feel like we’re missing, you know, this really 9 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 PLOS ONE Life course approach to the COVID-19 pandemic important group of people who could then continue and of course we’re looking into, you
know, reconciliation in trying to get people to take action. (10A) important group of people who could then continue and of course we’re looking into, you
know, reconciliation in trying to get people to take action. (10A) Parents transitioned to the role of grandparents without being able to meet their grandchil-
dren. While some people were able to see the silver lining of how the pandemic had affected
transitions in their lives (longer parental leaves, witnessing key milestones in children that they
may have otherwise missed due to being at work), overall, respondents experience of the
impact of the pandemic on transitions was negative. The Covid lockdowns came during the midst of my divorce. I believe it escalated it into a
higher conflict situation (Spryng 5791) There’s another kid who is also 16 and his dad kicked him out because he was smoking weed,
which is not uncommon for a 16-year-old, especially in that area. [..] So this one boy, who was
you know caught with the marijuana, he doesn’t want to go home anymore. And they don’t
want him home. So the youth counsellor had been working with them and they just decided
no that’s done, we don’t want to pursue that anymore. So we got transitional housing options
for him. (15B) Covid has left me and my boyfriend homeless. He then died from fentanyl because of all the
covid stress. (Spryng 5675) So we’ve already seen um, you know, domestic violence that has led to murder in during the
pandemic. Turning points ]COVID-19 itself may be looked back on as a turning point for many: a moment when lives
changed direction, deviating from previously set social pathways. Turning points ultimately
must be viewed retrospectively, since by definition they would be times one would look back
on as key moments of change. Nevertheless, our respondents identified life events which seem
likely to be looked back on as turning points in the future. These included divorces and separa-
tions of long-term partners, first experiences of homelessness, and deaths of loved ones (in our
responses there were no direct experiences of deaths from COVID-19, but respondents
reported other deaths including from drug overdose and homicide in the context of intimate
partner violence). The Covid lockdowns came during the midst of my divorce. I believe it escalated it into a
higher conflict situation (Spryng 5791) PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Principles Five core principles have been described within the Life Course theory that allow us to better
understand the trajectory components of the theory in the context of a cohort effect. These
principles can help us to understand how and why some people do better or worse given
superficially similar circumstances. Our data highlighted these principles, as outlined below. (9B) Long term, potentially irreversible, effects on the health and education of children were also
mentioned, including dropping out of high school, becoming obese, or experiencing new or
acute material deprivation. Older students failed to complete or opted to defer their plans for
higher education. Parents that don’t have a lot of resources, have decided to keep their kids home, they’re seden-
tary all day. So they’re on the screen or they’re watching TV, they’re gaining weight, and all
this is becoming entrenched. And so, even if everything went back to normal tomorrow–the
child that has gained 20 or 30 pounds during COVID, that’s a life-long. Now that that child
has obesity, that doesn’t go away, right. That’s a forever problem that’s type II diabetes as a
teenager problem. It’s significant, because even if all the restrictions were lifted tomorrow,
which they won’t be, even if they were, these new pattern of increased substance abuse and
sedentariness have long-lasting consequences. [. . .] And so I worry about my kids that were PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 10 / 20 PLOS ONE Life course approach to the COVID-19 pandemic starting grade 9 and grade 10 –this could be the end of high school for them. If they fall behind
in enough courses [. . .], they’re not going to catch up and they’re not going to be motivated to. (5B) While the vast majority of the turning points identified were negative, a few did report that
the respite provided by the Canadian Emergency Response Benefit allowed them to reconsider
their employment and move their lives in a new direction. While I was receiving CERB [Canadian Emergency Response Benefit] I had the time to start
my own business without worrying to much about what would happen if I wasn’t immediately
successful. Starting my own business had been something I wanted to do for a while and it was
a great experience and took off relatively quickly. (Spryng 5910) Some respondents identified these turning points as being directly related to the pandemic
and associated countermeasures, while this was not clear in other cases. In all cases, the pan-
demic affected people’s choices and strategies in how they managed or coped with the turning
points they experienced. Life-span development Our data illustrated how the COVID-19 pandemic and associated public health countermea-
sures impacted people across the lifespan. Participants’ responses tended to focus on children
and the elderly, but nevertheless illustrated the constant evolution and change experienced by
human beings, particularly in response to the unusual circumstances of the pandemic. My child is now 15 months old and has never played with another child. (Spryng 5797) My child is now 15 months old and has never played with another child. (Spryng 5797) One heart breaking change I have seen is that our 3 year old now knows to give people space. We were playing on the sidewalk outside of our house when a neighbor family was walking
down the street our daughter yelled “PEOPLE!” Got up and ran to the other side of our yard. (Spryng 4114) Oh for some of our Elders, [. . .] I know this directly from individuals affected that with social
distancing it means that there is a real reduction in physical activity. Like profound reduction
(13A) While many of the perceived changes were negative, there were positives as well particularly
for people who were able to improve their access to and ease with technology as a means to
reach out to the world and learn new things through platforms that were previously unavail-
able to them. The best impact of Covid-19 on my household (me) is that as an independent, disabled senior
on oxygen the world has opened up to me again through the computer. Instead of loading up
the car with my oxygen equipment and walker and going to the gym, my trainers now come to PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 11 / 20 PLOS ONE Life course approach to the COVID-19 pandemic me through zoom. Similarly, I now can attend political meetings, church, doctor’s appoint-
ments, interest courses, even concerts through the use of technology without having to expend
effort and money trying to get from point A to point B. (Spryng 5561) Agency Agency, or the lack thereof, was perhaps one of the most starkly differentiated themes in our
data. Findings ranged from people who obviously felt a significant sense of agency and/or who
witnessed agency, strength and resilience in others, to many who felt completely powerless in
the face of the pandemic. Whether or not people experienced a sense of agency seemed to have a particularly impor-
tant impact on their overall experience. While some quotes identify a sense of strength, self-
efficacy and resilience: Man, like when families work together they’re unstoppable. Like we need to encourage that. (2A) Man, like when families work together they’re unstoppable. Like we need to encourage that. (2A) (2A) So there’s been challenges, you know, certainly there’s been challenges, but at the same time
it’s been so much creativity that has been born, you know because you’ve got to make it
work. (3A) So there’s been challenges, you know, certainly there’s been challenges, but at the same time
it’s been so much creativity that has been born, you know because you’ve got to make it
work. (3A) PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Others echo fear and insecurity related to profound sense of fear and lack of control: Others echo fear and insecurity related to profound sense of fear and lack of control: The beginning of the pandemic was the worst. I didn’t know what we were up against, a lot of
unknowns. I was nervously eating a lot of bread & potatoes & foods that aren’t good for me. I’d almost be falling asleep at night & experience anxiety, fear etc. (Spryng 5568) I think they’ve settled into some kind of sense that, well this is my life and this is how it’s going
to be. And sort of in a dark place where we have no hope that it’s going to change. (11A) And they don’t seem to have the resilience on their own to find a way to fill that gap. I suspect
a lot of them are just watching tv or playing video games. (11A) For instance, some new mothers clearly felt disempowered, struggling with isolation, loss
related to lack of social and family support, missing out on celebrations and ceremony related
to their child’s birth and development. Others viewed the same situation as positive–time to
connect with their co-parent, an opportunity to demonstrate strength in “getting through this”
as their small nuclear family. I feel like myself and my spouse have been a good team. Our kids are happy and healthy,
active without much restriction (hikes, backyard play, themed learning weeks based on child-
lead interests, etc). Giving them positive memories and as little worry as possible regarding
Covid-19 is a top priority to us. (Spryng 4202) Agency cannot be viewed in isolation. Whether one has a sense of agency will depend in
part on other pre-existing conditions such as financial and material security, presence of a sup-
portive or abusive partner or social network, pre-existing mental health, social isolation, and
cultural grounding, to name a few. It’s about loss of control, which is often an impact of sexual violence in itself, right. (6B) 12 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 PLOS ONE Life course approach to the COVID-19 pandemic Time and place Time and place was the least explicitly addressed aspect of the life course model in our study
data. Since the majority of our respondents were from the Kingston area, we did not have a
wide geographic diversity. In our sample, the variable impacts of the public health counter-
measures on time and space were illustrated primarily in the urban-rural divide, and then spe-
cifically for people living in remote Indigenous communities. All of the webinars and stuff that we’ve been offering were then not accessible to the indige-
nous people that didn’t have the technology to be able to listen in on those webinars. [. . .] In
our [University Program] the lack of good internet or internet at all because we were going to
open a new [program] in [remote community name], like over half of the students were living
in communities that had dial-up. And you can’t do Zoom and those things with the dialup. (12A) I would add rural communities to that because for rural people, services are few and far
between to begin with. And then when you start taking away resources like maybe they’re not
making as much in their job. Maybe they’re not, you know they can’t travel to services now. And then maybe the rural services don’t have the enough of the digital virtual stuff going on
that they could provide virtual support. (2A) I know we had one situation where we had some equipment that was supposed to go to a child
up North and the family originally would have been coming down and would have taken it
home with them, after an appointment. They weren’t coming down so the child’s been without
the equipment. (2B) The majority of our respondents were from KFL&A, a region in southeastern Ontario with
both urban and rural communities, and with relatively low COVID-19 prevalence. While par-
ticipants reported a great deal of fear, as well as a great deal of self-imposed restriction on their
activities in addition to the restrictions imposed by public health authorities, no one in our
study reported the loss of, or even infection of a close friend or family member from COVID-
19, something that would have been very different in another geographic location, or even in
KFL&A at a different point in the pandemic. Timing Similar to life-span development, our data illustrated the differential impact of the pandemic
depending on the timing in which it occurred in a persons’ life. More than just a age related
construct (ie., this happened to me when I was X years old), respondents described how the
intersection of the pandemic with a certain moment in a person’s life (for instance, the impact
of a pandemic lockdown that occurred at the same time as an evolving custody battle and
change in employment, or the impact of elementary school closures on a mature student trying
to complete University studies while being a single parent) would significantly influence the
impact the events had on life course, being more significant for some, and less for others,
depending on the point of intersection. Generally, our respondents illustrated greatest concern
about the impact of the pandemic on the social, emotional, and educational lives of developing
children and youth. " kids are suffering" they are not getting the interaction that they need on so many different
levels. Emotional, mentally, physical contact with family and their friends. [. . .] Kids are start-
ing to worry about school. The 6 year old is afraid her friends won’t know her or she won’t 13 / 20 13 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 PLOS ONE Life course approach to the COVID-19 pandemic know them with masks on. The boys are afraid of their backpacks and coats on their chairs
will be too close. They won’t be able to play the same games, there won’t be hockey or all fun
with their friends, the 13 year is just afraid all together. (Spryng 5117) We’re seeing teens with um depression, anxiety, social isolation that’s getting worse. We’re see-
ing lots of kids that are really, I’m really worried about how they’re falling behind in school. They may have had special needs or individualized education plans, but now parents have
decided to keep them home probably based on not accurate health information. And now the
kids are not able to receive the specialized supports that they would have needed. And I’m
worried that they’re falling farther and farther behind. [. . .] And I worry that even when this
is all over, this kind of stuff is going to be long-term for these kids. Timing And that, that’s the part
that I that, like I said, that’s the part that is keeping me awake at night, it’s the kids. (5B) Linked lives Finally, the principle of linked lives was vividly illustrated throughout our findings. On the
positive side, participants identified increased time spent with loved ones and household mem-
bers as a benefit of the pandemic. It has strengthened our relationships in a way that can never be replicated (Spryng 5769) This crisis has allowed me more time to realize that family, love, and friendship are the most
important things in life and so much time spend online is a colossal waste of energy (Spryng
5775) On the negative side, previously fragile or tense relationships exploded under the added
strain of forced social isolation. Front line workers identified as a main source of stress the
risk of bringing infection home to their loved ones, again illustrating the impact of linked
lives. Workers who could not work from home observed others spending more time with
family in seemingly harmonious activities felt guilty or sad that this could not be available to
their own families. And people who were alone were lonelier, in some cases perhaps, than
ever before. I feel guilty when I come into work that I’m not home with my kids. I cry on my way into
work as I watch other families out together on bikes, going for walks, just being together. I feel
guilty that we’re not able to have that time together. (Spryng 4152) Covid-19 has impacted my household in a negative way, as it has significantly increased the
tension in my house. [. . .] Since we have been at home for the past couple of months my
parents have been fighting more, my parents have been on edge and taken their anger out on
us kids, and my siblings and I have been getting into more disagreements. (Spryng 4050) But the depth of this isolation, even if it is sporadic, I genuinely don’t think that we’re going to
understand it. We’ve, we’ve never gone through this kind of isolation, not really. And that’s a
blanket statement that applies to Canada and essentially the world. (8A) Clearly, stresses and disruptions in trajectories felt by one family member often had signifi-
cant impact on others. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Linked lives This is powerfully illustrated by a quote in which a child illustrates the
impact that new food insecurity has had on his mother in what he says to a worker bringing
them fresh fruit: 14 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 PLOS ONE Life course approach to the COVID-19 pandemic And then the children, when they would say–“I’m thankful that you’re here, my mom’s not
going to cry today”. (9A) And then the children, when they would say–“I’m thankful that you’re here, my mom’s not
going to cry today”. (9A) Additional theme: Culture While most of the themes emerging around culture could fit in original Life Course constructs,
gaps remained in capturing culture as core principle that affects a person’s life course, not
completely independently, but still distinct from the original constructs. In our data, the theme
of culture emerged predominantly around Indigenous culture, with the importance of cultural
belonging, spaces for engaging in cultural practices, and differential impacts of pandemic
restrictions depending on core components of cultural practice emerging. Additional culture-
related themes involved newcomers to Canada who articulated a cultural expectation that they
would provide care for their extended families. While all the quotes in this section relate to
Indigenous culture, we anticipate that a similar study done in a different setting, with more
cultural diversity, would uncover the importance of culture for other groups as well. It was really humbling to see finally some indigenous families not having to be in the colonial
structure of schools and school boards. That they were connecting to the land. They were
including their children in the baking. They were including their children in the measuring. They were they were going back to our traditional ways of how we teach our young. And it
wasn’t the western reading, writing, arithmetic. It was in the most holistic way. So if we’re
going to take something good out of it. (9A) And to just have that level of care, to know that we just have to keep going. You know, I just
think it really speaks to who we are as indigenous people. We’re resilient. We can get through
anything. (3A) From a cultural perspective. . . I think our ways of coming together have been total-
ly. . .constrained. So, and I think now people are trying to find ways of doing that, so trying to
figure out ways. So I’m starting to hear of things like people are doing little social distance
things to try and reconnect. So I heard about a Moon Ceremony recently. A little social dis-
tance Moon Ceremony. There’s been social distance Water ceremonies. There’s going to be a
social distance Sacred fire. (13A) PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Discussion Our findings illustrate that the pervasive impacts of the COVID-19 pandemic and associated
countermeasures echo the Life Course constructs as described by Elde [11]. We believe that
this framework provides an important lens in understanding how to consider the complex
and interrelated Life Course constructs when planning for recovery. The strength of Life
Course theory as applied to the COVID-19 pandemic is that it paints a complex and nuanced
picture of how the pandemic has affected the lives of individuals that may fundamentally
alter their life trajectories in the short, medium, and likely long term ways. Learning from
past major events which have been analyzed through the lens of Life Course theory, we
believe that this analysis can allow program developers, service providers, and policy makers
to think about how and which of these constructs need to be addressed by different initiatives
as we rebuild. Life Course theory-based analyses have demonstrated the long term impacts of major dis-
ruptions on peoples’ lives, such as that of the Great Depression on children in the United States PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 15 / 20 PLOS ONE Life course approach to the COVID-19 pandemic [12], as well as the impact of the Cultural Revolution in China [29]. These studies, completed
long after the fact, have shown how major disruptions can have impacts over the entire life-
span, both due to the initial insult, but also due to the pathway of cumulative advantage or dis-
advantage triggered by the initial event. Emerging studies are beginning to look at Life Course
impacts of COVID-19 pandemic, though they are limited by the timeframe which they are
studying. A German study exploring adolescent well-being and transitions to post-secondary
education [30] explored the impact of the pandemic on mental health, educational and career
plans. A second identified the importance of social relationships and coping skills on adult
quality of life [31] and made recommendations to mitigate the effects of these for future pan-
demics. A third study in pre-publication explores the concept of self-efficacy and valence as it
pertains to life satisfaction [32]. These constructs align closely with that of Agency. Discussion These stud-
ies will be necessarily have a near-sighted view of the impacts of the pandemic, but will add to
the call to reflect on how these constructs, and other Life Course constructs as defined by
Elder, will shape people’s lives, and therefore society, going forward, and how they can be lev-
eraged both to recover from the current pandemic, and to prepare for other pandemics or
major events which will inevitably occur. A construct which we believe is missing from Life Course theory and that was important in
understanding the experience of our participants is that of culture. Culture was a strength for
our study participants, which is consistent with other publications exploring the experience of
Canadian Indigenous culture during COVID-19 [33,34]. We know however that cultural prac-
tices, traditions and norms can play out differently in different situations, and the degrees and
ways in which an individual has a sense of belonging to their culture will affect their perspec-
tive, the responses they consider appropriate, decisions, and ultimately Life Course [10]. Other
authors have argued for an increased cross-cultural perspective to understanding the Life
Course [35,36]. Many Life Course analyses have been done within a specific country, which
may make the influence of culture less visible. In much of traditional Life Course theory, a
male European view predominates[35]. Our analysis, done within a single country, Canada,
highlights the importance of cultural differences between Indigenous Canadians and non-
Indigenous Canadians, and although anecdotal, differences emerged in our data between new
Canadians with different cultural backgrounds, and the dominant responses from euro-Cana-
dians as well. Life Course theory does not explicitly address individual or group identifiers
such as gender, race, religion, or culture which have cross cutting effects on all Life Course
constructs. When considering Life Course analysis of the COVID-19 pandemic, culture might
be particularly important given that the pandemic affected all areas of the globe, with the wide
cultural variation that implies. We propose adding “Culture” to the Life Course principles, or
alternatively explicitly including a cultural dimension to existing principles, as has sometimes
been done, including linked lives, and time and place, to ensure that the impact of an individu-
al’s experience of culture on Life Course is not lost. Implications for research and policy The program and policy implications of a Life Course understanding of the impacts of
COVID-19 are significant. First, a deeper understanding of the disruptions to transitions and
social pathways can help us understand short term impacts of the pandemic as we are seeing
them play out, including educational gaps, mental health struggles, substance use and youth
unemployment [1,2,4,9]. Second, an awareness of turning points experienced during the pandemic can help us antic-
ipate services that will be needed for certain particularly vulnerable groups in the short and
medium term. These could include specific training and support for child and youth workers PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 16 / 20 PLOS ONE Life course approach to the COVID-19 pandemic in school systems, child and family services, and youth mental health agencies as they try to
support children and youth whose trajectories have been disrupted, with meaningful impacts
on their socialization skills, physical and mental health, learning, development and identity
formation. Attention to aspects of life course development such as the development of agency,
impact of linked lives (specifically healthy relationships with others) should be considered. Marking transitions, even if retroactively, should be contemplated for children and youth, as
well as adults, who have not been able to mark important transitions with ceremony or who
have had these delayed. Third, an awareness of the pervasiveness of the impacts of the pandemic across geographic
locations, age groups and developmental stages can remind policy makers to anticipate longer
term impacts of the pandemic as they begin to emerge. These will include the life-long impacts
of adverse childhood experiences [2,9], as well as dysfunctional or missed transitions. Young
adults who have not been able to transition to independent roles, due to deferred or online
higher education, or through lack of employment opportunities, may need specific mentorship
and support programs to retroactively ensure these transitions happen in a good way. Educa-
tors, as well as health and social service providers, may need additional training and support to
help children and families who have experienced increased adversity during the pandemic to
heal and develop healthy relationships. Substantial long term resource allocation may be
required, which may challenge policy makers faced by competing demands, including those
presented by the equally important crisis of climate change. Implications for research and policy Fourth, understanding the importance of agency and identity as core constructs to good
physical, social and emotional health, and how these have been disrupted, may allow for spe-
cific programming particularly though not exclusively for children and youth. Considering
our data in light of the damaging impacts of the COVID-19 pandemic on individual agency
and the complex process of identity formation may be helpful in understanding some of the
long-term consequences of the pandemic and envisioning additional educational, mental
health, job creation, and other supports that need to be put in place to help people work their
way towards physically, socially and emotionally healthier life trajectories. Community agency
is also important, and community development strategies inspired by models that have already
demonstrate success [37,38] could be adapted to the ongoing pandemic context Ensuring that
people and communities have meaningful sense of agency and an ability to positively improve
their lives and that of those around them can provide an important resource to enhancing our
recovery from the current pandemic, and our preparedness for other challenges such as future
pandemics, natural disasters or social unrest. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 Limitations Our study, as those cited above, is limited by the timeframe with which we can currently exam-
ine the Life Course impacts of the pandemic. Many more findings will emerge concerning the
Life Course implications that have yet to unfold. Additionally, our study has some methodo-
logical limitations. Spryng.io micronarrative collection was both opportunistic in the first
instance, and then intentional. This combination carries a risk of bias. Approximately 6% of
the population of KFL&A identifies as Indigenous, and approximately 7% as “visible minority”
[17]. Our sample overrepresented Indigenous People, and underrepresented visible minorities
and men. As discussed in the introduction, the region where data were collected was relatively
spared during the first waves of the COVID-19 pandemic, (this has since changed [39]), and it
is likely that some of the implications of the pandemic on Life Course constructs could have
been illustrated differently and more profoundly in areas which were more acutely affected by
illness and death. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 17 / 20 PLOS ONE Life course approach to the COVID-19 pandemic Author Contributions Conceptualization: Eva Purkey, Imaan Bayoumi. Data curation: Eva Purkey, Imaan Bayoumi, Colleen M. Davison. Formal analysis: Eva Purkey. Data curation: Eva Purkey, Imaan Bayoumi, Colleen M. Davison. Formal analysis: Eva Purkey. Investigation: Eva Purkey, Imaan Bayoumi, Colleen M. Davison, Autumn Watson. Methodology: Eva Purkey, Imaan Bayoumi, Colleen M. Davison, Autumn Watson. Project administration: Eva Purkey, Imaan Bayoumi, Autumn Watson. Writing – original draft: Eva Purkey. Writing – review & editing: Imaan Bayoumi, Colleen M. Davison, Autumn Watson. Writing – review & editing: Imaan Bayoumi, Colleen M. Davison, Autumn Watson. Acknowledgments We would like the thank Eszter Papp and Minnie Fu who were instrumental in data collection,
as well as the Indigenous Health Council and members of the Research Advisory Committee
who helped guide our work as it pertains to Urban Indigenous Peoples. Conclusions Overall, an understanding of the implications of COVID-19 pandemic through a Life Course
lens is a reminder to service providers and policy makers that people will continue to experi-
ence the sequelae of the pandemic for decades to come. Acutely, this understanding must
inform the real time decisions policy makers continue to make about public health counter-
measures and special programming related to the ongoing pandemic and society’s recovery. For some, this fallout will be minimal, but for others it will radically alter their life course in
ways that will require long term creativity and care to support. Life Course theory can allow us
to engage with the complexities of the impacts of the pandemic and to develop a more nuanced
understanding of the implications of major disruptions, enabling us to come together as com-
munities to imagine how to truly be “all in this together” for the long-term response to and
recovery from COVID-19 and other pandemics which are sure to come. References 1. Aurini J. and Davies S., COVID-19 school closures and educational achievement gaps in Canada: Les-
sons from Ontario summer learning research. Canadian Review of Sociology/Revue canadienne de
sociologie, 2021. 58(2): p. 165–185. https://doi.org/10.1111/cars.12334 PMID: 34080780 1. Aurini J. and Davies S., COVID-19 school closures and educational achievement gaps in Canada: Les-
sons from Ontario summer learning research. Canadian Review of Sociology/Revue canadienne de
sociologie, 2021. 58(2): p. 165–185. https://doi.org/10.1111/cars.12334 PMID: 34080780 2. Ghosh R., et al., Impact of COVID -19 on children: special focus on the psychosocial aspect. Minerva
Pediatr, 2020. 72(3): p. 226–235. https://doi.org/10.23736/S0026-4946.20.05887-9 PMID: 32613821 2. Ghosh R., et al., Impact of COVID -19 on children: special focus on the psychosocial aspect. Minerva
Pediatr, 2020. 72(3): p. 226–235. https://doi.org/10.23736/S0026-4946.20.05887-9 PMID: 32613821 3. Nolan R., ‘We are all in this together!’ Covid-19 and the lie of solidarity. Irish Journal of Sociology, 2020. 29(1): p. 102–106. 3. Nolan R., ‘We are all in this together!’ Covid-19 and the lie of solidarity. Irish Journal of Sociology, 2020. 29(1): p. 102–106. 4. Mazza M., et al., Danger in danger: Interpersonal violence during COVID-19 quarantine. Psychiatry
Res, 2020. 289: p. 113046. https://doi.org/10.1016/j.psychres.2020.113046 PMID: 32387794 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240
December 16, 2022 18 / 20 PLOS ONE Life course approach to the COVID-19 pandemic 5. Cheung D.C., et al., "Bring the Hoses to Where the Fire Is!": Differential Impacts of Marginalization and
Socioeconomic Status on COVID-19 Case Counts and Healthcare Costs. Value Health, 2022. https://
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mun Dis Rep, 2021. 47(7–8): p. 300–304. https://doi.org/10.14745/ccdr.v47i78a02 PMID: 34421385 7. Sullivan W.F., et al., Ethics framework and recommendations to support capabilities of people with intel-
lectual and developmental disabilities during pandemics. J Policy Pract Intellect Disabil, 2022. 19(1): p. 116–124. https://doi.org/10.1111/jppi.12413 PMID: 35601010 8. Simonetti O., Martini M., and Armocida E., COVID-19 and Spanish flu-18: review of medical and social
parallelisms between two global pandemics. J Prev Med Hyg, 2021. 62(3): p. E613–e620. https://doi. org/10.15167/2421-4248/jpmh2021.62.3.2124 PMID: 34909487 9. Lawson M., Piel M.H., and Simon M., Child Maltreatment during the COVID-19 Pandemic: Conse-
quences of Parental Job Loss on Psychological and Physical Abuse Towards Children. Child Abuse
Negl, 2020. 110(Pt 2): p. 104709. https://doi.org/10.1016/j.chiabu.2020.104709 PMID: 32893003 10. Settersten R.A., et al., Understanding the effects of Covid-19 through a life course lens. References Advances in
Life Course Research, 2020. 45: p. 100360. 11. Elder G.H., Johnson M.K., and Crosnoe R. The Emergence and Development of Life Course Theory. 2003. 12. Elder, Children of the Great Depression: Social Change in Life Experience. 2018. 1– 13. Hung E.P.W. and Chiu S.W.K., The Lost Generation: Life Course Dynamics and Xiagang in China. Modern China, 2003. 29(2): p. 204–236. 14. Salinas-Miranda A.A., et al., Exploring the Life Course Perspective in Maternal and Child Health through
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December 16, 2022 PLOS ONE Life course approach to the COVID-19 pandemic 30. Sandner M., et al., The COVID-19 pandemic, well-being, and transitions to post-secondary education. Rev Econ Househ, 2022: p. 1–23. https://doi.org/10.1007/s11150-022-09623-9 PMID: 36187597 31. Cohrdes C., et al., Adult quality of life patterns and trajectories during the COVID-19 pandemic in Ger-
many. Curr Psychol, 2022: p. 1–13. https://doi.org/10.1007/s12144-022-03628-4 PMID: 36196377 32. de Vries J.H., Horstmann K.T., and Mussel P., Trajectories in life satisfaction before and during COVID-
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a response to the COVID-19 Pandemic. International Journal of Indigenous Health, 2022. 17(1): p. 111–121. 34. Yohathasan A.M.-P.C.S.S.S.T., What we heard: Indigenous Peoples and COVID-19. 2021, Waakebi-
ness-Bryce Institute for Indigenous Health, Dalla Lana School of Public Health, University of Toronto:
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Collective atomic scattering and motional effects in a dense coherent medium
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Nature communications
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cc-by
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ARTICLE Received 27 Oct 2015 | Accepted 15 Feb 2016 | Published 17 Mar 2016 Received 27 Oct 2015 | Accepted 15 Feb 2016 | Published 17 Mar 2016 Collective atomic scattering and motional effects
in a dense coherent medium S.L. Bromley1, B. Zhu1, M. Bishof1,w, X. Zhang1,w, T. Bothwell1, J. Schachenmayer1, T.L. Nicholson1,w, R. Kaiser2,
S.F. Yelin3,4, M.D. Lukin4, A.M. Rey1 & J. Ye1 S.L. Bromley1, B. Zhu1, M. Bishof1,w, X. Zhang1,w, T. Bothwell1, J. Schachenmayer1, T.L. Nicholson1,w, R. Kaiser2,
S.F. Yelin3,4, M.D. Lukin4, A.M. Rey1 & J. Ye1 We investigate collective emission from coherently driven ultracold 88Sr atoms. We perform
two sets of experiments using a strong and weak transition that are insensitive and sensitive,
respectively, to atomic motion at 1 mK. We observe highly directional forward emission with a
peak intensity that is enhanced, for the strong transition, by 4103 compared with that in the
transverse direction. This is accompanied by substantial broadening of spectral lines. For the
weak transition, the forward enhancement is substantially reduced due to motion. Meanwhile,
a density-dependent frequency shift of the weak transition (B10% of the natural linewidth)
is observed. In contrast, this shift is suppressed to o1% of the natural linewidth for the
strong transition. Along the transverse direction, we observe strong polarization dependences
of the fluorescence intensity and line broadening for both transitions. The measurements
are reproduced with a theoretical model treating the atoms as coherent, interacting
radiating dipoles. 1 JILA, NIST and Department of Physics, University of Colorado, 440 UCB, Boulder, Colorado 80309, USA. 2 Universite´ de Nice Sophia Antipolis, CNRS,
Institut Non-Line´aire de Nice, UMR 7335, F-06560 Valbonne, France. 3 Department of Physics, University of Connecticut, Storrs, Connecticut 06269, USA. 4 Department of Physics, Harvard University, Cambridge, Massachussetts 02138, USA. w Present addresses: Physics Division, Argonne National Laboratory,
Argonne, Illinois 60439, USA (M.B.); International Center for Quantum Materials, School of Physics, Peking University, Beijing 100871, China (X.Z.); Center
for Ultracold Atoms, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA (T.L.N.). Correspondence and requests for materials
should be addressed to A.M.R. (email: arey@jilau1.colorado.edu) or to J.Y. (email: Ye@jila.colorado.edu). 1 NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 U coherence prepared by the 461-nm light. To the contrary, the red
transition with a natural linewidth G ¼ 7.5 kHz is strongly
affected by atomic motion. By comparing the behaviours of
the same atomic ensemble observed at these two different
wavelengths (Fig. 1b), we can thus collect clear signatures of
motional effects on coherent scattering and dipolar coupling50,51. Collective atomic scattering and motional effects
in a dense coherent medium (b) In the coherent dipole model, photons are shared
between atoms. When the Doppler broadened linewidth becomes
comparable to the natural linewidth, dephasing must be considered. At our
B1 mK temperatures the Doppler broadening is E40 kHz, meaning
motional effects are important only for the red transition. (c) The three-
dimensional intensity distribution predicted for a blue probe beam. The
coupled dipole model predicts a strong 103 enhancement of the forward
intensity compared with other directions and a finite fluorescence along a
direction parallel to the incident polarization. The speckled pattern is due to
randomly positioned atoms and can be removed by averaging over multiple
atom configurations. Collective level shifts and line broadening arising from the real
and imaginary parts of dipole–dipole interactions have recently
been observed in both atomic19–23 and condensed matter24
systems. The modification of radiative decay dynamics at low
excitation levels has also been observed using short probe
pulses25–28, and interaction effects were manifested in coherent
backscattering29,30. Although
simple
models
of
incoherent
radiation transport have often been used to describe light
propagation through opaque media31,32 and radiation trapping
in laser cooling of dense atomic samples33, coherent effects
arising from atom–atom interactions, which are necessary to
capture correlated many-body quantum behaviour induced by
dipolar exchange, are beginning to play a central role. For
example, the dipole–dipole interaction is responsible for the
observed dipolar blockade and collective excitations in Rydberg
atoms34–41; it may also place a limit to the accuracy of an optical
lattice clock and will require non-trivial lattice geometries to
overcome the resulting frequency shift42. Previous theoretical
efforts have already shown that physical conditions such as finite
sample size, sample geometry and the simultaneous presence of
dispersive and radiative parts can play crucial roles in atomic
emission10–13,43–45. Collective atomic scattering and motional effects
in a dense coherent medium U
nderstanding interactions between light and matter in a
dense atomic medium is a long-standing problem in
physical science1,2 since the seminal work of Dicke3. In addition to their fundamental importance in optical physics,
such interactions play a central role in enabling a range of new
quantum technologies including optical lattice atomic clocks4 and
quantum networks5. We use the experimental scheme shown in Fig. 1a, to perform a
comprehensive set of measurements of fluorescence intensity
emitted by a dense sample of 88Sr atoms. The sample is released
from the trap and then illuminated with a weak probe laser. We
vary the atomic density, cloud geometry, observation direction
and polarization state of the laser field, and we report the system
characteristics using three key parameters as follows: the peak
scattered intensity, the linewidth broadening and the line centre
shift. For example, along the forward and transverse directions we
observe different values of intensity and linewidth broadening, as
well as their dependence on light polarization (see Fig. 1c). The key ingredient in a dense sample is dipole–dipole
interactions that arise from the exchange of virtual photons
with dispersive and radiative contributions, and their relative
magnitude varies between the near-field and far-field regimes. The dispersive (real) part is responsible for collective level shifts
and the radiative (imaginary) part gives rise to line broadening
and collective superradiant emission6–8. Intense theoretical
efforts have been undertaken over many years, to treat the
complex
interplay
between
the
dispersive
and
radiative
dynamics9–18. However,
experimental
demonstrations
that
provide a complete picture to clarify these interactions have
been elusive. DT
ˆz
ˆx
ˆy
k
1P1,3P1
1S0
Probe direction
DF
|m〉 = |0〉, | ± 1〉
Probe beam
Blue fluorescence intensity
5,000
1,000
100
10
1
y
z
= 461 nm
= 689 nm
= 32 MHz
= 7.5 kHz
k
a
b
c
Figure 1 | The experimental scheme and concept. (a) We weakly excite
the strontium atoms with a linearly polarized probe beam and measure the
fluorescence with two detectors: one in the forward direction, ^x, and the
other almost in the perpendicular direction, ^z. We probe two different J ¼ 0
to J’ ¼ 1 transitions. The first transition is a 1S0 1P1 blue transition with a
natural linewidth of G ¼ 32 MHz and the second is a 1S0 3P1 red transition
with G ¼ 7.5 kHz. Collective atomic scattering and motional effects
in a dense coherent medium DT
ˆz
ˆx
ˆy
k
1P1,3P1
1S0
Probe direction
DF
|m〉 = |0〉, | ± 1〉
Probe beam
Blue fluorescence intensity
5,000
1,000
100
10
1
y
z
= 461 nm
= 689 nm
= 32 MHz
= 7.5 kHz
k
a
b
c DT
ˆz
ˆx
ˆy
k
1P1,3P1
1S0
Probe direction
DF
|m〉 = |0〉, | ± 1〉
y
z
= 461 nm
= 689 nm
= 32 MHz
= 7.5 kHz
a
b a b Probe beam
Blue fluorescence intensity
5,000
1,000
100
10
1
k
c c In this work we use millions of Sr atoms in optically thick
ensembles, taking advantage of the unique level structure of Sr to
address
motional
effects,
to
study
these
radiative
and
dispersive parts simultaneously. We demonstrate that a single,
self-consistent,
microscopic
theory
model
can
provide
a
unifying picture for the majority of our observations. These
understandings can help underpin emerging applications based
on many-body quantum science, such as lattice-based optical
atomic
clocks4,46,47,
quantum
nonlinear
optics39,
quantum
simulations48 and atomic ensemble-based quantum memories49. Probe beam Figure 1 | The experimental scheme and concept. (a) We weakly excite
the strontium atoms with a linearly polarized probe beam and measure the
fluorescence with two detectors: one in the forward direction, ^x, and the
other almost in the perpendicular direction, ^z. We probe two different J ¼ 0
to J’ ¼ 1 transitions. The first transition is a 1S0 1P1 blue transition with a
natural linewidth of G ¼ 32 MHz and the second is a 1S0 3P1 red transition
with G ¼ 7.5 kHz. (b) In the coherent dipole model, photons are shared
between atoms. When the Doppler broadened linewidth becomes
comparable to the natural linewidth, dephasing must be considered. At our
B1 mK temperatures the Doppler broadening is E40 kHz, meaning
motional effects are important only for the red transition. (c) The three-
dimensional intensity distribution predicted for a blue probe beam. The
coupled dipole model predicts a strong 103 enhancement of the forward
intensity compared with other directions and a finite fluorescence along a
direction parallel to the incident polarization. The speckled pattern is due to
randomly positioned atoms and can be removed by averaging over multiple
atom configurations. NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 By scanning the probe frequency across resonance,
we extract the fluorescence linewidth, which is found to be
determined primarily by the OD of the cloud (open squares in
Fig. 2c). For the range of 0oODo20, the lineshape is Lorentzian
(see insets); however, the observed lineshape starts to flatten at
the centre for OD420. We have also varied the atom number by
a factor of four, and to an excellent approximation the linewidth
data are observed to collapse to the same curve when plotted as a
function of OD (open triangles). Forward observations. The coherent effect manifests itself
most clearly in the forward direction (Fig. 2). To separate the
forward fluorescence from the probe beam, we focus the probe
with a lens (L1) after it has passed through the atomic cloud and
then block it with a beam stopping blade, which can be translated
perpendicular to the probe beam (Fig. 2a inset). The same lens
(L1) also collimates the atomic fluorescence so that it can be
imaged onto DF. The position of the beam stopper can be used to
vary the angular range of collected fluorescence, characterized
by the angle (y) between ^x and the edge of the beam stopper
(see Methods). Using the maximum atom number available in
the experiment, the measured intensity Ix,0(y) is normalized to
that collected at ymax ¼ 7.5 mRad. Both the blue (square) and red
(triangle) transition results are displayed in Fig. 2a. For the
blue transition, we observe a 1,000-fold enhancement of the
normalized intensity for yo0.5 mRad. The enhancement is also
present for the red transition, but it is reduced by nearly two
orders of magnitude at small y due to the motional effect. On the Transverse observations. For independent emitters, the forward
fluorescence should have no dependence on the probe beam
polarization; however, the transverse fluorescence (along ^z)
should be highly sensitive to the probe polarization and it is even
classically forbidden if the probe is ^z polarized. However, multiple
scattering processes with dipolar interactions can completely
modify this picture by redistributing the atomic population in the
three excited magnetic states and thus scrambling the polarization
of the emitted fluorescence. Consequently, even for a ^z-polarized
probe there should be a finite emission along ^z (see Fig. 1c), with
an intensity that increases with increasing OD. Our experimental
investigation of the fluorescence properties along the transverse
direction is summarized in Fig. 3. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 We also observe motional effects on the red transition in contrast
to the same measurements on the blue transition. 88 We also observe motional effects on the red transition in contrast
to the same measurements on the blue transition. other hand, the wider angular area of enhancement is attributed
to the longer wavelength of the red transition. The forward
intensity strongly depends on the atom number. In Fig. 2b, we
present measurements of the forward intensity Ix versus the
transverse intensity Iz at a fixed y ¼ 2 mRad for different atom
numbers. The intensities are normalized to those obtained at the
peak atom number as used in Fig. 2a. To the first-order
approximation, the transverse fluorescence intensity scales line-
arly with the atom number. Hence, the forward intensity of both
the blue and red transitions scales approximately with the atom
number squared. In the experiment, up to 20 million 88Sr atoms are cooled to
B1 mK in a two-stage magneto-optical trap, the first based on the
blue transition and the second on the red transition. The
atomic cloud is then released from the magneto-optical trap and
allowed to expand for a variable time of flight (TOF), which
allows us to control its optical depth and density. They are
subsequently illuminated for 50(100) ms with a large-size probe
beam resonant with the blue (red) transition (Fig. 2a). The
resulting scattered light is measured with two detectors far away
from the cloud (see Fig. 1a). One detector is along the forward
direction ^x (detector DF) and the other along the transverse
direction ^z (detector DT, offset by B10). For a short TOF, the
atomic cloud is anisotropic and has an approximately Gaussian
distribution with an aspect ratio of Rx:Ry:Rz ¼ 2:2:1, where R{x,y,z}
are the root-mean-squared radii. We define OD as the on
resonance optical depth of the cloud, OD ¼
3N
2 kR? ð
Þ2, where R>
depends on the direction of observation with R>,T ¼ Rx ¼ Ry and
R>,F ¼ (RzRy)1/2 for the transverse and forward directions
respectively, N is the atom number and k is the laser wavevector
for the atomic transition (see Supplementary Note 1). In the forward direction, we have also investigated the
linewidth broadening of the blue transition as a function of the
atomic OD. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 Under the same OD along ^z,
the ^y-polarized probe beam (square) gives rise to a much more
broadened lineshape for the blue transition than the ^z-polarized
probe beam does (triangle), as shown in Fig. 3a. Meanwhile, the
peak intensity ratio of Iypol/Izpol decreases significantly with
an increasing OD, indicating the rapidly rising fluorescence with (mRad)
L2
L1
~2 mRad
1,000
1.0
120
OD = 1.8
Intensity
(MHz)
16
1
0
–75
75
0
–75
75
0
100
Linewidth (MHz)
80
60
40
20
0
5
10
Optical depth
15
20
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
–0.2
Blue probe
Red probe
Blue probe
Red probe
100
10
1
1
2
3
4
5
6
7
8
0
Ix,/Ix,,0
Ix,0(θ)/Ix,0(θmax)
Iz/Iz,0
a
b
c
Figure 2 | Forward scattering. (a) Comparison of forward scattering intensity versus angle using a red and blue probe beam. We use the setup shown in
the inset, to block the probe beam. After interacting with the atoms the probe beam is focused using a lens, which also collimates the fluorescence from the
atoms. We block the probe beam using a beam stopper, which we translate perpendicular to the probe beam, to change the angular range of fluorescence
collected by the detector, characterized by the angle (y) between ^x and the edge of the beam stopper (see Methods). The measured intensity, Ix,0(y), for
each probe beam is normalized to the intensity at ymax ¼ 7.5 mRad. The dephasing caused by motion reduces the forward intensity peak for the red
transition. (b) Comparison of intensity in the forward direction, Ix, versus intensity in the transverse direction, Iz. Both are varied by changing N. All
measurements are made at y ¼ 2 mRad (arrow in a) and normalized to the intensity, Ix,0, for the atom number used in a. (c) Linewidth broadening in the
forward direction measured by scanning the blue probe beam detuning, D, across resonance. Example lineshapes for different ODs are shown in the inset. Two different atom numbers are used, N ¼ 1.7(2) 107 (blue squares) and N/4 (cyan triangles). The dashed line represents G for reference. All solid curves
are based on the full theory of coupled dipoles and the band in c is for a ±20% atom number uncertainty. All error bars are for statistical uncertainties. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 (mRad)
L2
L1
1,000
Blue probe
Red probe
100
10
1
1
2
3
4
5
6
7
8
0
Ix,0(θ)/Ix,0(θmax)
a ~2 mRad
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
–0.2
Blue probe
Red probe
Ix,/Ix,,0
Iz/Iz,0
b b 120
OD = 1.8
Intensity
(MHz)
16
1
0
–75
75
0
–75
75
0
100
Linewidth (MHz)
80
60
40
20
0
5
10
Optical depth
15
20
c c a Linewidth (MHz) Optical depth Iz/Iz,0 Figure 2 | Forward scattering. (a) Comparison of forward scattering intensity versus angle using a red and blue probe beam. We use the setup shown in
the inset, to block the probe beam. After interacting with the atoms the probe beam is focused using a lens, which also collimates the fluorescence from the
atoms. We block the probe beam using a beam stopper, which we translate perpendicular to the probe beam, to change the angular range of fluorescence
collected by the detector, characterized by the angle (y) between ^x and the edge of the beam stopper (see Methods). The measured intensity, Ix,0(y), for
each probe beam is normalized to the intensity at ymax ¼ 7.5 mRad. The dephasing caused by motion reduces the forward intensity peak for the red
transition. (b) Comparison of intensity in the forward direction, Ix, versus intensity in the transverse direction, Iz. Both are varied by changing N. All
measurements are made at y ¼ 2 mRad (arrow in a) and normalized to the intensity, Ix,0, for the atom number used in a. (c) Linewidth broadening in the
forward direction measured by scanning the blue probe beam detuning, D, across resonance. Example lineshapes for different ODs are shown in the inset. Two different atom numbers are used, N ¼ 1.7(2) 107 (blue squares) and N/4 (cyan triangles). The dashed line represents G for reference. All solid curves
are based on the full theory of coupled dipoles and the band in c is for a ±20% atom number uncertainty. All error bars are for statistical uncertainties. Figure 2 | Forward scattering. (a) Comparison of forward scattering intensity versus angle using a red and blue probe beam. We use the setup shown in
the inset, to block the probe beam. After interacting with the atoms the probe beam is focused using a lens, which also collimates the fluorescence from the
atoms. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 We block the probe beam using a beam stopper, which we translate perpendicular to the probe beam, to change the angular range of fluorescence
collected by the detector, characterized by the angle (y) between ^x and the edge of the beam stopper (see Methods). The measured intensity, Ix,0(y), for
each probe beam is normalized to the intensity at ymax ¼ 7.5 mRad. The dephasing caused by motion reduces the forward intensity peak for the red
transition. (b) Comparison of intensity in the forward direction, Ix, versus intensity in the transverse direction, Iz. Both are varied by changing N. All
measurements are made at y ¼ 2 mRad (arrow in a) and normalized to the intensity, Ix,0, for the atom number used in a. (c) Linewidth broadening in the
forward direction measured by scanning the blue probe beam detuning, D, across resonance. Example lineshapes for different ODs are shown in the inset. Two different atom numbers are used, N ¼ 1.7(2) 107 (blue squares) and N/4 (cyan triangles). The dashed line represents G for reference. All solid curves
are based on the full theory of coupled dipoles and the band in c is for a ±20% atom number uncertainty. All error bars are for statistical uncertainties. Figure 2 | Forward scattering. (a) Comparison of forward scattering intensity versus angle using a red and blue probe beam. We use the setup shown in
the inset, to block the probe beam. After interacting with the atoms the probe beam is focused using a lens, which also collimates the fluorescence from the
atoms. We block the probe beam using a beam stopper, which we translate perpendicular to the probe beam, to change the angular range of fluorescence
collected by the detector, characterized by the angle (y) between ^x and the edge of the beam stopper (see Methods). The measured intensity, Ix,0(y), for
each probe beam is normalized to the intensity at ymax ¼ 7.5 mRad. The dephasing caused by motion reduces the forward intensity peak for the red transition. (b) Comparison of intensity in the forward direction, Ix, versus intensity in the transverse direction, Iz. Both are varied by changing N. All
measurements are made at y ¼ 2 mRad (arrow in a) and normalized to the intensity, Ix,0, for the atom number used in a. Results
E
i Experimental setup. Bosonic alkaline-earth atoms with zero
nuclear spin have simple atomic structure compared with the
more complex hyperfine structure present in typical alkali
metal atoms that complicates the modelling and interpretation
of the experimental observations. For example, 88Sr atoms have
both a strong 1S0 1P1 blue transition (l ¼ 461 nm) and a
spin-forbidden weak 1S0 3P1 red transition (l ¼ 689 nm), with a
strict four-level geometry (Fig. 1a). When the atoms are cooled to
a temperature of B1 mK, Doppler broadening at 461 nm is
B55 kHz, which is almost three orders of magnitude smaller
than the blue transition natural linewidth, G ¼ 32 MHz. To an
excellent approximation, atomic motion is negligible for atomic 2 ARTICLE NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 yp
p
icle scattering is dominant and for single particle scattering almost zero intensity is predicted for ^z-polarized
directly into the detector. (c) Linewidth broadening for the red transition in the transverse direction for ^y-polarized
e transition. This transition is more sensitive to magnetic fields; thus, a large magnetic field is applied to probe only
curves are based on the full theory of coupled dipoles and the band in a, b and c is for a ±20% atom number
tical uncertainties. used. For low optical depths single particle scattering is dominant and for single particle scattering almost zero intensity is predicted for ^z-polarized
fluorescence, as this polarization points directly into the detector. (c) Linewidth broadening for the red transition in the transverse direction for ^y-polarized
light, showing a similar trend to the blue transition. This transition is more sensitive to magnetic fields; thus, a large magnetic field is applied to probe only
the m ¼ 0 to m’ ¼ 0 transition. All solid curves are based on the full theory of coupled dipoles and the band in a, b and c is for a ±20% atom number
uncertainty. All error bars are for statistical uncertainties. a ^z-polarized probe when OD increases (Fig. 3b). For the red
transition, the existence of Doppler broadening requires the
lineshape data to be fitted to a Voigt profile. With the Doppler
linewidth DD fixed from the thermal velocity measured in free
expansion, the Voigt profile determines the line centre and the
Lorentzian linewidth with the Gaussian linewidth determined by
the temperature. Figure 3c displays the Lorentzian linewidth
obtained with a ^y-polarized red probe showing a strong increase
of the linewidth with OD. 0.1
Blue probe
Red probe
0.0
–0.1
Frequency shift /
–0.2
–0.3
0.0
0.5
1.0
Peak density (1012 cm–3)
1.5
2.0 0.1
Blue probe
Red probe
0.0
–0.1
Frequency shift /
–0.2
–0.3
0.0
0.5
1.0
Peak density (1012 cm–3)
1.5
2.0
Figure 4 | Frequency shift. Comparison of frequency shift normalized to
the corresponding natural linewidth for the blue and red transitions. The
blue frequency shift is consistent with 0–0.004 of G at an atomic density of
1012 cm 3. The red shift, on the other hand, shows more than 0.1G at
densities up to 0.7 1012 cm 3. Spectral broadening and shift. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 To a good approximation, the
dependence of the linewidth on OD along the forward and
transverse directions (for the classically allowed ^y polarization in
the single scattering limit) is similar. However, owing to the
anisotropic aspect ratio of the cloud, for the same TOF, the OD is
lower along ^z than along ^x. This is responsible for the smaller
broadenings measured along ^z than along ^x. The classically
forbidden polarization direction, on the other hand, exhibits a
different scaling with OD, which is understandable given that the
emission in this case comes only from multiple scattering events
with dipolar interactions. The transverse linewidth broadening
for the red transition is similar to that of the blue, and it does not
depend sensitively on motional effects. This behaviour is in stark
contrast to another important observation: the shift of the
transition centre frequency. Figure 4 contrasts the linecentre
frequency shift observed for 1S0 1P1 (square) and 1S0 3P1
(triangle, with original data reported in ref. 52 and see
Supplementary Fig. 1). The blue transition frequency shift is
consistent with zero at the level of 0.004G using an atomic density
of 1012 cm 3. However, the measured density shift for the red
transition (normalized to the transition linewidth) is more than
one order of magnitude larger. This density-related frequency
shift significantly exceeds the predicted value based on general
S-matrix
calculations
of
s-wave
collisions52
(2.18 10 10
Hz cm3 if the unitary limit is used). Figure 4 | Frequency shift. Comparison of frequency shift normalized to
the corresponding natural linewidth for the blue and red transitions. The
blue frequency shift is consistent with 0–0.004 of G at an atomic density of
1012 cm 3. The red shift, on the other hand, shows more than 0.1G at
densities up to 0.7 1012 cm 3. This simple semiclassical model recovers the linear dependence of
the forward width for small OD and predicts a nonlinear
dependence of the linewidth for large OD and a flattening of the
line centre. However, we find that this semiclassical approach
cannot provide explanations for most aspects of the experimental
observations. The full microscopic model builds on a set of coherently
coupled dipoles. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 (c) Linewidth broadening in the
forward direction measured by scanning the blue probe beam detuning, D, across resonance. Example lineshapes for different ODs are shown in the inset. Two different atom numbers are used, N ¼ 1.7(2) 107 (blue squares) and N/4 (cyan triangles). The dashed line represents G for reference. All solid curves
are based on the full theory of coupled dipoles and the band in c is for a ±20% atom number uncertainty. All error bars are for statistical uncertainties. 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 60
8
6
4
2
y Polarization
Linewidth (kHz)
Optical depth
5
10
15
20
25
z Polarization
55
50
45
40
35
30
30
25
20
15
10
5
0
Linewidth (MHz)
Iypol/Izpol
Optical depth
Optical depth
2
0
5
10
15
4
6
8
10
12
a
b
c
Figure 3 | Transverse scattering. (a) Linewidth broadening for the blue transition in the transverse direction for ^y polarization (open squares) and ^z
polarization (open triangles). (b) Intensity ratio, Iypol/Izpol, of ^y polarization to ^z polarization measured in the transverse direction when a blue probe beam is
used. For low optical depths single particle scattering is dominant and for single particle scattering almost zero intensity is predicted for ^z-polarized
fluorescence, as this polarization points directly into the detector. (c) Linewidth broadening for the red transition in the transverse direction for ^y-polarized
light, showing a similar trend to the blue transition. This transition is more sensitive to magnetic fields; thus, a large magnetic field is applied to probe only
the m ¼ 0 to m’ ¼ 0 transition. All solid curves are based on the full theory of coupled dipoles and the band in a, b and c is for a ±20% atom number
uncertainty. All error bars are for statistical uncertainties. 8
6
4
2
Iypol/Izpol
Optical depth
0
5
10
15
b 60
y Polarization
z Polarization
55
50
45
40
35
30
Linewidth (MHz)
Optical depth
2
4
6
8
10
12
a Linewidth (kHz)
Optical depth
5
10
15
20
25
30
25
20
15
10
5
0
c b c a Figure 3 | Transverse scattering. (a) Linewidth broadening for the blue transition in the transverse direction for ^y polarization (open squares) and ^z
polarization (open triangles). NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 (b) Intensity ratio, Iypol/Izpol, of ^y polarization to ^z polarization measured in the transverse direction when a blue probe beam is
used. For low optical depths single particle scattering is dominant and for single particle scattering almost zero intensity is predicted for ^z-polarized
fluorescence, as this polarization points directly into the detector. (c) Linewidth broadening for the red transition in the transverse direction for ^y-polarized
light, showing a similar trend to the blue transition. This transition is more sensitive to magnetic fields; thus, a large magnetic field is applied to probe only
the m ¼ 0 to m’ ¼ 0 transition. All solid curves are based on the full theory of coupled dipoles and the band in a, b and c is for a ±20% atom number
uncertainty. All error bars are for statistical uncertainties. a) Linewidth broadening for the blue transition in the transverse direction for ^y polarization (open squares) and ^z
ensity ratio Iypol/Izpol of ^y polarization to ^z polarization measured in the transverse direction when a blue probe beam is tering. (a) Linewidth broadening for the blue transition in the transverse direction for ^y polarization (open squares) a
) (b) I t
it
ti
I
/I
f ^
l
i
ti
t ^
l
i
ti
d i th t
di
ti
h
bl
b Figure 3 | Transverse scattering. (a) Linewidth broadening for the blue transition in the transverse direction for y polarization (open squares) and z
polarization (open triangles). (b) Intensity ratio, Iypol/Izpol, of ^y polarization to ^z polarization measured in the transverse direction when a blue probe beam is
used. For low optical depths single particle scattering is dominant and for single particle scattering almost zero intensity is predicted for ^z-polarized
fluorescence, as this polarization points directly into the detector. (c) Linewidth broadening for the red transition in the transverse direction for ^y-polarized
light, showing a similar trend to the blue transition. This transition is more sensitive to magnetic fields; thus, a large magnetic field is applied to probe only
the m ¼ 0 to m’ ¼ 0 transition. All solid curves are based on the full theory of coupled dipoles and the band in a, b and c is for a ±20% atom number
uncertainty. All error bars are for statistical uncertainties. Discussion To
understand
the
forward
enhancement
we
first
consider
non-interacting
atoms
under
the
zeroth
order
approximation. The
atomic
coherence
is
driven
only
by
the probe field that imprints its phase and polarization onto
the atoms: ba 0
ð Þ
j
¼ ida;xOxeikrj =2
iD G=2 , where D ¼ Da. The corresponding
intensity, I rs
ð Þ¼ 9‘ 2G2
16k2m2r2s
Ox
j
j
2
4 D2 þ G2=4
ð
Þ ðN þ N2e ks k0
j
j2R2
?;FÞ has a
2 ð
Þ
Lorentzian profile. It also exhibits an N2 scaling and an enhanced
forward emission lobe, with an angular width given by the ratio
between the transition wavelength and the transverse size of the
sample Dy 1= kR?;F
. The forward lobe reflects the construc-
tive interference of the coherently emitted radiation stimulated by
the laser. Outside the coherent lobe the constructive interference
is quickly reduced due to the random position of atoms28,59,61. The longer wavelength of the red transition corresponds
to a wider angular width of the forward lobe for the red
fluorescence. p
g
So far, we have shown the observed effects on linewidth and
fluorescence intensity are uniquely determined by OD. However,
following the arguments discussed above, the frequency shift
arising from the dipolar coupling is expected to scale with atomic
density, D
=G / n0k 3, which includes both the collective Lamb
shift and the Lorentz–Lorenz shift50,65. For our experimental
density, this effect is t10 3, which is consistent with the
observed frequency shift for the blue transition (Fig. 4). (It is
noteworthy that the role of multiple scattering processes is to
further suppress this frequency shift mechanism50.) In contrast,
for the red transition the measured density shift (normalized to
G) is significantly larger than what is predicted from the current
treatment of interacting dipoles; it is also much bigger than the
unitarity limit of s-wave scattering. Qualitatively, we expect that
as the atoms move and approach each other, the long-lived
ground-excited state coherence in the red transition can be
significantly modified by the collisional process and open higher
partial wave channels. We can thus expect a larger collisional
phase shift. This process can be further complicated by atomic
recoil, light forces and Doppler dephasing66. Simple considerations can also give rise to a qualitative
understanding of atomic motion-related effects on forward
enhancement. with m is the atomic transition dipole moment and ks¼k^rs. with m is the atomic transition dipole moment and ks¼k^rs. p
Meanwhile, for the linewidth broadening observed in the
forward direction, it becomes evident that the scaling of the
linewidth versus OD turns nonlinear at large values of OD. The
experimental data falls within the shaded area in Fig. 2c, which
represents the full solution with a 20% uncertainty in the
experimental atom number. Multiple scattering processes are also
key to the explanation of the measured fluorescence along the
transverse direction, especially for the classically forbidden
polarization
^z. Indeed,
for
both
intensity
and
linewidth
broadening observed in the transverse direction, under either ^y
or ^z probe polarization, the full model (shown as shaded areas
in both Fig. 3a,b) reproduces well the experimental results
on
1S0 1P1. Taking
into
account
motional
dephasing
(see Supplementary Note 2), the transverse broadening for
1S0 3P1 is also well reproduced as shown in Fig. 3c. Discussion Again for the red transition, the Doppler effect
introduces random phases accumulated by df kv=G. Here, v is
the thermal velocity. The dephasing reduces coherent photon
emission and gives rise to a net suppression of the forward
emission intensity. The suppression becomes stronger with DD/G,
with DD¼
ffiffiffiffiffiffiffiffiffiffiffiffiffi
kBT
8ml2 ln 2
q
the Doppler width. Such a suppression is
clearly observed for the red transition. To address the linewidth broadening we now consider atoms
coupled by dipolar interactions, which tend to emit collectively in
an optically dense cloud. The collective emission manifests itself
with a broader fluorescence linewidth. Moving to the first-
order approximation, we note that the atomic coherence
acquires contributions not only from the probe beam but
also from the surrounding atoms, with ba
j ba 0
ð Þ
j
þ ba 1
ð Þ
j
. Here,
ba 1
ð Þ
j
¼
iOxG=2
iD G=2
ð
Þ2 Kj
a;xeikxj and Kj
a;a0¼ P
m 6¼ j Ga;a0 rj rm
eik xm xj
ð
Þ:
For a relatively dilute cloud with average interparticle distance
r 1=k, the far-field interactions dominate; thus, higher-order
terms beyond 1/r can be neglected. Dipolar interactions modify
the fluorescence lineshape, with consequences of both a frequency
shift that depends on the cloud peak density n0 and a line
broadening
that
is
proportional
to
OD:
D ! D þ D
and
G ! G þ G, with D¼ 3 ffiffi
2
p
pn0k 3
16
G and G¼ OD
4 G. Thus, the
first-order approximation provides an intuitive picture about the
role of dipolar effects on the lineshape. We have shown that a coherent dipole model describes light
scattering in a dense atomic medium with collective effects and
multiple scatterings. The model captures the quantitative features
of the experimental observations. Motional effects, as manifested
in dephasing, can be captured in the model as well. Our results
provide useful guides for further developments of optical atomic
clocks and other applications involving dense atomic ensembles. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 Here, each four-level atom is treated as a discrete
radiating dipole located at a frozen position, but coupled with
retarded dipole radiation, and it is driven with a weak incident
laser
beam. The
atomic
ensemble
follows
the
Gaussian
distribution observed in the experiment with the appropriate
aspect ratio. By solving the master equation in the steady state, we
find that the coherence, ba
j ¼ Tr½ gj ihea
j j^r, of atom j, located at rj is
modified by other atoms as18,54–60: ba
j ¼
Oxeikrj
2 Da þ i G
2
da;x þ G
X
a0;m 6¼ j
Ga;a0 rj rm
iDa G
2
ba0
m:
ð1Þ ð1Þ Theory model. Before we turn to a microscopic model to obtain a
full and consistent understanding of all these related experimental
observations, we note that semiclassical models53 treating the
atomic cloud as a continuous medium of an appropriate
refractive index can give an intuitive explanation of the
linewidth broadening in the forward direction. Classically, an
incoming electric field is attenuated as it propagates through the
medium according to the Beer–Lambert law and the forward
fluorescence intensity is determined by the same mechanism. Here,
gj i¼1S0,
ea
j i corresponds to the three excited states
of 1P1 or 3P1, with aA{x, y, z} representing the Cartesian
states. In addition, ^r is the reduced density matrix of the
atoms and dg,g0 is the Kronecker Delta. The driving laser’s linear
polarization state x is along ^y or ^z, with wavevector k along ^x,
Rabi frequency Ox and detuned by Da from the
gj i ! ea
j
i
transition. The function Ga,a0(r) accounts for the retarded NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 pairwise
dipolar
interactions
and
is
given
by18,48,56
Ga;a0ðrÞ¼ i 3
4
eikr
kr ½ da;a0 ^ra^ra0
þ da;a0 3^ra^ra0
ð i
kr
1
ðkrÞ2Þ. The
fluorescence
intensity
I rs
ð Þ¼h^E
þ
ð
Þ rs
ð Þ^E
ð
Þ rs
ð Þi,
detected
at position rs, can be determined17,18 as a function of ba
j , arise from the forward fluorescence intensity, where its naive
N2 scaling is reduced with an increasing atom number as a
consequence of multiple scattering processes. This effect is
observed in both red and blue calculations, and it is expected to
be more pronounced on the red transition due to its longer
wavelength. However, atomic motion leads to a lower effective
OD, which tends to suppress multiple scattering processes and
thus helps to partially recover the collective enhancement. The
solid
lines
in
Fig. 2a,b
represent
such
quantitative
theory calculations for both transitions, which agree with the
experiment. I rs
ð Þ
9‘ 2G2
16k2m2r2
s
X
j;m
bj b
m bj ^rs
b
m ^rs
eiks rj rm
ð
Þ;
ð2Þ
with m is the atomic transition dipole moment and ks¼k^rs. I rs
ð Þ
9‘ 2G2
16k2m2r2
s
X
j;m
bj b
m bj ^rs
b
m ^rs
eiks rj rm
ð
Þ;
ð2Þ ð2Þ Methods
Coherent d Coherent dipole model. Here we present the derivation of equation (1). The
fundamental assumption is to treat the atoms as frozen during the interrogation. This is an excellent approximation if :G is much faster than other energy scales in
the problem. The latter condition is always satisfied in the case of the blue
transition. For the J ¼ 0 to J ¼ 1 configuration exhibited by 88Sr, we can label the
J ¼ 0 ground state as gj i and the excited J ¼ 1 states using a Cartesian basis
ez
j i¼ 0j i, ex
j
i¼
1
j
i þ 1
j
i
ð
Þ=
ffiffiffi
2
p
, ey
j i¼i
1
j
i þ þ 1
j
i
ð
Þ=
ffiffiffi
2
p
. Here, the |0, ±1i
states are the standard angular momentum ones. In the Cartesian basis,
the vector transition operator for the j atom located at rj can be written as
ba
j ¼^x^bx
j þ^y^by
j þ^z^bz
j . Here ^ba
j ¼ gj ij ea
h
j. On this basis, the master equation
governing the evolution of the reduced density matrix of the N atom ensemble,
^r, in the presence of an incident laser beam with linear polarization x can be However, in a cloud with an increasingly large OD, dipolar
interactions are stronger and multiple scattering processes
become relevant. The first-order perturbative analysis then breaks
down62–64. The full solution of equation (1) based on the
coherent coupled dipole model becomes necessary to account for
multiple scattering processes (see Methods). The first signatures 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 written as18: where the first lens with a 15-cm focal length collimates the fluorescence. In
numerical calculations, the CCD camera is simulated as a ring area centred around
the forward direction and the average intensity collected over the ring is
determined. The external radius is set to be large enough to reach the angular
region outside the interference cone and the inner angular radius ysim, simulating
the blocking of the signal by the beam stopper, is varied accordingly to the
experiment. To account for the difference between ssim and the experiment cloud
size, ysim is rescaled so that we satisfy the experimental observation that at ymax the
enhancement factor drops to 1. References Jennewein, S., Sortais, Y. R. P., Greffet, J.-J. & Browaeys, A. Propagation of p
y
y
21. Jennewein, S., Sortais, Y. R. P., Greffet, J.-J. & Browaeys, A. Propagation of
Light Through Small Clouds of Cold Interacting Atoms. http://arxiv.org/abs/
1511.08527 (2015). Light Through Small Clouds of Cold Interacting Atoms. http://arxiv.org/abs/
1511.08527 (2015).
h
i
h
i
As we are interested in the situation of a weak probe limit, Ox G, we expand
the density matrix in successive orders of Ox/G, ^r¼^r 0
ð Þ þ ^r 1
ð Þ þ ^r 2
ð Þ þ . . . , and
keep the first-order terms. At this level of approximation, rG,G ¼ 1, rja,mb ¼ 0 and
only the optical coherences ba
j
rja;G evolve in time accordingly to the following
set of linear equations: 22. Jennewein, S. et al. Observation of the Failure of Lorentz Local Field Theory in
the Optical Response of Dense and Cold Atomic Systems. http://arxiv.org/abs/
1510.08041 (2015). 23. Jenkins, S. D. et al. Optical Resonance Shifts in the Fluorescence Imaging of
Thermal and Cold Rubidium Atomic Gases. http://arxiv.org/abs/1602.01037
(2016). dba
j
dt ¼
iDa G
2
ba
j i
2 Ojda;x i
X
m 6¼ j;b
gab
jm if ab
jm
bb
m:
ð10Þ ð10Þ 24. Ro¨hlsberger, R., Schlage, K., Sahoo, B., Couet, S. & Ru¨ffer, R. Collective lamb
shift in single-photon superradiance. Science 328, 1248–1251 (2010). Here, Ga,b(r) ¼ (fa,b(r) þ iga,b(r))/G. The steady-state solution can be
dba obtained by setting
dba
j
dt ¼0 and then solving the subsequent 3N linear equations. 25. Balik, S., Win, A. L., Havey, M. D., Sokolov, I. M. & Kupriyanov, D. V. Near-resonance light scattering from a high-density ultracold atomic 87Rb gas
Phys. Rev. A 87, 053817 (2013). Measure the enhancement of forward fluorescence. To measure the scattered
light in the forward direction, we use the setup shown in the inset of Fig. 2a, to
tightly focus and block the probe beam, while still collecting most of the atomic
fluorescence on the CCD (charge-coupled device) camera. We focus the probe
beam, after it interacts with the atoms, to a small spot with 15 mm root-mean-
squared radius and block it using a beam stopping blade. Methods
Coherent d d^r
dt ¼ i
2
P
j;a
da;x Oj^bay
j
þ O
j ^ba
j ; ^r
h
i
i P
j;m 6¼ j
a;b
gab
jm^bay
j ^bb
j ; ^r
h
i
þ i P
j;a
Da½^bay
j ^ba
j ; ^r þ P
j;m
a;b
f ab
jm
2^ba
j ^r^bby
m
^bay
j ^bb
m; ^r
n
o
; ð3Þ where Oj¼Oxeikrj is the Rabi frequecy of the incident field, polarized along x
ð^x k¼0Þ and detuned by Da from the atomic transition gj i ! ea
j
i. The
parameters gab
jm¼ga;b rj rm
and f ab
jm ¼fa;b rj rm
are the components of the
elastic and inelastic dipolar interactions between a pair of atoms at position rj and
rm, respectively, and are given by where Oj¼Oxeikrj is the Rabi frequecy of the incident field, polarized along x
ð^x k¼0Þ and detuned by Da from the atomic transition gj i ! ea
j
i. The
parameters gab
jm¼ga;b rj rm
and f ab
jm ¼fa;b rj rm
are the components of the
elastic and inelastic dipolar interactions between a pair of atoms at position rj and
rm, respectively, and are given by ðx k¼0Þ and detuned by Da from the atomic transition gj i ! ea
j
i. The
parameters gab
jm¼ga;b rj rm
and f ab
jm ¼fa;b rj rm
are the components of the
elastic and inelastic dipolar interactions between a pair of atoms at position rj and
rm, respectively, and are given by References 1. Gross, M. & Haroche, S. Superradiance: an essay on the theory of collective
spontaneous emission. Phys. Rep. 93, 301–396 (1982). ga;b rð Þ¼ 3G
4
y0 kr
ð
Þ y1 kr
ð
Þ
kr
da;b þ y2 kr
ð
Þ^ra^rb
;
ð4Þ
fa;b rð Þ¼ 3G
4
j0 kr
ð
Þ j1 kr
ð
Þ
kr
da;b þ j2 kr
ð
Þ^ra^rb
;
ð5Þ y
p
2. Andreev, A. V., Emel’yanov, V. I. & Il’inskii, Y. A. Collective spontaneous
emission (Dicke superradiance). Sov. Phys. Usp. 23, 493–514 (1980). 3. Dicke, R. H. Coherence in spontaneous radiation processes. Phys. Rev. 93,
99–110 (1954). 4. Bloom, B. J. et al. A new generation of atomic clocks: accuracy and stability at
the 10 18 level. Nature 506, 71–75 (2014). where r¼ rj j ¼ rj rm
, yn(x), jn(x) are the spherical Bessel functions of the
d
d fi
k
d
l
l
b where r¼ rj j ¼ rj rm
, yn(x), jn(x) are the spherical Bessel functions of the
second and first kind, respectively. Here, also a, b ¼ x, y or z represent
Cartesian components. The symbol dg,g0 is the Kronecker Delta. In the low-
intensity limit, we can project the density matrix into a state space including the
ground state G
j i
g1; g2; :::gN
j
i and states with only one excitation57–59 such
as ja
j i
g1; :::ea
j ; :::gN
E
. In this reduced space, the relevant equations of motion
simplify to where r¼ rj j ¼ rj rm
, yn(x), jn(x) are the spherical Bessel functions of the
second and first kind, respectively. Here, also a, b ¼ x, y or z represent where r¼ rj j ¼ rj rm
, yn(x), jn(x) are the spherical Bessel functions of the
second and first kind, respectively. Here, also a, b ¼ x, y or z represent
Cartesian components. The symbol dg,g0 is the Kronecker Delta. In the low-
intensity limit, we can project the density matrix into a state space including the
ground state G
j i
g1; g2; :::gN
j
i and states with only one excitation57–59 such
as ja
j i
g1; :::ea
j ; :::gN
E
. In this reduced space, the relevant equations of motion
simplify to 5. Kimble, H. J. The quantum internet. References drja;G
dt
¼ i
2
Ojda;xrG;G P
m
Omrja;mx
i P
m 6¼ j;b
gab
jm if ab
jm
rmb;G
þ iDa G
2
rja;G; 11. Friedberg, R. & Hartmann, S. R. Temporal evolution of superradiance in a small sphere. Phys. Rev. A 10, 1728–1739 (1974). ð7Þ ð7Þ 12. Lewenstein, M. & Rzazewski, K. Collective radiation and the near-zone field. J. Phys. A Math. Gen 13, 743–756 (1980). 13. Steudel, H. & Richter, T. Radiation properties of a continuously pumped
two-atom system. Ann. Phys. (Berlin) 490, 122–136 (1978). drja;mb
dt
¼ i
2 Ojda;xrG;mb O
mdx;brja;G
þ iðDa DbÞrja;mb
i
P
l 6¼ j;n
rln;mbga;n
jl P
l 6¼ m;n
rja;lngb;n
lm
! Grja;mb
P
l 6¼ j;n
rln;mbf an
jl þ
P
l 6¼ m;n
rja;lnf bn
lm
! ;
drG;G
dt
¼ i
2
P
j;a
O
j rja;G OjrG;ja
! þ G 1 rG;G
þ
P
m;j 6¼ m
a;b
f ab
jm
rja;mb þ rmb;ja
: 14. Rzaz˙ewski, K. & Z˙akowicz, W. Initial value problem for two oscillators
interacting with electromagnetic field. J. Math. Phys. 21, 378–388 (1980). 15. Ruostekoski, J. & Javanainen, J. Quantum field theory of cooperative atom
response: low light intensity. Phys. Rev. A 55, 513–526 (1997). ð8Þ 16. Scully, M. O. Collective Lamb shift in single photon dicke superradiance. Phys. Rev. Lett. 102, 143601 (2009). y
17. Lehmberg, R. H. Radiation from an N-atom system. I. General formalism. Phys. Rev. A 2, 883–888 (1970). y
18. James, D. F. V. Frequency shifts in spontaneous emission from two interacting
atoms. Phys. Rev. A 47, 1336–1346 (1993). ð9Þ 19. Keaveney, J. et al. Cooperative lamb shift in an atomic vapor layer of nanometer
thickness. Phys. Rev. Lett. 108, 173601 (2012). 20. Meir, Z., Schwartz, O., Shahmoon, E., Oron, D. & Ozeri, R. Cooperative lamb
shift in a mesoscopic atomic array. Phys. Rev. Lett. 113, 193002 (2014). where rG;G¼ Tr ^r G
j i G
h j
½
, rja;mb¼ Tr ^r ^bby
m ^ba
j
h
i
and rja;G¼ Tr ^ba
j ^r
h
i
. where rG;G¼ Tr ^r G
j i G
h j
½
, rja;mb¼ Tr ^r ^bby
m ^ba
j
h
i
and rja;G¼ Tr ^ba
j ^r
h
i
. p
y
y
21. References Nature 453, 1023–1030 (2008). 6. Skribanowitz, N., Herman, I. P., MacGillivray, J. C. & Feld, M. S. Observation
of Dicke superradiance in optically pumped hf gas. Phys. Rev. Lett. 30, 309–312
(1973). Cartesian components. The symbol dg,g0 is the Kronecker Delta. In the low-
intensity limit, we can project the density matrix into a state space including the
ground state G
j i
g1; g2; :::gN
j
i and states with only one excitation57–59 such
as ja
j i
g1; :::ea
j ; :::gN
E
. In this reduced space, the relevant equations of motion
simplify to 7. Pavolini, D., Crubellier, A., Pillet, P., Cabaret, L. & Liberman, S. Experimental
evidence for subradiance. Phys. Rev. Lett. 54, 1917–1920 (1985). 8. Wang, T. et al. Superradiance in ultracold rydberg gases. Phys. Rev. A 75,
033802 (2007). drja;ja
dt
¼ i
2 Ojda;xrG;ja O
j da;xrja;G
i P
m 6¼ j;b
gab
jm rmb;ja rja;mb
P
m 6¼ j;b
f ab
jm
rmb;ja þ rja;mb
Grja;ja;
drja;G
dt
¼ i
2
Ojda;xrG;G P
m
Omrja;mx
i P
m 6¼ j;b
gab
jm if ab
jm
rmb;G
þ iDa G
2
rja;G;
drja;mb
dt
¼ i
2 Ojda;xrG;mb O
mdx;brja;G
þ iðDa DbÞrja;mb
i
P
l 6¼ j;n
rln;mbga;n
jl P
l 6¼ m;n
rja;lngb;n
lm
! Grja;mb
P
l 6¼ j;n
rln;mbf an
jl þ
P
l 6¼ m;n
rja;lnf bn
lm
! ;
drG;G
dt
¼ i
2
P
j;a
O
j rja;G OjrG;ja
! þ G 1 rG;G
þ
P
m;j 6¼ m
a;b
f ab
jm
rja;mb þ rmb;ja
: 9. Agarwal, G. S., Saxena, R., Narducci, L. M., Feng, D. H. & Gilmore, R. Analytical solution for the spectrum of resonance fluorescence of a cooperative
system of two atoms and the existence of additional sidebands. Phys. Rev. A 21,
257–259 (1980). ð6Þ 10. Friedberg, R. & Hartmann, S. Superradiant stability in specially shaped small
samples. Opt. Commun. 10, 298–301 (1974). 11. Friedberg, R. & Hartmann, S. R. Temporal evolution of superradiance in a
small sphere Phys Rev A 10 1728–1739 (1974) 10. Friedberg, R. & Hartmann, S. Superradiant stability in specially shaped small
samples. Opt. Commun. 10, 298–301 (1974). NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11039 58. Bienaime´, T., Bachelard, R., Piovella, N. & Kaiser, R. Cooperativity in light
scattering by cold atoms. Fortschr. Phys. 61, 377–392 (2013). 31. de Vries, P., van Coevorden, D. V. & Lagendijk, A. Point scatterers for classical
waves. Rev. Mod. Phys. 70, 447–466 (1998). g
y
y
59. Bienaime´, T. et al. Interplay between radiation pressure force and scattered light
intensity in the cooperative scattering by cold atoms. J. Mod. Opt. 61, 18–24
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waves: microscopy, mesoscopy, and diffusion. Rev. Mod. Phys. 71, 313–371
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neutral atoms. Phys. Rev. Lett. 64, 408–411 (1990). 60. Mu¨ller, C. A. & Miniatura, C. Multiple scattering of light by atoms with internal
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34. Heidemann, R. et al. Evidence for coherent collective Rydberg excitation in the
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y
35. Lukin, M. D. et al. Dipole blockade and quantum information processing in
mesoscopic atomic ensembles. Phys. Rev. Lett. 87, 037901 (2001). 62. Pellegrino, J. et al. Observation of suppression of light scattering induced by
dipole-dipole interactions in a cold-atom ensemble. Phys. Rev. Lett. 113, 133602
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atoms. Rev. Mod. Phys. 82, 2313–2363 (2010). 63. Fleischhauer, M. & Yelin, S. F. Radiative atom-atom interactions in optically
dense media: quantum corrections to the lorentz-lorenz formula. Phys. Rev. A
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37. Lahaye, T., Menotti, C., Santos, L., Lewenstein, M. & Pfau, T. The physics of
dipolar bosonic quantum gases. Rep. Prog. Phys. 72, 126401 (2009). 64. Lin, G.-D. & Yelin, S. F. in Advances in Atomic, Molecular, and Optical Physics
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absorption by resonant systems ot two-level atoms. Phys. Rep. 7, 101–179
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their important insights and stimulating discussions. This research is supported by NIST,
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total uncertainty. Nat. Commun. 6, 6896 (2015). S.L.B., M.B., X.Z., T.B., T.L.N. and J.Y. contributed to the executions of the experiments. B.Z., J.S., R.K., S.F.Y., M.D.L. and A.M.R. contributed to the development of the theory
model. All authors discussed the results, contributed to the data analysis and worked
together on the manuscript. 47. Ushijima, I., Takamoto, M., Das, M., Ohkubo, T. & Katori, H. Cryogenic optical
lattice clocks. Nat. Photon. 9, 185–189 (2015). 48. Olmos, B. et al. Long-range interacting many-body systems with alkaline-earth-
metal atoms. Phys. Rev. Lett. 110, 143602 (2013). 49. Specht, H. P. et al. A single-atom quantum memory. Nature 473, 190–193
(2011). References We then translate the
beam stopper perpendicular to the probe beam by a distance Dx from our reference
point of x ¼ 0, which we define as the position of the beam stopper where we see
the greatest fluorescence without saturating the CCD camera with the probe beam. As only the forward direction is particularly sensitive to positional changes, we
convert this change in position to a change in angle simply using y¼ arctan Dx
15cm, y
26. Chalony, M., Pierrat, R., Delande, D. & Wilkowski, D. Coherent flash of light
emitted by a cold atomic cloud. Phys. Rev. A 84, 011401 (2011). 27. Guerin, W., Araujo, M. O. & Kaiser, R. Subradiance in a large cloud of cold
atoms. Phys. Rev. Lett. 116, 083601 (2016). 28. Kwong, C. C. et al. Cooperative emission of a coherent superflash of light. Phys. Rev. Lett. 113, 223601 (2014). 29. Labeyrie, G. et al. Coherent backscattering of light by cold atoms. Phys. Rev. Lett. 83, 5266–5269 (1999). 30. Kulatunga, P. et al. Measurement of correlated multiple light scattering in
ultracold atomic 85Rb. Phys. Rev. A 68, 033816 (2003). NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications 6 Additional information 50. Javanainen, J., Ruostekoski, J., Li, Y. & Yoo, S.-M. Shifts of a resonance line in a
dense atomic sample. Phys. Rev. Lett. 112, 113603 (2014). Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications p
y
51. Labeyrie, G., Delande, D., Kaiser, R. & Miniatura, C. Light transport in cold
atoms and thermal decoherence. Phys. Rev. Lett. 97, 013004 (2006). 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 no competing financial interests. 52. Ido, T. et al. Precision spectroscopy and density-dependent frequency shifts in
ultracold Sr. Phys. Rev. Lett. 94, 153001 (2005). Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ 53. Lagendijk, A. & Van Tiggelen, B. A. Resonant multiple scattering of light. Phys. Rep 270, 143–215 (1996). How to cite this article: Bromley, S. L. et al. Collective atomic scattering and motional
effects in a dense coherent medium. Nat. Commun. 7:11039 doi: 10.1038/ncomms11039
(2016). How to cite this article: Bromley, S. L. et al. Collective atomic scattering and motional
effects in a dense coherent medium. Nat. Commun. 7:11039 doi: 10.1038/ncomms11039
(2016). 54. Morice, O., Castin, Y. & Dalibard, J. Refractive index of a dilute Bose gas. Phys. Rev. A 51, 3896–3901 (1995). 55. Rouabah, M.-T. et al. Coherence effects in scattering order expansion of light by
atomic clouds. J. Opt. Soc. Am. A 31, 1031–1039 (2014). atomic clouds. J. Opt. Soc. Am. A 31, 1031–1039 (2014). This work is licensed under a Creative Commons Attribution 4.0
International License. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise
in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 56. Chomaz, L., Corman, L., Yefsah, T., Desbuquois, R. & Dalibard, J. Absorption
imaging of a quasi-two-dimensional gas: a multiple scattering analysis. New J. Phys. 14, 055001 (2012). 57. Bienaime´, T., Petruzzo, M., Bigerni, D., Piovella, N. & Kaiser, R. Atom and
photon measurement in cooperative scattering by cold atoms. J. Mod. Opt. 58,
1942–1950 (2011). 7 NATURE COMMUNICATIONS | 7:11039 | DOI: 10.1038/ncomms11039 | www.nature.com/naturecommunications
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https://openalex.org/W2152887453
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English
| null |
The influence of bevacizumab on platelet function
|
Schweizerische medizinische Wochenschrift
| 2,011
|
cc-by
| 5,512
|
Summary Systemic treatment with bevacizumab is associated with
increased rates of arterial and venous thromboembolism
and haemorrhage. In order to investigate the patho-
physiological mechanism involved, platelet adhesive and
aggregatory functions were tested with a platelet function
analyser (PFA-100®) in an in vitro study and in a longitud-
inal clinical observation study. For the in vitro study, blood
from ten healthy volunteers was incubated with different
concentrations of bevacizumab (0–1000 μg/ml plasma) and
vascular endothelial growth factor (0–500 μg/ml). In the
clinical observation study, PFA-100® closure times (CTs)
and soluble P-selectin (sP-selectin) serum levels as a sero-
logical marker of platelet activation were assessed in 20 pa-
tients with metastatic cancer who were treated with bevaci-
zumab in addition to cytotoxic chemotherapy. No signific-
ant changes of PFA-100® CTs were observed in the in vitro
study. In the clinical observation study, mean PFA-100®
CTs after treatment with bevacizumab were unchanged. sP-
selectin was decreased after bevacizumab infusion by 18%
(p = 0.045), which could suggest an inhibitory action on
platelets. Our data do not support the view that increased
platelet activation or increased platelet adhesiveness and
aggregation by bevacizumab are relevant mechanisms for
thrombus formation in clinical practice. Key words: Bevacizumab; platelet function; haemorrhage;
thrombosis; PFA-100; soluble P-selectin; von Willebrand
antigen The pathophysiological mechanisms leading to these side
effects are poorly understood. Data from in vitro experi-
ments and animal models point to a possible influence of
bevacizumab in primary haemostasis and platelet function. Recently VEGF and VEGF receptors (VEGF-R) have been
found to be relevant mediators of platelet aggregation [10,
11]. Both of these targets represent potential sites at which
bevacizumab could potentially interact with primary hae-
mostasis. The presence of VEGF-R messenger-RNA and
the membranous localisation of VEGF on platelets have
been known for some years [12, 13]. In addition, the pres-
ence and functions of VEGF-R1 (fms-like tyrosine kinase
receptor, Flt-1) and VEGF-R2 (kinase-insert domain re-
gion, KDR) on human platelets has been demonstrated. In vitro tests have shown a stimulatory effect of VEGF
on thrombin-induced platelet activation. This suggests that Original article | Published 30 July 2011, doi:10.4414/smw.2011.13243
Cite this as: Swiss Med Wkly. 2011;141:w13243 Original article | Published 30 July 2011, doi:10.4414/smw.2011.13243 Original article | Published 30 July 2011, doi:10.4414/smw.2011.13243
Cite this as: Swiss Med Wkly. 2011;141:w13243 The influence of bevacizumab on platelet function rtin Fehra, Sereina Catschegnb, Walter H. Reinhartb, Jerzy Madonc, Lars Asmisc, Richard Cathomasa, Roger von Moo a Division of Oncology, Kantonsspital Graubünden, Chur, Switzerland
b Department of Medicine, Kantonsspital Graubünden, Chur, Switzerland
c Division of Haematology, University Hospital, Zürich, Switzerland cancer patients has recently been demonstrated [4]. The
use of bevacizumab has also been associated with an in-
creased risk of developing venous and arterial thromboem-
bolic events and haemorrhage. In a meta-analysis, patients
treated with bevacizumab had an increased risk of venous
thromboembolism with a relative risk (RR) of 1.33 and
an overall incidence rate of 11.9% [5]. The risk of arterial
thromboembolic events such as a myocardial infarction or
stroke was found to be increased 2-fold with an event rate
of 5.5 per 100 person-years [6]. Another recently published
meta-analysis of 20 randomized trials confirmed that be-
vacizumab was associated with an increased risk of arter-
ial ischaemia with an overall RR of 1.46 [7]. In a large
observational treatment study in patients with metastatic
colorectal cancer the incidence rates of clinically signific-
ant bleeding associated with bevacizumab was 2.4% [8]. In
a randomized phase III trial of patients with advanced non-
small-cell lung cancer significant haemorrhage was repor-
ted in 4.4% of patients treated with bevacizumab [2]. In-
creased treatment related mortality of bevacizumab in com-
bination with chemotherapy or biological therapy (RR of
1.43) as compared to chemotherapy alone has been demon-
strated in a recently published meta-analysis of 16 random-
ized controlled trials with incidence rates of fatal adverse
events (FAE) of 2.5% versus 1.7%. Haemorrhage was the
most common FAE linked to the use of bevacizumab with
23.5% of all FAEs [9]. In vitro study Blood was drawn atraumatically from an antecubital vein
from ten healthy, non-smoking Caucasian volunteers (four
females, six males, age 20–59 years), using a standard tour-
niquet with a cuff pressure of 40 mm Hg, an 18 gauge but-
terfly needle and a standard vacutainer system with 1 part
0.126 M sodium citrate for 9 parts of blood. Six of the 20 patients included had a previously implanted
central venous access device with a small reservoir
(CVAD; “Port-a-cath“), which was used for blood drawing
and administration of drugs. In the remaining patients
blood was drawn in a similar way as described above be-
fore and 15 minutes after the first i.v. application of be-
vacizumab and again after 2-3 infusions of bevacizumab,
depending on the schedule (6 weeks of treatment). The dur-
ation of the first infusion was 90 minutes. No other medic-
ation was given between the two blood samples. A portion
of the blood samples was processed on the same day ac-
cording to current operational standards for the PFA-100®
[14]. Another portion of the blood samples was immedi-
ately centrifuged and the serum was stored at –80° Celsius
until being further processed for the sP-selectin measure-
ments. SP-selectin and vWF:Ag measurements were per-
formed at the Laboratory for Coagulation Studies at the
University Hospital of Zürich, Switzerland, using com-
mercially available immunoassay-kits (R&D Systems, Inc.,
Minneapolis, MN, USA and Asserachrom vWF:Ag, Dia-
gnostica Stago Inc., France). The researchers performing Up to at least one week prior to the blood being taken, the
volunteers had not taken any medication known to influen-
ce platelet function (e.g. acetylsalicylic acid, non-steroidal
anti-inflammatory drugs). After measuring the haematocrit
level, the exact volume of citrated whole blood per aliquot
was adjusted in order to obtain exactly 3 ml plasma per ali-
quot. The aliquots were incubated with different amounts
of bevacizumab (Genentech Inc, San Francisco, CA, USA)
diluted in phosphate buffered saline, resulting in the fol-
lowing plasma concentrations of bevacizumab: 0 μg/ml
(control), 125 μg/ml, 250 μg/ml, 500 μg/ml, and 1000 μg/
ml. After an incubation period of 30 minutes the platelet
function was analysed. Clinical observation study In the clinical part of the study all patients with advanced
metastatic disease, who received a first infusion of be-
vacizumab
and
chemotherapy
at
the
Kantonsspital
Graubünden, Switzerland, were considered eligible. Pa-
tients taking acetylsalicylic acid were excluded. Patients
receiving anticoagulation in a therapeutic dose were not
included, because safety data for this group of patients
were lacking at the time of the trial. Informed consent
was obtained from all patients. The study was approved
by the local ethical committee, was conducted according
to good clinical practice guidelines (GCP) and the Declar-
ation of Helsinki (National Cancer Institute Registration:
NCT00898794). Introduction Angiogenesis is a key element in growth and metastasis of
cancer. Vascular endothelial growth factor (VEGF), its re-
ceptors and signalling pathways are the target of several
novel anti-cancer drugs. Bevacizumab is a recombinant hu-
manised monoclonal neutralizing antibody against VEGF,
which has shown clinical benefits and efficacy in several
types of malignancies including metastatic colorectal can-
cer, advanced breast and lung cancer [1–3]. The inhibition
of VEGF has been noted to cause serious adverse events
such as wound dehiscence, bowel perforation and, in addi-
tion, an increased rate of congestive heart failure in breast Swiss Medical Weekly · PDF of the online version · www.smw.ch Page 1 of 6 Page 1 of 6 Original article Swiss Med Wkly. 2011;141:w13243 Platelet function was analysed after an incubation period of
30 minutes. the endogenously secreted platelet VEGF may function
as a positive feedback regulator during platelet activation
[10]. Theoretically, the interaction of bevacizumab with the
platelet VEGF during platelet activation could result in im-
paired primary haemostasis, increasing the risk of haemor-
rhage. Analysis of platelet function Platelet adhesive and aggregatory function was tested with
a platelet function analyser (PFA-100®, Dade Behring,
Düdingen, Switzerland). In this instrument blood is aspir-
ated at a constant negative pressure through a capillary with
a diameter of 200 μm at a high shear rate of 5000–6000 s-1
into a 150 μm membrane pore coated with collagen type I
and either epinephrine bitartrate (EPI) or adenosine diphos-
phate (ADP) as an additional platelet activator. Platelets ad-
here to the membrane pore, become activated, then aggreg-
ate and form an occluding plug in the pore thus stopping
blood flow. The time measured from the start of the test
until the formation of an occluding platelet plug is called
closure time (CT). Consequently, the duration of the CT is
inversely related to the platelet adhesiveness and aggreg-
ation. All measurements were done in duplicate and the
mean value was calculated [14]. Recently, the activation of the platelet FcgammaRIIa re-
ceptor and induction of platelet aggregation, degranulation
and thrombosis through immune-complex formation of be-
vacizumab with VEGF and unfractionated heparin has
been demonstrated in a transgenic mouse model [11]. However, platelet activation was not observed with beva-
cizumab alone, with VEGF alone, with bevacizumab and
VEGF alone, or when using a VEGF variant lacking the
heparin binding domain [11]. So far, this preclinical ob-
servation serves as a possible explanation of the increased
rate of arterial and venous thrombus formation in mice. However, its clinical relevance in humans remains unclear. On the basis of these preclinical data we hypothesized
that primary haemostasis is a possible site of interaction
of bevacizumab and VEGF resulting in and explaining
some of its adverse effects. We decided to test whether
the effects observed in experimental settings could be con-
firmed by observations in clinical practice. To test for dis-
orders in platelet function the platelet function analyser
PFA-100® was employed to obtain quantitative data on
primary haemostasis in vitro and in a clinical observational
study. In addition, soluble P-selectin (sP-selectin) serum
levels and von Willebrand Factor antigen (vWF:Ag) were
measured as a serological marker of platelet activation and
endothelial activation in the clinical part of the study. Routine analysis of haemoglobin levels, haematocrit and
platelet count were performed with an electronic particle
counter (Sysmex K-1000, Digitana AG, Horgen, Switzer-
land). Statistics For the in vitro study sample sizes were calculated to detect
a difference of 20 seconds (s) in collagen/ADP CTs and of
35 sec in collagen/EPI CTs which can be equated with an
excess of the normal limits of the PFA-100® CTs (68–121
s for collagen/ADP and 84–160 s for collagen/EPI) with
a power of 90% and a level of significance of 5%. For
the clinical observation study sample size calculations were
based on the assumption of a larger standard deviation in
the patient population and take a drop out rate of 20% into
account. The median CTs before and after the first bevacizumab ap-
plication and after six weeks of treatment are shown in
table 3. No statistically significant changes of CTs were ob-
served (p >0.05). Six of 20 patients, who had a previously implanted CVAD
were exposed to small amounts of unfractioned heparin,
because the small reservoir of this device was filled with
3–4 ml of heparin containing solution after its use. In a
post hoc analysis comparing those patients exposed to un-
fractioned heparin via the CVAD, with the other patients,
who had no exposure to unfractioned heparin, no statistic-
ally significant difference was observed. CT-EPI difference
from baseline were –1.3 s (range –26.9 – 24.3) and – 1.8 s
(–16.4 – 12.7), CT-ADP difference from baseline were + 5
s (range –11.9 – 21.9) and + 3.1 s (–3.6 – 9.9) for heparin
exposed (n = 6) and heparin-non-exposed (n = 12) patients,
respectively (p >0.05). Statistical analysis were performed with the help of Statist-
ica® 8.0 and Instat® 3.1a computer software (SoftStat, Inc.,
OK, USA and GraphPad Software, Inc., CA, USA) using
repeated measures analysis of variance (ANOVA), Bonfer-
roni Multiple Comparisons Test and Friedman-ANOVA. A
p-value <0.05 was considered as statistically significant. The median sP-selectin and vWF:Ag serum levels before
and after the first bevacizumab application and after six
weeks of treatment are also shown in table 3. Mean sP-se-
lectin serum levels were significantly reduced by 18% (p =
0.045) after the first bevacizumab application. The changes
in mean vWF:Ag after the first bevacizumab application
and after six weeks of treatment were not statistically sig-
nificant. Note that the normal reference range for vWF:Ag
is 40–200%. In vitro study The results of the in vitro experiments are shown in table
1. Note that the normal range of the PFA-100® closure
times (CTs) for collagen/EPI is 84–160 s and for collagen/
ADP 68–121s. Increasing bevacizumab concentrations up
to 1000 μg/ml did not affect CTs with either epinephrine
or ADP as a platelet activator, i.e. there was neither a CT
prolongation indicating a platelet inhibition, nor CT short-
ening indicating platelet activation. Moreover the increase
of the physiological VEGF plasma concentration by 100 or
500 μg/l had no effect on platelet function. The combina-
tion of VEGF and bevacizumab was also without an effect
(table 1). In vitro study In a second series of experiments aliquots of citrated whole
blood prepared as described above were incubated with
VEGF (Santa Cruz, Biotechnology) diluted in phosphate
buffered saline resulting in VEGF plasma concentrations
of 0 μg/l (control), 100 μg/l, 500 μg/l and a combination
of 500 μg/l VEGF followed by 500 μg/ml bevacizumab. Swiss Medical Weekly · PDF of the online version · www.smw.ch Page 2 of 6 Original article Swiss Med Wkly. 2011;141:w13243 the sP-selectin and vWF:Ag analysis were blinded for the
PFA-100® results. vWF:Ag due to a technical sampling error. After six weeks
the results of 15 patients were evaluable (2 technical errors,
2 continued treatment at another institution, 1 drop out due
to disease progression). Discussion Our hypothesis, based on preclinical data [10, 11], was that
bevacizumab could influence human platelet function by
either inhibiting or stimulating primary haemostasis. Our
data indicate however, that this is not the case, i.e. bevaci-
zumab did not affect platelet aggregation as assessed by the
platelet function analyser PFA-100® in either direction. Swiss Medical Weekly · PDF of the online version · www.smw.ch Clinical observation study Twenty patients, who were treated with bevacizumab for
the first time, were recruited. The clinical characteristics of
these patients are shown in table 2. Steady state bevacizumab concentrations after one infusion
are in the order of 200 μg/ml (e.g., 500 mg diluted in 2.5
l plasma). Hence with in vitro concentrations of 0–1000
μg/ml we covered a sufficiently broad range of clinically
relevant bevacizumab concentrations. In the in vitro study
no dose effect was observed, which corresponds to certain
clinical observations. In a randomized phase III trial with
two different bevacizumab doses in metastatic breast can- Of the 20 patients, the results of 18 (90%) were considered
evaluable for statistical analysis. One technical sampling
error and one error in processing led to the exclusion of two
patients for the evaluation of PFA-100® CTs. One patient
was excluded from the evaluation of sP-selectin levels and Table 1: PFA-100® closure times using either collagen/epinephrine (EPI) or collagen/adenosine diphosphate (ADP) as a platelet activator in in vitro experiments with
bevacizumab, VEGF, or a combination thereof (mean ± SD, n = 10; n.s. = not significant). Closure times (s)
EPI
ADP
0
120.8 ± 25.7
92.8 ± 7.7
125
106.2 ± 21.7
88.5 ± 9.9
250
123.2 ± 31.9
88.4 ± 18.2
500
121.3 ± 23.5
93.5 ± 17.5
Bevacizumab (μg/ml)
1000
111.9 ± 24.2
100.2 ± 17.2
0
112.2 ± 35.8
93.7 ± 11.8
100
117.6 ± 24.5
92.6 ± 26.7
VEGF (μg/l)
500
113.7 ± 27.3
98.3 ± 13.8
VEGF (500 μg/l) + Bevacizumab (500 μg/ml)
108 ± 23.4
91.8 ± 10.0
n.s. n.s. Table 1: PFA-100® closure times using either collagen/epinephrine (EPI) or collagen/adenosine diphosphate (ADP) as a platelet activator in
bevacizumab, VEGF, or a combination thereof (mean ± SD, n = 10; n.s. = not significant). Swiss Medical Weekly · PDF of the online version · www.smw.ch Page 3 of 6 Original article Swiss Med Wkly. 2011;141:w13243 Given the complexity of platelet function testing we con-
sidered it to be appropriate to additionally employ sP-se-
lectin as a serological marker of platelet activation. P-se-
lectin is a member of the selectin family localised in the
membranes of α-granules of platelets and the Weibel-
Palade bodies of endothelial cells [28]. A soluble form of
P-selectin can be found in the serum as a circulating pro-
tein [29]. Clinical observation study Elevated levels of sP-selectin may reflect platelet
activation [30], because P-selectin is proteolytically shed
from the plasma membrane in vivo shortly after activation
[31, 32]. Therefore, serum levels of sP-selectin have been
considered a useful tool to predict thrombotic consumptive
platelet disorders [33–36]. Physiological functions of sP-
selectin have not been elucidated to a large extent, but pre-
clinical data support the view that sP-selectin may also be
a direct inducer of pro-coagulant activity associated with
vascular and thrombotic disease [37]. cer the rates of side effects amongst the two different treat-
ment groups were not statistically different [15]. In a meta-
analysis, the rate of venous thrombosis was also not de-
pendent on the bevacizumab dose [5]. The target of bevacizumab, VEGF, given alone had no ef-
fect on platelet aggregation despite the fact that platelets
have VEGF receptors on their surface [10]. Apparently, a
binding of VEGF to its platelet receptor is not a signalling
event for epinephrine – or ADP-induced platelet aggrega-
tion tested in this study. This does not exclude some other
form of platelet activation as described for other pathways
such as thrombin [10] or immunocomplex-induced activ-
ation [11]. However, considering that sP-selectin serum
levels were not increased after the application of bevacizu-
mab, such other forms of platelet activation seem unlikely. Our results are in agreement with the work of Meyer et
al. [11], who did not observe platelet activation with be-
vacizumab alone, VEGF alone or bevacizumab and VEGF
alone. In the presence of unfractionated heparin, however,
immune complexes consisting of bevacizumab, VEGF and
heparin were formed, which activated platelets via Fc
gamma receptors very similarly to platelet activation in
heparin-induced thrombocytopenia caused by heparin-
platelet factor 4 complexes. Our post-hoc analysis of six
patients who had their heparin-containing Port-a-cath sys-
tem rinsed shortly before bevacizumab application sug-
gests that this amount of heparin was not sufficient to trig-
ger this elegantly shown mechanism nor that it is clinically
relevant. We have found an 18% decrease of sP-selectin serum levels
after bevacizumab infusion. This decrease is unlikely to be
due to an expansion of the plasma volume and a dilution
effect by the concomitant saline infusion, because no sig-
nificant effect on the haematocrit was observed. Since the
half-life of sP-selectin is approximately two hours [38], a
true decrease of sP-selectin is possible. Clinical observation study This would sug-
gest a decreased rather than an increased platelet activation. However, it seems unlikely that this has a lasting effect on
primary haemostasis in view of the unchanged platelet ag-
gregation and thrombus formation in our study. SP-selectin may also be shed from endothelial cells upon
their activation [39, 40], an observation which has,
however, not been confirmed by others [41]. Therefore,
measurement of vWF:Ag was additionally employed as
a marker of endothelial activation. In our patient cohort
vWF:Ag was not significantly changed after the first be-
vacizumab administration and after six weeks of treatment. This points to different origins of vWF (endothelium) and
sP-selectin (platelets). However the study was not designed
to address the interaction of bevacizumab with endothelial
function and therefore the results of the vWF:Ag alone
should be interpreted with caution. The PFA-100® has a good sensitivity and specificity for
the detection of primary haemostatic disorders [16]. It has
replaced the classic bleeding time test in clinical practice in
view of improved reproducibility and better test perform-
ance [14, 17, 18]. The test is highly sensitive for the detec-
tion of platelet disorders [19] and von Willebrand disease
[14]. The range of additional clinical and research applica-
tions is also expanding [20–23]. Whilst the PFA-100® has
been designed to detect an impaired haemostatic function
of platelets, it has become apparent that it is also capable
of detecting an increased adhesive and aggregatory platelet
function [24–27]. Table 2: Patient characteristics in the clinical observation study. Age
Median: 64.4 years (range 37–82)
Females: 7/20 (35%)
Gender
Males: 13/20 (65%)
Bevacizumab dose
Median: 8.25 mg/kg bodyweight (range 5–15)
Metastatic colorectal cancer: 14/20 (70%)
Metastatic breast cancer: 4/20 (20%)
Disease
Other: 2/20 (10%)
1 pulmonary embolism after 2 months of treatment
Adverse events
No bleeding events
Table 3: Results from the clinical observation study, PFA-100® closure times, soluble P-selectin serum levels and haematological parameters (mean value with 95%
confidence interval); n.s. = not significant. Before first bevacizumab
After first bevacizumab
After 6 weeks of treatment
EPI
112.6 (99.8 – 125.4)
110.9 (98.5 – 123.3)
110.8 (94 – 127.7)
n.s. PFA-100® closure times (s)
ADP
79.9 (71.9 – 86.7)
82.1 (72.7 – 90.8)
81 (72.8 – 89.2)
n.s. Swiss Medical Weekly · PDF of the online version · www.smw.ch Clinical observation study 5 Nalluri SR, Chu D, Kerszetes R, Zhu X, Wu S. Risk of venous throm-
boembolism with the angiogenesis inhibitor bevacicumab in cancer pa-
tients. JAMA. 2008;300(19):2277–85. 6 Scappaticci FA, Skillings JR, Holden SN, Gerber HP, Miller K, Kab-
binavar F, et al. Arterial thromboembolic events in patients with meta-
static carcinoma treated with chemotherapy and bevacizumab. J Natl
Cancer Inst. 2007;99(16):1232–9. 7 Schutz FAB, Je Y, Azzi GR, Nguyen PL, Choueiri TK. Bevacizumab
increases the risk of arterial ischemia: a large study in cancer patients
with a focus on different subgroup outcomes. Ann Oncol. Nov 29. 2010
[Epub ahead of print]. 8 Grothey A, Sugrue MM, Purdie DM, Dong W, Sargent D, Hedrick E, et
al. Bevacizumab beyond first progression is associated with prolonged
overall survival in metastatic colorectal cancer: Results from a large ob-
servational cohort study (BRiTE). J Clin Oncol. 2008;26(33):5326–34. servational cohort study (BRiTE). J Clin Oncol. 2008;26(33):5326–34. 9 Ranpura V, Hapani S, Wu S. Treatment-related mortality with bevacizu-
mab in cancer patients. A meta-analysis. JAMA. 2011;305(5):487–94. 10 Selheim F, Holmsen H, Vassbotn FS. Identification of functional VEGF
receptors on human platelets. FEBS Lett. 2002;512(1-3):107–10. To conclude, our in vitro and clinical observation data
taken together suggest that the interaction of bevacizumab
with platelet aggregation can not be regarded as a major
contributing factor to either thrombotic events or bleeding. Consequently other mechanisms must be involved, such as
the interaction of bevacizumab with endothelial cell func-
tion and the plasma coagulation cascade. It is therefore
worthwhile considering these other potential mechanisms
of interactions in further studies. 11 Meyer T, Robles-Carrillo L, Robson T, Langer F, Desai H, Davila M,
et al. Bevacizumab immune complexes activate platelets and induce
thrombosis
in
FCGR2A
transgenic
mice. J
Thromb
Haemost. 2009;7(1):171–81. 12 Salgado R, Benoy I, Bogers J, Weytjens R, Vermeulen P, Dirix L, et al. Platelets and vascular endothelial growth factor (VEGF): A morpholo-
gical and functional study. Angiogenesis. 2001;4(1):37–43. 13 Katoh O, Tauchi H, Kawaishi K, KImura A, Satow Y. Expression of
the vascular endothelial growth factor (VEGF) receptor gene, KDR,
in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell
death caused by ionizing radiation. Cancer Res. 1995;55(23):5687–92. We are very grateful to Ms. Gillian Roberts for editing and
reviewing the manuscript. We thank Thomas Schultzki,
M.D. for his contribution to the statistical analysis. 14 Wuillemin WA, Gasser KM, Zeerleder, Lämmle B. Clinical observation study Soluble P-selectin plasma level (ng/ml)
49.6 (39.5 – 59.6)
40.8 (31.7 – 49.8)*
40.5 (29.5 – 51.6)
*p = 0.045
vWF:Ag (%)
189.7 (146.2 – 233.2)
196.9 (130.4 – 263.4)
221.9 (168.4 – 275.4)
n.s. Thrombocyte count (G/l)
190 (149 – 232)
188 (150 – 225)
159 (121 – 195)
n.s. Haematocrit (%)
38.2 (35.2 – 41.1)
36.8 (34.2 – 39.4)
36.0 (32.7 – 39.4)
n.s. Table 3: Results from the clinical observation study, PFA-100® closure times, soluble P-selectin serum levels and haematological parameters (mean value with 95%
confidence interval); n.s. = not significant. Before first bevacizumab
After first bevacizumab
After 6 weeks of treatment
EPI
112.6 (99.8 – 125.4)
110.9 (98.5 – 123.3)
110.8 (94 – 127.7)
n.s. PFA-100® closure times (s)
ADP
79.9 (71.9 – 86.7)
82.1 (72.7 – 90.8)
81 (72.8 – 89.2)
n.s. Soluble P-selectin plasma level (ng/ml)
49.6 (39.5 – 59.6)
40.8 (31.7 – 49.8)*
40.5 (29.5 – 51.6)
*p = 0.045
vWF:Ag (%)
189.7 (146.2 – 233.2)
196.9 (130.4 – 263.4)
221.9 (168.4 – 275.4)
n.s. Thrombocyte count (G/l)
190 (149 – 232)
188 (150 – 225)
159 (121 – 195)
n.s. Haematocrit (%)
38.2 (35.2 – 41.1)
36.8 (34.2 – 39.4)
36.0 (32.7 – 39.4)
n.s. Page 4 of 6 Original article Swiss Med Wkly. 2011;141:w13243 This study has some further limitations. It was designed
as a clinical observation study without a control group be-
cause the design of any control group (e.g. patients not
receiving bevacizumab or healthy volunteers receiving an
anti-cancer drug) was considered unethical and impossible. The sample size appears small but was adequately calcu-
lated for changes of PFA-100® CTs exceeding the normal
limits with sufficient power. However, we refrained from
increasing the number of patients to observe smaller ef-
fects on PFA-100® CTs in order to minimise the burden
on patients and resources. The study was not designed to
compare differences in platelet aggregation after bevacizu-
mab treatment in patients exposed to unfractioned heparin
with patients who were not exposed to heparin. However,
we consequently aimed at addressing this question by the
means of a small post hoc analysis after the work of Meyer
et al. was published when our patient accrual was almost
completed [11]. 4 Choueiri TK, Mayer EL, Je Y, Rosenberg JE, Nguyen PL, Azzi GR,
et al. Congestive Heart Failure Risk in Patients With Breast Cancer
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been supported in part by the Bonizzi-Theler Foundation,
Switzerland (industry independent). The funding source
had no role in the study design and conduct, data
collection, analysis or interpretation and preparation of the
manuscript. Financial disclosures: Richard Cathomas
reports having received consulting fees form Roche,
Switzerland. Roger von Moos reports having received
consulting fees and an unrestricted research grant from
Roche, Switzerland. The other authors report no
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coagulant state resulting from high levels of soluble P-selectin in blood. Pro-coagulant state resulting from high levels of soluble P-selectin in
blood. Proc Natl Acad Sci USA. 2000; 97(25):13835–40. 28 Wagner DD. The Weibel-Palade body: the storage granule for von Wil-
lebrand factor and P-selectin. Thromb Haemost. 1993;70(1):105–10. 38 Fägerstam JP, Whiss PA, Ström M, Andersson RG. Expression of plate-
let P-selectin and detection of soluble P-selectin, NPY and RANTES
in
patients
with
inflammatory
bowel
disease. Inflamm
Res. 2000;49(6):466–72. 29 Dunlop LC, Skinner MP, Bendall LJ, Favaloro EJ, Castaldi PA, Gorman
JJ, et al. Characterization of GMP-140 (P-selectin) as a circulating
plasma protein. J Exp Med.1992;175(4):1147–50. 39 Frijns CJ, Kappelle LJ, van Gijn J, Nieuwenhuis HK, Sixma JJ, Fijnheer
R. Soluble adhesion molecules reflect endothelial cell activation in
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stroke
and
in
carotid
atherosclerosis. Stroke. 1997;28(11):2214–8. 30 Fijnheer R, Frijns CJ, Korteweg J, Rommes H, Peters JH, Sixma JJ,
et al. The origin of P-selectin as a circulating plasma protein. Thromb
Haemost. 1997;77(6):1081–5. 31 Berger G, Hartwell DW, Wagner DD. P-Selectin and platelet clearance. Blood. 1998:92(11):4446–52. 40 Verhaar MC, Betler JJ, Gaillard CA, Koomans HA, Fijnheer R, Ra-
belink TJ. Progressive vascular damage in hypertension is associated
with increased levels of circulating P-selectin. J Hypertension. 1998;16(1):45–50. 32 Michelson AD, Barnard MR, Hechtmann HB, MacGregor H, Conolly
RJ, Loscalzo J, et al. In vivo tracking of platelets: circulating degran-
ulated platelets rapidly lose surface P-selectin but continue to circulate
and function. Proc Natl Acad Sci USA. 1996;93(21):11877–82. 41 Jilma B, Eichler HG, Vondrovec B, Breiteneder H, Kyrle PA, Kitzweger
E, et al. Effects of desmopressin on circulating P-selectin. Br J Haemat-
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The androgen receptor is a therapeutic target in desmoplastic small round cell sarcoma
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Nature communications
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cc-by
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1 Sarcoma Medical Oncology Department, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 2 Department of Genomic
Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 3 Department of Surgery, Breast surgical Oncology, Baylor
College of Medicine, Houston, TX 77030, USA. 4 Texas Children’s Cancer & Hematology Centers, Houston, TX 77384, USA. 5 Division of Pathology, The
University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 6 Division of Pediatrics, The University of Texas MD Anderson Cancer Center,
Houston, TX 77030, USA. 7 Optical Microscopy Facility, Rice University, Houston, TX 77030, USA. 8 Genitourinary Medical Oncology Department, The
University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 9 Experimental Therapeutics Department, The University of Texas MD
Anderson Cancer Center, Houston, TX 77030, USA. 10 Ionis Pharmaceuticals, Carlsbad, CA 92010, USA. 11 Lineberger Comprehensive Cancer Center, UNC,
Chapel Hill, NC 27599, USA. 12These authors contributed equally: Salah-Eddine Lamhamedi-Cherradi, Mayinuer Maitituoheti. 13These authors jointly
supervised this work: Salah-Eddine Lamhamedi-Cherradi, Kunal Rai, Joseph A. Ludwig. ✉email: SLamhamedi@mdanderson.org; Krai@mdanderson.org;
jaludwig@mdanderson.org ARTICLE The androgen receptor is a therapeutic target in
desmoplastic small round cell sarcoma NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z D double-hierarchical
clustering
correctly
separated
normal
mesenteric tissues from DSRCT, which overexpressed Akt, Syk,
PKC-α, and other proteins. Next, we identified proteins enriched
in DSRCT compared to another malignancy, using ES as the
closest molecular cousin that shares an N-terminus EWSR1
fusion partner. Interestingly, AR and Syk proteins were upregu-
lated in most DSRCT specimens but nearly undetectable in
ES (Fig. 1a, b). The RPPA data, validated by western blots
(WB), provides the first screen of proteins enriched in DSRCT
(Fig. 1c, d). D
esmoplastic small round cell tumor (DSRCT) is an
aggressive soft-tissue malignancy that usually presents in
post-pubertal adolescents and young adults as a large
intra-abdominal mass together with a widespread coating of
serosal and subdiaphragmatic surfaces by hundreds to thousands
of malignant nodules. Given the inconspicuous tumor location,
nearly all patients present in an advanced stage with abdominal
pain or distention, nausea, constipation, and weight loss1. p
p
g
DSRCT’s cell of origin is unknown, and these tumors uni-
versally exhibit a high-grade, poorly differentiated state char-
acterized by cellular nests of unclear lineage, seeming to show
epithelial, muscular, mesenchymal, and neural differentiation
admixed with prominent desmoplastic stroma2–4. Given its rarity,
with an age-adjusted incidence peak incidence of 0.3–0.74 cases
per million5, it was not until 1989 that Gerald and Rosai first
described DSRCT as a unique clinicopathologic disease6. Shortly
thereafter,
cytogenetic
analyses
demonstrated
that
DSRCT
tumors harbor a pathognomonic t(11;22)(p13:q12) chromosomal
translocation that pairs the Ewing sarcoma (ES) gene (EWSR1)
with the Wilms tumor suppressor gene (WT1)6–9. The resulting
chimeric 59 kDa fusion protein (FP), in concert with the het-
erozygous functional loss of the WT1 tumor suppressor protein,
promotes an oncogenic effect that reinforces tumor survival and
growth10. Expression of AR in DSRCT primary tumors. Since AR activity
requires androgen-mediated nuclear translocation, we created a
DSRCT-specific tissue microarray (TMA) from 60 cases treated at
MD Anderson Cancer Center (MDACC) to determine the pre-
valence and cellular distribution of AR staining in patients treated
at a single institution. Seventy-five percent of the cores available
for analysis were positive for nuclear AR by immunohistochem-
istry (Fig. 2a, b and Supplementary Fig. S2a–c), usually more
prominently in the epithelioid cells rather than the desmoplastic
stromal cells. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Of the AR-positive samples, 7% showed focal AR
expression in 10–50% rare, scattered cells and could be of
any intensity (low positive AR expression), 3.3% had low AR
expression where only 1–10% of cells are positive for AR
expression (focal expression), and 25% demonstrated negative
AR expression (where only 0–1% of cells are positive). 65%
showed high AR expression, defined by AR-positivity in >50% of
a sample’s cells (Fig. 2b and Supplementary Fig. S2c). High-
intensity staining was defined as completely obscuring the nuclear
hematoxylin counterstain, while moderate staining allowed
visualization of the stain. Weak staining required examination of
the cells at least 200× to detect staining reliably. As an additional
metric of AR expression, we evaluated an additional 12 DSRCT
patient tumors for AR expression by western blotting: 42% of the
tumors showed high AR expression, 33% had moderate AR
expression, and 25% were AR-negative (Fig. 2c, d). As IHC and
western blotting revealed moderate to high AR expression in
~three-quarters of the DSRCT cases assessed, this seemed to
substantiate the RPPA results. g
The PI3K/AKT and androgen receptor (AR) signaling cascades
are among the most frequently activated in cancer11,12. Given the
striking observation that 90% of DSRCT cases occur in post-
pubertal males (with an average age at diagnosis of 21.4 years), we
investigated
how
AR
contributes
to
tumorigenesis
and
survival4,13,14. A potential connection between the AR and
DSRCT, first reported by Fine et al. in 2006, studied a series of
twenty-seven advanced-stage DSRCT patients who had pro-
gressed through at least two chemotherapy regimens15. In that
retrospective multi-center analysis, 37% of the samples were ≥2+
by immunohistochemistry, and surprisingly, three of six AR+
patients transiently benefited from second-generation combined
androgen blockade (CAB) using Lupron and bicalutamide. Despite that promising signal of activity, AR targeting has not
been pursued further in the clinic, and at the time of this writing,
no additional studies were reported in the literature. In the present work, we use DSRCT xenografts and patient-
derived tumor explants (PDXs) to extend the findings by Fine
et al. to modern-day AR-targeted therapies, such as enzalutamide,
that form the backbone of prostate cancer (PC) treatment16. In
addition, we present promising efficacy data using an experi-
mental AR-targeted antisense oligonucleotide (ASO) that sig-
nificantly delayed tumor growth by suppressing AR expression. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Finally, we present chromatin immunoprecipitation sequencing
(ChIP-seq) results that suggest a new mechanistic understanding
of AR’s role in DSRCT tumorgenicity. Our research findings
substantiate DSRCT as a second AR-driven malignancy and
implicitly suggest a path toward clinical trials that center on AR-
directed treatment options for this otherwise intractable pediatric
cancer. Genomic profiling of DSRCT, PC, and other sarcoma primary
tumor samples. Given the male predominance of DSRCT, new
AR protein expression data, and known abundance of AR-
targeted therapies used for PC, we elected to pursue AR as a
potential therapeutic target in DSRCT. The case report by Fine
et al. hinted that DSRCT patients can respond briefly to a first-
generation CAB, which heightened our enthusiasm to investigate
how AR signaling contributes to DSRCT biology15. g
g
gy
Given the AR’s central role in PC growth and survival, and
deep mechanistic understanding of that malignancy20–23, we
performed a gene expression analysis comparing 22 DSRCT
samples to 12 PC samples and a group of other diverse sarcoma
subtypes, including 7 chondrosarcomas, 7 well-differentiated
liposarcomas, 10 dedifferentiated liposarcoma, and 47 osteosar-
coma samples, which served as negative controls. As expected,
DSRCT demonstrated significant AR upregulation compared to
these other sarcoma samples from chondrosarcoma, well-
differentiated and dedifferentiated liposarcoma, and osteosar-
coma (p < 0.01–p < 0.0001) but did not surpass the levels observed
in PC (Fig. 2e, f). The androgen receptor is a therapeutic target in
desmoplastic small round cell sarcoma Salah-Eddine Lamhamedi-Cherradi
1,12,13✉, Mayinuer Maitituoheti2,12, Brian A. Menegaz
3,
Sandhya Krishnan
1, Amelia M. Vetter1, Pamela Camacho4, Chia-Chin Wu
2, Hannah C. Beird
2,
Robert W. Porter
1, Davis R. Ingram5, Vandhana Ramamoorthy2, Sana Mohiuddin6, David McCall
6,
Danh D. Truong
1, Branko Cuglievan6, P. Andrew Futreal
5, Alejandra Ruiz Velasco1,
Nazanin Esmaeili Anvar
5, Budi Utama7, Mark Titus8, Alexander J. Lazar
5, Wei-Lien Wang
5,
Cristian Rodriguez-Aguayo
9, Ravin Ratan
1, J. Andrew Livingston
1, Kunal Rai
2,13✉,
A. Robert MacLeod10, Najat C. Daw
6, Andrea Hayes-Jordan11 & Joseph A. Ludwig
1,13✉ Desmoplastic small round cell tumor (DSRCT) is an aggressive, usually incurable sarcoma
subtype that predominantly occurs in post-pubertal young males. Recent evidence suggests
that the androgen receptor (AR) can promote tumor progression in DSRCTs. However, the
mechanism of AR-induced oncogenic stimulation remains undetermined. Herein, we
demonstrate that enzalutamide and AR-directed antisense oligonucleotides (AR-ASO) block
5α-dihydrotestosterone (DHT)-induced DSRCT cell proliferation and reduce xenograft tumor
burden. Gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq)
were performed to elucidate how AR signaling regulates cellular epigenetic programs. Remarkably, ChIP-seq revealed novel DSRCT-specific AR DNA binding sites adjacent to key
oncogenic regulators, including WT1 (the C-terminal partner of the pathognomonic fusion
protein) and FOXF1. Additionally, AR occupied enhancer sites that regulate the Wnt pathway,
neural differentiation, and embryonic organ development, implicating AR in dysfunctional cell
lineage commitment. Our findings have direct clinical implications given the widespread
availability of FDA-approved androgen-targeted agents used for prostate cancer. 1 ARTICLE URE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z b
Fig. 1 Proteomic comparison of DSRCT and ES. a The protein lysates from DSRCT (red; n = 16) and ES (blue; n = 6) were subjected to RPPA analysis for
151 proteins and phosphoproteins (red, increased signal; green, decreased signal). Unsupervised double-hierarchical clustering using the Pearson
correlation distance metric between proteins (rows) and Centroid linkage (a clustering method) separated the 22 samples into two groups by tumor type
(columns). Of the 22 proteins, 8 had expression that differed significantly between ES and DSRCT (p ≤0.05; fold-change ≥2). b The mean expression
intensity values of the 8 proteins associated with DSRCT or ES and their statistical significance after normalization for global protein expression by median
centering across 151 antibodies in the RPPA panel. c Western blotting was used to validate the proteins identified by RPPA as being differentially expressed
between DSRCT and ES. d Normalized protein expression is relative to β-actin. Data points in b and d represent mean ± SD. n is the number of samples
analyzed for each sarcoma subtype. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z
ARTICLE b Fig. 1 Proteomic comparison of DSRCT and ES. a The protein lysates from DSRCT (red; n = 16) and ES (blue; n = 6) were subjected to RPPA analysis for
151 proteins and phosphoproteins (red, increased signal; green, decreased signal). Unsupervised double-hierarchical clustering using the Pearson
correlation distance metric between proteins (rows) and Centroid linkage (a clustering method) separated the 22 samples into two groups by tumor type
(columns). Of the 22 proteins, 8 had expression that differed significantly between ES and DSRCT (p ≤0.05; fold-change ≥2). b The mean expression
intensity values of the 8 proteins associated with DSRCT or ES and their statistical significance after normalization for global protein expression by median
centering across 151 antibodies in the RPPA panel. c Western blotting was used to validate the proteins identified by RPPA as being differentially expressed
between DSRCT and ES. d Normalized protein expression is relative to β-actin. Data points in b and d represent mean ± SD. n is the number of samples
analyzed for each sarcoma subtype. androgen signaling (Supplementary Fig. S2f). Notably, PC and
DSRCT clustered together on their own branch, apart from other
sarcoma subtypes, based upon the 1500 most varied genes among
all samples (Fig. 3). Pathway analysis revealed other cancer
pathways enriched in DSRCT compared to other sarcoma
subtypes. Results Protein expression in DSRCT differs substantially from ES. Given the differences in clinical presentation, tumor biology, and
response to biologically targeted therapies, we conducted a
reverse-phase protein lysate array (RPPA) to identify proteins
enriched in DSRCT. To determine tumor-specific proteins, we
compared protein lysates from DSRCT nodules and paired
adjacent normal-appearing mesenteric tissue from the same
patients using a well-described RPPA platform enriched for
known oncoproteins (Supplementary Fig. S1)17–19. Unsupervised Compared to other sarcoma subtypes, most DSRCT samples
clustered together based upon their expression of 89 genes
associated with the androgen pathway, as defined by KEGG
(Supplementary Fig. S2d). Similarly, DSRCT samples clustered
together by their expression of 55 genes linked to canonical NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Fig. 2 DSRCT TMA and frozen specimen profiling for AR and PSA expression. a A histogram showing the AR IHC expression levels of 60 human DSRCT
tumors grouped by intensity (low, moderate, and high). The demographic data, including the corresponding gender (red: male or green: female), the age at
diagnosis, and the pre/post-chemotherapy treatment to the surgery of each primary or metastatic resected tumor patient, are displayed at the left of each
histogram. b AR expression level interpretation on DSRCT TMA IHC-stain and percentage scoring of tumoral labeling (positive (>50%), low positive
(10–50%), focal (1–10%), and negative (0–1%)). c Western blotting analyses of AR expression in 11 DSRCT snap-frozen primary tumors. AR expression
P = positive, N = negative, or M = moderate. d Relative AR levels across samples shown in c. Bars show mean ± SD. e The principal components analysis
plot performed on gene expression from prostate cancer (PC), DSRCT, and additional type of sarcomas samples. f Boxplot for the AR gene expression leve
across DSRCT, prostate cancer, and four other sarcoma types. The Wilcoxon rank-sum test performed to compare the AR levels between DSRCT (n = 22)
and each of the other cancer types. PC = prostate cancer (n = 12); CS = chondrosarcoma (n = 7); OS = osteosarcoma (n = 47); WDLPS = well-
differentiated liposarcoma (n = 7), and DDLPS = dedifferentiated liposarcoma (n = 10). ***p value < 0.001, **p value < 0.01, and *p value < 0.05. Fig. 2 DSRCT TMA and frozen specimen profiling for AR and PSA expression. a A histogram showing the AR IHC expression levels of 60 human DSRCT
tumors grouped by intensity (low, moderate, and high). The demographic data, including the corresponding gender (red: male or green: female), the age at
diagnosis, and the pre/post-chemotherapy treatment to the surgery of each primary or metastatic resected tumor patient, are displayed at the left of each
histogram. b AR expression level interpretation on DSRCT TMA IHC-stain and percentage scoring of tumoral labeling (positive (>50%), low positive
(10–50%), focal (1–10%), and negative (0–1%)). c Western blotting analyses of AR expression in 11 DSRCT snap-frozen primary tumors. AR expression:
P = positive, N = negative, or M = moderate. d Relative AR levels across samples shown in c. Bars show mean ± SD. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z e The principal components analysis
plot performed on gene expression from prostate cancer (PC), DSRCT, and additional type of sarcomas samples. f Boxplot for the AR gene expression level
across DSRCT, prostate cancer, and four other sarcoma types. The Wilcoxon rank-sum test performed to compare the AR levels between DSRCT (n = 22)
and each of the other cancer types. PC = prostate cancer (n = 12); CS = chondrosarcoma (n = 7); OS = osteosarcoma (n = 47); WDLPS = well-
differentiated liposarcoma (n = 7), and DDLPS = dedifferentiated liposarcoma (n = 10). ***p value < 0.001, **p value < 0.01, and *p value < 0.05. (Supplementary Fig. S3b). However, the JN-DSRCT cells showed
low expression of NCOA1/2 and equivalent expression of NCOA2
versus
LNCaP
PC
cells
(Supplementary
Fig. S3c). Further
investigation with a larger sample set will be required to determine
how
the
AR-dependent
integrin/NCOA-dependent
pathway
impacts DSRCT cell migration and death. PC3 PC cells, and ES TC71 cells that were used as positive or
negative controls. As hypothesized, DHT stimulation increased
cell proliferation of JN-DSRCT and LNCaP cells compared to
PC3 and ES cells (Fig. 4a). As measured by western blotting, we
confirmed strong AR expression by LNCaP and JN-DSRCT cell
lines following DHT stimulation in contrast to its absence in the
TC71 ES and PC3 PC cells (Fig. 4b). Next, we performed confocal
immunofluorescence staining of these cells to determine if (and
how quickly) DHT-mediated stimulation would facilitate AR
transmigration from the cytoplasm into the cell nucleus. Our
results suggest that AR-nuclear translocation begins within 5 h of
DHT exposure and peaks within 24 h (Fig. 4c, d). (Supplementary Fig. S3b). However, the JN-DSRCT cells showed
low expression of NCOA1/2 and equivalent expression of NCOA2
versus
LNCaP
PC
cells
(Supplementary
Fig. S3c). Further
investigation with a larger sample set will be required to determine
how
the
AR-dependent
integrin/NCOA-dependent
pathway
impacts DSRCT cell migration and death. In vitro stimulation and inhibition of DSRCT proliferation via
AR. Though AR activation by testosterone and DHT leads to
brisk
PC
cell
proliferation38,
it
was
uncertain
whether
DSRCT cells similarly relied upon AR signaling for proliferation,
growth, and survival. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z This included the cell-adhesion molecule communica-
tion pathway that regulates cell invasion through a coordinated
balance between adhesion and detachment of cells24,25 and the
extracellular matrix (ECM) interaction pathway, known to
initiate cell motility across the ECM barrier26. the initial twelve samples expressed AR-V by IHC, we elected not
to examine this further in the broader sample set. As AR activity can be influenced by integrins and transcrip-
tional co-regulators30,31, we evaluated these AR activators within
the same cohort of 11 DSRCT tumors at the proteomic level using
a western blotting analysis. Among three alpha integrin (ITGAV,
ITGA4, and ITGA5) and beta integrin (ITGB1, ITGB3, and
ITGB5) subunits commonly observed in mesenchymal tissues,
protein expression varied considerably and did not correlate with
AR expression (Supplementary Fig. S3a). Since the epigenetic effects of AR can be modified by cofactor
binding and matrix metalloproteins, we assessed whether steroid
receptor coactivators NCOA1/2/3 or MMP2/13 contribute to the
development of DSRCT through AR-dependent mechanisms32–37. To accomplish this, we performed a WB of 11 primary DSRCT
tumors and the JN-DSRCT and LNCaP PC cell lines. The three-
NCOA biomarkers demonstrated heterogeneous expression in the
DSRCT clinical samples proportional to their AR expression Characterizing extranuclear partners of AR and its nuclear
cofactors. AR splice variants (AR-Vs) have been implicated in PC
tumor progression, an increased risk of biochemical relapse, and
inferior overall survival outcomes27–29. To determine if variant
forms of AR exist in DSRCT, we assessed AR-V7 expression
within a cohort of twelve consecutive DSRCT patients. As none of 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Altogether, these data
indicate a vital role for DHT-stimulated AR expression in DSRCT
cell proliferation and conclusively demonstrates a potent anti-
neoplastic effect of AR antagonists. AR-ASO
(IONIS
560131;
formerly
AZD5312)
significantly
slowed DSRCT cell proliferation at two weeks (Fig. 4f) and
reduced
AR
expression
(Fig. 4g). However,
the
in
vitro
antiproliferative effect was four-fold more effective in the cells
treated with the AR-targeted antisense blockade (Fig. 4e, f). Notably, this antineoplastic effect required concurrent adminis-
tration with DHT (Supplementary Fig. S4). Altogether, these data
indicate a vital role for DHT-stimulated AR expression in DSRCT
cell proliferation and conclusively demonstrates a potent anti-
neoplastic effect of AR antagonists. bearing a DSRCT PDX (Fig. 5d–f). As expected, tumor growth
and Kaplan–Meier curves revealed that tumors treated with AR-
ASO have significantly reduced tumor burden and improved
survival compared to control ASO group (p = 0.0097 and
p < 0.0001, respectively). p
y
Though both agents delayed tumor growth, AR-ASOs were more
effective than enzalutamide in both preclinical models. Therefore,
our pharmacodynamic analysis focused primarily on the effect of
AR-ASO treatment. Proteomic profiling by RPPA (Fig. 6a), western
blotting
(Fig. 6b,
c),
immunofluorescence
(Fig. 6d,
e
and
Supplementary Fig. S5a–c), and immunohistochemistry (Fig. 6f–h
and Supplementary Fig. S5d–f) validated the AR-ASO mediated
knockdown of AR expression in the xenograft and PDX. To further
characterize how the AR-ASO differed from enzalutamide mechan-
istically,
we
performed
liquid
chromatography-tandem
mass
spectrometry analysis of 38 collected preclinical animal specimens
shown in Fig. 5. Consistent with prior literature in PC, loss of AR
following AR-ASO treatment destabilized testosterone and reduced
its intratumoral expression (Supplementary Fig. S6)39,40. As a
negative control, the corticosterone levels were unchanged by AR
blockade. In addition, since the antineoplastic action of enzaluta-
mide works by preventing ligand-AR binding, reducing AR shuttling
to the nucleus, and impairing AR DNA binding affinity—instead of
reducing AR levels (Supplementary Fig. S7a–e)—enzalutamide
treatment did not significantly lower intratumoral testosterone. Preclinical efficacy of AR-based targeted therapy for the
treatment of DSRCT. Since only one DSRCT cell line exists, we
extended our evaluation of the AR antagonists to the in vivo
setting using the JN-DSRCT xenograft and available DSRCT
PDXs. Immunocompromised NSG mice bearing JN-DSRCT
xenograft tumors treated with enzalutamide or AR-ASO sig-
nificantly reduced tumor burden and improved survival with the
same efficacy, compared to placebo or control groups during the
first two months of treatment (Fig. 5a, b). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z To evaluate this, we performed in vitro cell
proliferation assays following DHT-mediated AR stimulation in
JN-DSRCT; AR-expressing LNCaP PC cells, AR-non-expressing p
p
g
Having shown that DHT stimulates DSRCT cells, we explored
whether FDA-approved and experimental AR antagonists had an
antiproliferative effect. Both enzalutamide (Fig. 4e) and the novel NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Fig. 3 Double-hierarchical clustering of PC (n = 12), DSRCT (n = 22), and other sarcoma subtypes (n = 71). The top 1500 most variable genes across all
samples were used to compare DSRCT to PC and other sarcoma subtypes. Unsupervised double-hierarchical clustering placed DSRCT next to PC on
branch 1. Other sarcoma subtypes clustered on branch 2. Distinct blocks within the heatmap indicate genes overexpressed (A) or underexpressed (B) in PC
and DSRCT compared to other sarcoma subtypes. Within the branch 1, some genes were upregulated strictly in PC (C) compared to DSRCT (D). PC = prostate cancer (n = 12); OS = osteosarcoma (n = 47); CS = chondrosarcoma (n = 7); WDLPS = well-differentiated liposarcoma (n = 7), and
DDLPS = dedifferentiated liposarcoma (n = 10). Fig. 3 Double-hierarchical clustering of PC (n = 12), DSRCT (n = 22), and other sarcoma subtypes (n = 71). The top 1500 most variable genes across all
samples were used to compare DSRCT to PC and other sarcoma subtypes. Unsupervised double-hierarchical clustering placed DSRCT next to PC on
branch 1. Other sarcoma subtypes clustered on branch 2. Distinct blocks within the heatmap indicate genes overexpressed (A) or underexpressed (B) in PC
and DSRCT compared to other sarcoma subtypes. Within the branch 1, some genes were upregulated strictly in PC (C) compared to DSRCT (D). PC = prostate cancer (n = 12); OS = osteosarcoma (n = 47); CS = chondrosarcoma (n = 7); WDLPS = well-differentiated liposarcoma (n = 7), and
DDLPS = dedifferentiated liposarcoma (n = 10). AR-ASO
(IONIS
560131;
formerly
AZD5312)
significantly
slowed DSRCT cell proliferation at two weeks (Fig. 4f) and
reduced
AR
expression
(Fig. 4g). However,
the
in
vitro
antiproliferative effect was four-fold more effective in the cells
treated with the AR-targeted antisense blockade (Fig. 4e, f). Notably, this antineoplastic effect required concurrent adminis-
tration with DHT (Supplementary Fig. S4). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z At 2 months, tumor
growth began to accelerate in the enzalutamide-treated mice,
whereas growth suppression continued in the mice treated with
either 25 or 50 mg/kg of the AR-ASO (p < 0.0001; Fig. 5a). Compared to enzalutamide, the AR-targeted ASO (25 and 50 mg/
kg) demonstrated superior antineoplastic activity (Fig. 5a;
p < 0.0001 or p = 0.006, respectively). The effects of AR-ASO and
control ASOs were also assessed in NSG mice (5 mice/group) To gain a preliminary understanding of the short-term
pharmacodynamic
effects
of
AR
suppression,
a group
of
JN-DSRCT xenografts and DSRCT PDXs was collected 10 days 5 5 TURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications Fig. 4 In vitro stimulation and inhibition of DSRCT proliferation via AR. a JN-DSRCT, TC71, LNCaP, and PC3 cell proliferation assays after treating
with an AR agonist hormone, dihydrotestosterone (DHT) in a dose-dependent manner. n is the number of experimental replicates. b Profiling JN-D
TC71, and LNCaP cells for their AR expression by western blotting and histogram presentation of relative AR levels across each cell line. c Profiling
DSRCT cells for AR protein expression (green) by immunofluorescence analysis with DAPI-labeled nuclei (blue), d Quantitative scatter plot represen
of the ratio Nuclear/Cytoplasmic AR mean intensity reported within a single cell at 0, 5 and 24 h of DHT post-treatment. e JN-DSRCT cells are rel
less sensitive to enzalutamide than (f) AR antisense oligonucleotides treatment, as shown by the in vitro WST1-Proliferation cell-based assay. g W
blot analysis of AR expression in JN-DSRCT cells untreated or after Control-ASO and AR-ASO treatments. Histogram presentation of relative AR
across each cell line after GAPDH normalization. Data points in a, e, and f represent mean ± SEM using three experimental replicates for each cell line
in b (bottom panel), d and g (bottom panel) represent mean ± standard deviation. P values calculated by unpaired two-tailed t-test. ARTICLE
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Fig. 4 In vitro stimulation and inhibition of DSRCT proliferation via AR. a JN-DSRCT, TC71, LNCaP, and PC3 cell proliferation assays after treating them
with an AR agonist hormone, dihydrotestosterone (DHT) in a dose-dependent manner. n is the number of experimental replicates. b Profiling JN-DSRCT,
TC71, and LNCaP cells for their AR expression by western blotting and histogram presentation of relative AR levels across each cell line. c Profiling of JN-
DSRCT cells for AR protein expression (green) by immunofluorescence analysis with DAPI-labeled nuclei (blue), d Quantitative scatter plot representation
of the ratio Nuclear/Cytoplasmic AR mean intensity reported within a single cell at 0, 5 and 24 h of DHT post-treatment. e JN-DSRCT cells are relatively
less sensitive to enzalutamide than (f) AR antisense oligonucleotides treatment, as shown by the in vitro WST1-Proliferation cell-based assay. g Western
blot analysis of AR expression in JN-DSRCT cells untreated or after Control-ASO and AR-ASO treatments. Histogram presentation of relative AR levels
across each cell line after GAPDH normalization. Data points in a, e, and f represent mean ± SEM using three experimental replicates for each cell line. Data
in b (bottom panel), d and g (bottom panel) represent mean ± standard deviation. P values calculated by unpaired two-tailed t-test. increase in AKT signaling can occur following AR inhibition42. Notably, the same AR-ASO (AZD5312) used in our preclinical
experiments was well tolerated when administered to PC patients
(NCT03300505). Therefore, one could theoretically investigate
this AR-ASO drug candidate in DSRCT-specific phase 2 trials
without delay. Given the limited nature of our preclinical studies,
future studies with enzalutamide are also of interest. into their AR-ASO treatment (Fig. 6a, AR-ASO PD) for analysis
by RPPA to assess early compensatory pharmacodynamic
changes. pS6, Akt, estrogen receptor (ER), PD-1L, pAKT, and
other proteins (Fig. 6a and Supplementary Fig. S7f–g) were
upregulated. Others have reported that the PI3K-AKT pathway
has pleiotropic effects on survival, proliferation, metabolism, and
growth pathways of several malignancies41, and its blockade has
long been of interest in managing PC, where a compensatory NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z
ART reclinical efficacy of AR antisense-based therapy for the treatment of DSRCT. a–c Therapeutic effect of AR blockade in JN-DSRCT xen
three replicates. Tumor-bearing mice volumes and survival were reported after treatment with enzalutamide (25 mg/kg, orange), AR-AS
kg, regular red; 50 mg/kg, dotted red), control ASO (gray), or placebo (black). The top panel (a) shows the individual data for each mou
anel (b) shows a smoothed grouped median of relative tumor volumes; the lower panel (c) shows the survival Kaplan–Meier curves of
group of mice. d–f Similar data is shown for a DSRCT PDX treated with the same agents. d Individual PDX data, e smoothed PDX data,
–Meier curves for the DSRCT PDX. P values reported for the smoothed tumor growth curves (b and e) were calculated by a two-tailed unp
plan–Meier P values were calculated by the log-rank (Mantel–Cox) test. n is the number of mice treated in each treatment group. Fig. 5 Preclinical efficacy of AR antisense-based therapy for the treatment of DSRCT. a–c Therapeutic effect o al efficacy of AR antisense-based therapy for the treatment of DSRCT. a–c Therapeutic effect of AR blockade in JN Fig. 5 Preclinical efficacy of AR antisense-based therapy for the treatment of DSRCT. a–c Therapeutic effect of AR blockade in JN-DSRCT xenografts
done in three replicates. Tumor-bearing mice volumes and survival were reported after treatment with enzalutamide (25 mg/kg, orange), AR-ASO
(25 mg/kg, regular red; 50 mg/kg, dotted red), control ASO (gray), or placebo (black). The top panel (a) shows the individual data for each mouse; the
middle panel (b) shows a smoothed grouped median of relative tumor volumes; the lower panel (c) shows the survival Kaplan–Meier curves of each
treated group of mice. d–f Similar data is shown for a DSRCT PDX treated with the same agents. d Individual PDX data, e smoothed PDX data, and
f Kaplan–Meier curves for the DSRCT PDX. P values reported for the smoothed tumor growth curves (b and e) were calculated by a two-tailed unpaired t-
test. Kaplan–Meier P values were calculated by the log-rank (Mantel–Cox) test. n is the number of mice treated in each treatment group. Fig. 5 Preclinical efficacy of AR antisense-based therapy for the treatment of DSRCT. a–c Therapeutic effect of AR blockade in JN-DSRCT xenografts
done in three replicates. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710 NATURE COMMUNICATIO R directly regulates important oncogenic regulators in
SRCT. To model the AR transcriptional program in a human
-DSRCT cell line, we determined the genome-wide AR binding
ofiles using ChIP-Seq experiments in unstimulated or DHT-
mulated JN-DSRCT cells treated with control ASO or AR-ASO. expected, DHT treatment enhanced AR binding to the chro-
atin as assessed by the average intensity plot on all significant
peaks (p < 1e−7) and heatmap (Fig. 7a). DHT stimulation led t
~4000 new peaks that were suppressed by treatment with AR
ASO (Fig. 7b and Supplemental Data 1). These binding sites wer
enriched at known AR response elements (AREs) and in sites fo
FOXA1, a transcription factor known to open compacted DNA
and cooperate with AR in PC43 (Fig. 7c and Supplementa
Data 1). Consistent with DSRCT’s pathogenesis, we also note
NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications AR directly regulates important oncogenic regulators in
DSRCT. To model the AR transcriptional program in a human
JN-DSRCT cell line, we determined the genome-wide AR binding
profiles using ChIP-Seq experiments in unstimulated or DHT-
stimulated JN-DSRCT cells treated with control ASO or AR-ASO. As expected, DHT treatment enhanced AR binding to the chro-
matin as assessed by the average intensity plot on all significant
peaks (p < 1e−7) and heatmap (Fig. 7a). DHT stimulation led to
~4000 new peaks that were suppressed by treatment with AR-
ASO (Fig. 7b and Supplemental Data 1). These binding sites were
enriched at known AR response elements (AREs) and in sites for
FOXA1, a transcription factor known to open compacted DNA
and cooperate with AR in PC43 (Fig. 7c and Supplemental
Data 1). Consistent with DSRCT’s pathogenesis, we also noted
8
NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications AR directly regulates important oncogenic regulators in
DSRCT. To model the AR transcriptional program in a human
JN-DSRCT cell line, we determined the genome-wide AR binding
profiles using ChIP-Seq experiments in unstimulated or DHT-
stimulated JN-DSRCT cells treated with control ASO or AR-ASO. As expected, DHT treatment enhanced AR binding to the chro-
matin as assessed by the average intensity plot on all significant peaks (p < 1e−7) and heatmap (Fig. 7a). DHT stimulation led to
~4000 new peaks that were suppressed by treatment with AR-
ASO (Fig. 7b and Supplemental Data 1). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Tumor-bearing mice volumes and survival were reported after treatment with enzalutamide (25 mg/kg, orange), AR-ASO
(25 mg/kg, regular red; 50 mg/kg, dotted red), control ASO (gray), or placebo (black). The top panel (a) shows the individual data for each mouse; the
middle panel (b) shows a smoothed grouped median of relative tumor volumes; the lower panel (c) shows the survival Kaplan–Meier curves of each
treated group of mice. d–f Similar data is shown for a DSRCT PDX treated with the same agents. d Individual PDX data, e smoothed PDX data, and
f Kaplan–Meier curves for the DSRCT PDX. P values reported for the smoothed tumor growth curves (b and e) were calculated by a two-tailed unpaired t-
test. Kaplan–Meier P values were calculated by the log-rank (Mantel–Cox) test. n is the number of mice treated in each treatment group. NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications 7 7 ARTICLE ARTICLE SEs
in control ASO-treated cells marked important oncogenes such as
AKT3 and GRHL2, whereas SEs in AR-ASO-treated cells marked
tumor suppressor genes such as RUNX1 and CUX1 (Supple-
mentary Fig. S10c, d and Supplemental Data 3), that potentially
regulate the AR-driven transcriptome. Overall, our results suggest
that AR activation reprograms typical enhancers and SE to reg-
ulate key oncogenic signaling pathways in DSRCT. enrichment of WT1 binding motifs within AR binding peaks
(Fig. 7c) suggesting potential interactions between AR, FOXA1,
and WT1 in JN-DSRCT cells. To further characterize the genes
adjacent to AR binding site peaks, we performed a pathway
analysis using 700 genes that are direct targets of AR. Upregulated
pathways included the TNFα pathway, Hippo signaling, and
pluripotency regulators (Fig. 7d and Supplemental Data 1), and
key genes included WT1, CTNNB1, SOX2, GLI2, FOXF1, and
GATA6 (Fig. 7e, Supplementary Fig. S8d, and Supplemental
Data 1). After evaluating the effects of androgen stimulation and
withdrawal in JN-DSRCT cells, we next compared DSRCT to
data from PC cells. Significant overlap existed at sites for AR
binding at AREs (Supplementary Fig. S8a), FOXA1 motifs
(Supplementary Fig. S8b), and sites that regulate key cancer
pathways, including WNT, TGFβ, PI3K, MAPK, Hippo signaling,
TNFα, and epithelial-to-mesenchymal transformation (Supple-
mentary Fig. S8c). To further evaluate the AR regulatory function
in DSRCT tumor mouse models, we performed ChIP-seq on
DSRCT-xenograft and PDX samples. Consistent with the cell line
data, we observed suppressed AR binding to the chromatin by the
treatment with AR-ASO (Supplementary Fig. S9a, b). Similarly,
pathway analysis of the top 5000 lost AR binding sites targeted
genes showed enrichment of MAPK pathway, Hippo signaling,
Wnt signaling, and pluripotency regulators (Supplementary
Fig. S9c, d). We also noted enrichment of AR and FOX family
binding motifs within AR binding peaks in both DSRCT-
xenograft and PDX samples (Supplementary Fig. S9e). Genes
adjacent to AR binding site peaks also showed high overlap with
DSRCT-specific genes in both models (Supplementary Fig. S9f). Key genes from cell line data (Fig. 7e) also showed AR signal
reduction after AR-ASO treatment (Supplementary Fig. S9g). Interestingly, in preclinical tumor samples, we also observed
similar enhancer reprogramming. AR-ASO treatment of the
DSRCT-xenograft significantly increased the active enhancer and
promoter binding sites compared to control ASO treatment,
whereas PDX samples showed a moderate increase (Supplemen-
tary Fig. S11a–d). We also observed AR-dependent active
enhancers regulating PI3K-AKT-mTOR, WNT signaling, cell-
adhesion pathways (Supplementary Fig. ARTICLE ARTICLE Fig. 6 Proteomic evaluation of AR expression in JN-DSRCT and PDX tumors after AR-based antisense therapy. a The principal components analysis
plot and reverse-phase protein lysate array (RPPA) evaluations of JN-DSRCT and PDX tumors after therapies, separated the 32 samples into four groups
and identified 37 proteins statistically significantly associated with the treatment at a false discovery rate (FDR) of 0.05. b Immunoblotting evaluation of
JN-DSRCT xenograft and PDX-DSRCT tumors after AR-ASO treatment. c AR normalization relative to GAPDH within the preclinical tumor samples. AR
biomarker was significantly reduced in mice treated with AR-ASO compared to the control ASO group (p = 0.01). d Representative AR
immunofluorescence confocal microscopy quantification of the preclinical JN-DSRCT and PDX tumor samples, within the single cell or e the averaged
treated samples (placebo, control ASO, and AR-ASO). f Immunohistochemical evaluation images of preclinical JN-DSRCT and PDX1 tumor samples. IHC
stains for AR in primary tumors of JN-DSRCT and PDX-DSRCT mice after treatment with AR-ASO, control ASO, and placebo. 100 μm scale bars are shown. g Representative IHC AR mean intensity quantification of the preclinical JN-DSRCT and PDX tumor samples, within the single cell or h the averaged treated
samples (placebo, control ASO, and AR-ASO). All tumors analyzed by RPPA were collected at tumor progression or the experiment’s conclusion, except
for the AR-ASO PD group, which was collected 10 days after initiating therapy to enable pharmacodynamic analysis. Data in c, d, e, g, and h represent
mean ± standard deviation. P values calculated by unpaired two-tailed t-test. marking in PC45. To identify which enhancers were likely derived
by AR binding and potential recruitment of enhancer-marking
proteins, we overlapped the AR and H3K27ac peaks in DHT-
treated cells (Supplementary Fig. S10b and Supplemental Data 2). We then intersected these AR-targeted enhancer peaks with
highly expressed genes in DSRCT (Fig. 8c and Supplemental
Data 2) (FC > 1.5, adjusted p value < 0.05 in comparison to other
sarcoma subtypes). There, we identified WNT signaling and cell-
adhesion as major drivers that are regulated at the chromatin
level by AR-dependent active enhancer programs (Fig. 8d). The
genes with direct AR binding and enhancer gains included
important oncogenes such as AXIN2 and CDK6 (Fig. 8e). In
addition, we investigated alterations in super-enhancer (SE)
regions that harbor a high density of TF binding motifs46–48. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z These binding sites were
enriched at known AR response elements (AREs) and in sites for
FOXA1, a transcription factor known to open compacted DNA
and cooperate with AR in PC43 (Fig. 7c and Supplemental
Data 1). Consistent with DSRCT’s pathogenesis, we also noted NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications 8 ARTICLE S11e, f), and key
oncogenes (Fig. 8e), with direct AR binding and enhancer gains
in both DSRCT-xenograft and PDX models (Supplementary
Fig. S11g). AR-dependent enhancer reprogramming activates oncogenic
pathways in DSRCT. Several studies have shown that AR
establishes a pro-tumorigenic transcriptome by reprogramming
the active enhancer landscape (assessed by H3K27ac profiles) in
PC progression44. Therefore, we asked if AR plays similar roles in
DSRCT by examining genome-wide profiles for H3K27ac marks
in unstimulated or DHT-stimulated JN-DSRCT cells treated with
control ASO or AR-ASO. We noted that unstimulated cells
treated with AR-ASO showed a higher intensity and a higher
number of H3K27ac peaks compared to control ASO-treated cells
(Fig. 8a, b, Supplementary Fig. S10a, and Supplemental Data 2). Similarly, AR-ASO treatment in DHT-treated cells also increased
the active enhancer peaks compared to control ASO treatment
(Fig. 8a, b, Supplementary Fig. S10a, and Supplemental Data 2). This observation is contrary to those in PCs where active
enhancer
peaks
are
positively
associated
with
higher
AR
activity44. It has been previously shown that AR recruits the MLL
complex and CBP/p300, which is responsible for active enhancer Discussion Ever since Ladanyi and Gerald discovered the EWSR1-WT1
chromosomal translocation8, DSRCT has been treated with the
same chemotherapy regimens used for ES. The recent exceptions
include ES-specific agents like TK-216 that target c-terminus ETS
genes (e.g., FLI1 or ERG), or pazopanib, which demonstrates
preferential activity in DSRCT and other soft-tissue sarcomas49. Phase II studies testing neoantigen targeted monoclonal anti-
bodies, for example, 8H9 in DSRCT, are also directed at unique
sarcoma subtypes50. As three-quarters of all DSRCT patients typically succumb to
their malignancy within 5-years, our RPPA study intended to
define new molecular targets for DSRCT and expand our ther-
apeutic arsenal of biologically targeted therapies that engage them
(Fig. 1). Surprisingly, of 151 proteins assessed in the RPPA, SYK
and AR were the most differentially expressed. The SYK protein
—not previously reported in DSRCT—is a non-receptor tyrosine
kinase (also known as spleen tyrosine kinase) commonly found in TURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Fig. 7 AR binding in JN-DSRCT cells. a Heatmaps (left panels) and average intensity curves (right panels) of ChIP-seq reads (RPKM; reads per kilobase o
transcript per million mapped reads) for AR binding regions. AR binding sites are shown in a 10-kb window (centered on the middle of the binding site) in
Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples. b Venn diagram showing the overlap of all AR peaks between Control ASO
DHT + Control ASO, and DHT + AR-ASO samples to identify the AR-unique or shared binding sites. c List of enriched transcription factor (TF) motifs in
AR-specific binding sites. Motifs are identified using HOMER (Binomial test). d Dot plot showing significantly enriched pathways for AR-specific binding
sites. Dot size represents gene ratio, and colors represent adjusted p values (Fisher’s exact test). e IGV images showing enrichment of AR peaks around
WT1, SOX2, CTNNB1, GATA6, FOXF1, and GLI2 genes using aggregate ChIP-seq profiles of Control ASO, DHT + Control ASO, and DHT + AR-ASO samples
10
NATURE COMMUNICATIONS | (2022)13:3057 | https://doi org/10 1038/s41467-022-30710-z | www nature com/naturecommunications Fig. 7 AR binding in JN-DSRCT cells. a Heatmaps (left panels) and average intensity curves (right panels) of ChIP-seq reads (RPKM; reads per kilobase of
transcript per million mapped reads) for AR binding regions. AR binding sites are shown in a 10-kb window (centered on the middle of the binding site) in
Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples. b Venn diagram showing the overlap of all AR peaks between Control ASO, Fig. 7 AR binding in JN-DSRCT cells. a Heatmaps (left panels) and average intensity curves (right panels) of ChIP-seq reads (RPKM; reads per kilobase of
transcript per million mapped reads) for AR binding regions. AR binding sites are shown in a 10-kb window (centered on the middle of the binding site) in
Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples. b Venn diagram showing the overlap of all AR peaks between Control ASO,
DHT + Control ASO, and DHT + AR-ASO samples to identify the AR-unique or shared binding sites. c List of enriched transcription factor (TF) motifs in
AR-specific binding sites. Motifs are identified using HOMER (Binomial test). d Dot plot showing significantly enriched pathways for AR-specific binding
sites. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z a Heatmaps (left panels) and average intensity curves (right panels) of ChIP-seq reads (RPKM;
reads per kilobase of transcript per million mapped reads) for typical enhancer regions. Enhancer regions are shown in a 10-kb window (centered on the
middle of the binding site) in Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples. b Venn diagram showing the overlap of all
enhancer peaks between Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples to identify the AR-unique or shared enhancer
reprogramming. c Venn diagram showing the overlap of annotated genes for AR-specific gained enhancer peaks and upregulated gene list for DSRCT
tumors vs. other sarcoma tumors to identify the AR-unique enhancer reprogramming associated transcription upregulation. d Bar plot showing significantly
enriched pathways for AR-specific enhancer reprogramming associated transcription upregulation. Bar length represents gene numbers, and colors Fig. 8 Enhancer reprogramming by AR in JN-DSRCT cells. a Heatmaps (left panels) and average intensity curv Fig. 8 Enhancer reprogramming by AR in JN-DSRCT cells. a Heatmaps (left panels) and average intensity curves (right panels) of ChIP-seq reads (RPKM;
reads per kilobase of transcript per million mapped reads) for typical enhancer regions. Enhancer regions are shown in a 10-kb window (centered on the
middle of the binding site) in Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples. b Venn diagram showing the overlap of all
enhancer peaks between Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples to identify the AR-unique or shared enhancer
reprogramming. c Venn diagram showing the overlap of annotated genes for AR-specific gained enhancer peaks and upregulated gene list for DSRCT
tumors vs. other sarcoma tumors to identify the AR-unique enhancer reprogramming associated transcription upregulation. d Bar plot showing significantly
enriched pathways for AR-specific enhancer reprogramming associated transcription upregulation. Bar length represents gene numbers, and colors
represent adjusted p values (Fisher’s exact test). e IGV images showing enrichment of H3K27Ac peaks around AGRE2, AXIN2, CDK6, and MYH10 genes
using aggregate ChIP-seq profiles of Control ASO, DHT + Control ASO, and DHT + AR-ASO samples. have not yet had the opportunity to evaluate these relatively new
drugs within our preclinical DSRCT models. hematological tissues. Its constitutive activation has been shown
to induce malignant transformation of B-cells to lymphomas or
leukemias. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Dot size represents gene ratio, and colors represent adjusted p values (Fisher’s exact test). e IGV images showing enrichment of AR peaks around
WT1, SOX2, CTNNB1, GATA6, FOXF1, and GLI2 genes using aggregate ChIP-seq profiles of Control ASO, DHT + Control ASO, and DHT + AR-ASO samples. Fig. 7 AR binding in JN-DSRCT cells. a Heatmaps (left panels) and average intensity curves (right panels) of ChIP-seq reads (RPKM; reads per kilobase of
transcript per million mapped reads) for AR binding regions. AR binding sites are shown in a 10-kb window (centered on the middle of the binding site) in
Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples. b Venn diagram showing the overlap of all AR peaks between Control ASO,
DHT + Control ASO, and DHT + AR-ASO samples to identify the AR-unique or shared binding sites. c List of enriched transcription factor (TF) motifs in
AR-specific binding sites. Motifs are identified using HOMER (Binomial test). d Dot plot showing significantly enriched pathways for AR-specific binding
sites. Dot size represents gene ratio, and colors represent adjusted p values (Fisher’s exact test). e IGV images showing enrichment of AR peaks around
WT1, SOX2, CTNNB1, GATA6, FOXF1, and GLI2 genes using aggregate ChIP-seq profiles of Control ASO, DHT + Control ASO, and DHT + AR-ASO samples. NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications 10 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z ARTICLE Fig. 8 Enhancer reprogramming by AR in JN-DSRCT cells. a Heatmaps (left panels) and average intensity curves (right panels) of ChIP-seq reads (RPKM;
reads per kilobase of transcript per million mapped reads) for typical enhancer regions. Enhancer regions are shown in a 10-kb window (centered on the
middle of the binding site) in Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples. b Venn diagram showing the overlap of all
enhancer peaks between Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples to identify the AR-unique or shared enhancer
reprogramming. c Venn diagram showing the overlap of annotated genes for AR-specific gained enhancer peaks and upregulated gene list for DSRCT
tumors vs. other sarcoma tumors to identify the AR-unique enhancer reprogramming associated transcription upregulation. d Bar plot showing significantly
enriched pathways for AR-specific enhancer reprogramming associated transcription upregulation. Bar length represents gene numbers, and colors
represent adjusted p values (Fisher’s exact test). e IGV images showing enrichment of H3K27Ac peaks around AGRE2, AXIN2, CDK6, and MYH10 genes
using aggregate ChIP-seq profiles of Control ASO, DHT + Control ASO, and DHT + AR-ASO samples. Fig. 8 Enhancer reprogramming by AR in JN-DSRCT cells. a Heatmaps (left panels) and average intensity curves (right panels) of ChIP-seq reads (RPKM;
reads per kilobase of transcript per million mapped reads) for typical enhancer regions. Enhancer regions are shown in a 10-kb window (centered on the
middle of the binding site) in Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples. b Venn diagram showing the overlap of all
enhancer peaks between Control ASO, AR-ASO, DHT + Control ASO, and DHT + AR-ASO samples to identify the AR-unique or shared enhancer
reprogramming. c Venn diagram showing the overlap of annotated genes for AR-specific gained enhancer peaks and upregulated gene list for DSRCT
tumors vs. other sarcoma tumors to identify the AR-unique enhancer reprogramming associated transcription upregulation. d Bar plot showing significantly
enriched pathways for AR-specific enhancer reprogramming associated transcription upregulation. Bar length represents gene numbers, and colors
represent adjusted p values (Fisher’s exact test). e IGV images showing enrichment of H3K27Ac peaks around AGRE2, AXIN2, CDK6, and MYH10 genes
using aggregate ChIP-seq profiles of Control ASO, DHT + Control ASO, and DHT + AR-ASO samples. Fig. 8 Enhancer reprogramming by AR in JN-DSRCT cells. ARTICLE Lacking suitable
ChIP-seq validated WT1-specific antibodies ATAC-seq might be
used before and after WT1 RNA silencing, though the inter-
pretation of that experiment would not be as straightforward
given the absence of selective antagonism of WT1 or EWS-WT1. 11 58 59 Interestingly, as occurs in castration-resistant PC11,58,59, our
pharmacodynamic
studies
revealed
an
inverse
relationship
between AR and the Akt/PI3K/mTOR pathway. With numerous
inhibitors of PI3K and mTOR already FDA approved, an obvious
next step would be to investigate whether co-targeting AR and
either PI3K or mTOR results in synergistic anti-cancer activity. Though not explored in the present study, the ER was also highly
expressed following AR-ASO treatment. Given the shared bind-
ing DNA motifs that ER, AR, and other steroid hormone
receptors have in common, this observation suggests that ER-
targeted drugs might prove useful for patients with castrate-
resistant DSRCT and, plausibly, the small minority of women that
acquire this rare cancer type. Of course, further research is
required to determine how AR and ER pathway switching affects
tumor
growth
and
survival,
both
in
DSRCT
and
other
hormonally-driven malignancies60. pp
g
Meeting the second of Koch’s postulates, a 72-h cell pro-
liferation assay demonstrated a significant increase in JN-DSRCT
cell proliferation following exposure to physiological levels of
DHT (Fig. 4a), though lower than LNCaP PC cells. As one would
expect in androgen-sensitive cells, DHT also promoted the
nuclear shuttling of AR into the nucleus where it would function
as a transcriptional regulator of its target genes (Fig. 4c, d). As our
results rely upon data from a single cell line (the only one in
existence at the time), we remain vigilant to avoid over-
interpreting them. Nevertheless, our results echo similar find-
ings by Fine et al., where they reported a DHT-induced two-fold
increase in cell proliferation in a transient DSRCT cell line
obtained from ascitic fluid18. y
g
Collectively, though morphologically and phenotypically dis-
tinct from PC, our data suggest that DSRCT is a second
androgen-stimulated malignancy (third, if one considers the AR-
positive molecular subset of triple-negative breast cancer). Shared
dependence upon AR for tumor growth and survival provides an
exciting opportunity to study AR signaling in a different cancer
type and within a younger DSRCT-stricken patient population. Preclinical data using enzalutamide and AR-ASO raises the tan-
talizing possibility that AR-targeted drugs used for PC may also
find utility to combat DSRCT. ARTICLE Fulfilling the third requirement that defines an AR-driven
malignancy, our team again bolsters the work by Fine et al., which
had taken a prescient step more than a decade ago to evaluate
CAB—in that case, using Lupron and bicalutamide in six DSRCT
patients that were AR-positive (3+ or 4+ by IHC)18. Interest-
ingly, in their limited pilot trial, non-castrate level baseline tes-
tosterone levels were associated with modest responses lasting
3–4 months. Admittedly, having tested several DSRCT patients
with the same drug combination between 2006 and 2015, well
before the advent of modern-day androgen deprivation therapies
such as abiraterone and enzalutamide, our team observed limited
clinical benefits lasting <3 months. Our renewed enthusiasm for
AR targeting in DSRCT stemmed from the RPPA expression
results, accompanied by the in vitro DHT stimulation studies and
in vivo data using enzalutamide and the AR-ASO (Fig. 5). ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z including those directly tying AR to AREs, lends further cred-
ibility that DSRCT is a second AR-driven malignancy. binding of AR to specific genomic loci in PC53–55, and that
control AR-mediated transcriptional regulation of prostate genes
(such as PSA)56, FOXA1 was the most enriched MOTIF in JN-
DSRCT cells (Fig. 7c). Shared activation of the androgen signaling
cascade in DSRCT and PC may explain the close transcriptomic
clustering observed in Fig. 2e. Despite their similarities, ChIP-seq
also identified notable differences in AR’s epigenetic regulation at
enhancer (Fig. 8) and SE (Supplementary Fig. S9c) binding sites. y
g
y
To date, the first criterion—requiring AR expression—has been
reported by two prior teams that recognized the striking pre-
dilection of DSRCT for young males15,51. As discussed briefly in
the Introduction, Fine et al. evaluated protein expression of AR,
c-Kit, EGFR, and other proteins by WB and IHC, scored using a
5-point scale that ranged between 0 (no staining) to 4+ (highly
positive)15. Ten of 27 (37%) DSRCT patients in their case series
scored 2+ or higher, but we highlight that 15 demonstrated no
AR expression (Fig. 2), which suggests prospective studies may
wish to stratify for response by AR status to determine whether
AR expression correlates with therapeutic efficacy. A more recent
study published in 2018 by Bulbul et al. at U.C.S.D., used IHC
and next-generation sequencing on tumors from 35 DSRCT
patients (86% who were males); 59% were AR-positive using a
dichotomous cut-off that required ≥1+ staining in ≥10% of the
cells51. In the present study, we report the most extensive series of
DSRCT patients to have undergone protein and transcriptomic
profiling. Though enriched in oncoproteins, our RPPA array
ranked AR as the most differentially expressed protein compared
to ES, its closest molecular sarcoma subtype (Fig. 1). Our sub-
sequent confirmation of the RPPA results by WB, and later semi-
quantitative analysis by IHC, agrees with earlier reports and
appears to substantiate AR as a bona fide target in DSRCT. Though the subject of future research, we suspect the hetero-
typic loss of WT1 or aberrant EWS-WT1 FP may recruit a specific
set of chromatin modifiers at binding sites that differ from PC. Others have performed ChIP-seq in DSRCT patient specimens
using WT1-specific antibodies, but the Santa Cruz antibody used
in that publication57 has been discontinued. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z As such, the oral SYK inhibitors cerdulatinib (Portola
Pharmaceutical) and entospletinib (Gilead Sciences) are under
active clinical investigation for the treatment of certain lympho-
mas, chronic lymphocytic leukemia, and acute myeloid leukemia
(NCT01994382 and NCT02457598). An orally active SYK inhi-
bitor, fostamatinib, has already received FDA approval as a
treatment for immune thrombocytopenia and continues to be
investigated as an experimental therapy for hematological
malignancies (NCT00446095). Though tantalizing to consider
that SYK hyperactivation plays an oncogenic role in DSRCT, we In contrast to SYK, numerous FDA-approved and experi-
mental AR antagonists were available for immediate preclinical
evaluation, and potentially available to patients via compassionate
access or early-phase clinical trials. Though our RPPA data and
9:1 male-to-female ratio hinted that DSRCT is an AR-driven
malignancy, to prove this explicitly we proposed several criteria,
akin to Koch’s postulates: (a) tumors must adequately express
AR, (b) DHT must stimulate DSRCT cell proliferation, and (c)
AR antagonists should curtail the tumor-promoting effects of
androgen stimulation. The inclusion of mechanistic studies, TURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications 11 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Raw log2 intensity values were normalized for
global protein expression by median centering across 151 antibodies tested. Indi-
vidual protein expressions in DSRCT specimens, ES specimens, and mesenteric
normal tissue specimens were compared using two-sided unpaired t-tests with the
GeneSpring GX software program version 12.6.1-GX (Agilent Technologies). For
multiple comparison testing, proteins whose expression levels between specimen
types were two-fold and significantly different (p ≤0.05) were subjected to unsu-
pervised hierarchical clustering. Differentially expressed proteins identified by
RPPA were confirmed by WB analysis. The RPPA data for the proteomic com-
parison of DSRCT patient tumors, normal mesenteric tissues, and ES patient
tumors are available from the Gene Expression Omnibus repository under
GSE108687 series. The preparation of extracted protein from cells, DSRCT patient tumors, or
xenograft animal tumors for western blotting validations was prepared as described
previously62. Lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl,
1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM Na pyrophosphate, 1 mM
Na3VO4, 10% glycerol) containing freshly added protease and phosphatase
inhibitors (Roche Applied Sciences) was applied to lyse cellular washed pellets via
cold incubation. Next, protein extraction from xenograft tumors was performed by
homogenizing approximately 10 mg of frozen tissue in 500 μL of the lysis buffer
using an electric tissue homogenizer (PRO Scientific). The homogenized tumors
were incubated at +4 °C for 2 h to complete their dissociation and lysis. Altogether,
the total lysed proteins from tumors were collected after centrifugation, quantified
using BCA protein assay kit (Thermo Fisher Scientific), and stored at −80 °C until
further analyses. WBs were performed as previously described by our group61,62. Protein detection was performed using a list of antibodies provided in the above
supplementary table of antibodies used for WBs. The immune-reactive proteins
were captured using horseradish peroxidase-conjugated secondary anti-rabbit IgG
or anti-mouse IgG antibodies (Cell Signaling Technology), amplified using the
SuperSignal West Dura chemiluminescent substrate (Thermo Fisher Scientific),
detected using the Chemi-Doc system (Bio-Rad), and quantified for their
densitometry using the ImageJ Gel Analysis tool (NIH, Bethesda, MD). Generation of DSRCT xenograft/PDX mouse models and drug evaluation. All
experiments were conducted per protocols and conditions approved by the Uni-
versity of Texas MDACC (Houston, TX) Institutional Animal Care and Use
Committee (eACUF Protocols #00000712-RN03). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z control (sterile vehicle buffer). Tumor volumes were measured using digital calipers
at study initiation and 2–5 times per week after that for up to 85 days, or until their
tumors reached 1500 mm3, whichever came first. Per institutional requirements,
tumor size never exceeded 2 cm in maximal linear dimension. A Kaplan–Meier
analysis was performed to assess drug efficacy. Statistical analyses between the
control and treated groups or between different treated groups were performed
with the log-rank (Mantel–Cox) test using GraphPad Prism 8.0. RNA sequencing, gene expression analysis, and fusion detection. Total RNA
from primary tumor samples was extracted and libraries were made from cDNA
using the NuGEN Ovation Ultralow Library System V2 (San Carlos, CA). RNA
sequencing reads of the samples were mapped to the hg19 reference genome using
the STAR aligner64. For calculation of gene expression, each gene’s raw count data
were first obtained using HTSeq65, and are normalized by scaling the library size
using calcNormFactors in the edgeR package66. Then, Voom transformation was
applied to normalized counts and a linear model fit to the data for differential
expression analysis using the Limma package67. Pathway analyses of differentially
expressed genes between two sample clusters were performed using Gene Set
Enrichment Analysis68. Fusion transcripts were detected from RNA-seq data using
MapSplice69. Mass spectroscopy-based determination of intratumoral hormone levels. Testosterone and corticosterone quantification was determined using Agilent’s
Infinity II UHPLC in line with a 6495 triple quadrupole mass spectrometer and
MassHunter workstation software (8.0.8.23.5). Briefly, DSRCT xenograft and PDX
samples were homogenized using water containing internal standard (Cerilliant,
T070) extracted with tert-butyl methyl ether (Sigma 34875), dried under nitrogen,
and derivatized using hydroxylamine hydrochloride (Sigma 431362). The recov-
ered ketoxime steroids were reconstituted in methanol/water (1:1 v/v) and injected
into the Infinity II UHPLC. Ketoxime steroids were separated using a Chromolith
reverse-phase column (RP-18 endcapped 100–2 mm, Sigma 152006) and intro-
duced into a JetStream source (Agilent) for triple quadrupole analysis. Data were
analyzed and quantified using MassHunter software (Agilent)39,40. TMA preparation and immunohistochemistry analyses. A TMA was con-
structed from archival surgical pathology materials comprising 60 formalin-fixed,
paraffin-embedded (FFPE) tissues from 60 DSRCT patients. Areas of the viable
tumor were selected by pathologist review of whole slide H&E-stained sections. Selected areas were punched and transferred, in duplicate, to a recipient block
using an ATA-100 Advanced Tissue Arrayer (Chemicon International). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z All human
specimens were utilized under an Institutional Review Board-approved research
protocol (LAB04-0890) allowing for the retrospective sampling and analysis of
existing archival materials collected during standard patient care. Immunohisto-
chemical studies were performed using an autostainer (Bond‐Max; Leica Micro-
systems, Buffalo Grove, IL, USA) with anti‐AR (1:30; clone AR441, Dako#M3562)
antibody. Additional details about TMA slide preparation and IHC analyses are
provided in the Supplementary Methods. ChIP-seq assays. Chromatin immunoprecipitation was performed as described
earlier70 with optimized shearing conditions and minor modifications for JN-
DSRCT cells. The antibodies used were: H3K27ac (Abcam ab4729) and AR
(CST#5153). Briefly, 3 million cells per sample were cross-linked using 1% for-
maldehyde for 10 min at 37 °C. After quenching with 150 mM glycine for 5 min at
37 °C, cells were washed twice with ice-cold PBS and frozen at −80 °C for further
processing. Later, cells were thawed on ice and lysed with ChIP harvest buffer
(12 mM Tris-HCl, 0.1 × PBS, 6 mM EDTA, 0.5% sodium dodecyl sulfate [SDS]) for
30 min on ice. Lysed cells were sonicated with Bioruptor (Diagenode) to obtain
chromatin fragments. Antibody-dynabead mixtures were incubated for 1 h at 4 °C
and cellular extracts were then incubated overnight with these mixtures. After
overnight incubation, immune complexes were washed five times with RIPA buffer,
twice with RIPA-500 (RIPA with 500 mM NaCl) and twice with LiCl wash buffer
(10 mM Tris-HCl pH8.0, 1 mM EDTA pH8.0, 250 mM LiCl, 0.5% NP-40, 0.1%
deoxycholate). For reverse-crosslinking and elution, immune complexes were
incubated overnight at 65 °C in elution buffer (10 mM Tris-HCl pH8.0, 5 mM
EDTA, 300 mM NaCl, 0.5% SDS). Eluted DNA was then treated with proteinase K
(20 mg/ml) and RNase A and DNA clean-up was done using SPRI beads (Beck-
man-Coulter). ChIP libraries were amplified and barcoded with use of the NEB-
Next® Ultra™II DNA library preparation kit (New England Biolabs). After library
amplification, DNA fragments were size-selected (200–500 bp) using AMPure XP
beads (Beckman Coulter) and assessed using high sensitivity D1000 screen tape on
the Bioanalyzer (Agilent Technologies). Libraries were multiplexed together and
sequenced in HiSeq2000 (Illumina). WST1 cell proliferation assays. The JN-DSRCT, LNCaP, and TC71 cells were
tested for their proliferation capacity in vitro using a colorimetric assay in 96-Well
plates with WST1 reagent (Roche). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z The cells were seeded at 3000 cells/well in
triplicates with 10% FBS DMEM (JN-DSRCT) or RPMI (TC71 and LNCaP)
complete media. Additional details about WST1 cell proliferation assays are pro-
vided in the Supplementary Methods. JN-DSRCT and TC71 cell lines were sup-
plied by JL. LNCaP and PC3 were obtained through MDACC’s cell line repository. Immunostaining of JN-DSRCT cells and xenograft animal tumors. JN-DSRCT
cell line exhibiting a pathognomonic t(11;22)(p13;q12) translocation was gener-
ously provided from Dr M. Kikuchi’s laboratory (Fukuoka University, Fukuoka,
Japan). In addition, PC3, LnCaP, and TC71 cell lines are provided by the MDA cell
lines core facility. All available cell lines in Dr Ludwig’s lab are registered within the
MDA characterized cell line core (CCLC). Each cell line identity is validated twice
per year in MDA CCLC using short-tandem repeat fingerprinting with an
AmpFLSTR Identifier kit. Furthermore, according to the manufacturer’s protocol,
all our cell lines are tested twice per year for mycoplasma contamination using the
MycoAlert Detection Kit (Lonza Group Ltd.). In addition, cell lines are sent for
third-party mycoplasma testing using a sensitive PCR testing approach any time a
collection of cells are cryopreserved. y
Monolayer JN-DSRCT cell culture in 8 chamber slides was fixed for 10 min at
room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS). The primary JN-DSRCT xenograft and PDX tumors were harvested, fixed in 10%
formalin, embedded in paraffin (FFPE), and then sliced in 5-μm sections before
processing them for antigen retrieval using 0.1 M citrate buffer for 20 min and in a
vegetable steamer. Altogether, monolayer and primary tumor slides were
permeabilized and blocked with superblock buffer (Thermo Fisher Scientific, #37535)
for 1 h at room temperature. Slides were then incubated consecutively with primary
antibodies to AR (Cell Signaling Technologies, #5153), (overnight at 4 °C) and Alexa
Fluor 488-labeled Goat-anti-Rabbit (Thermo Fisher Scientific, #A11037) (for 1 h at
room temperature). The nuclei were visualized using Hoechst (Thermo Fisher
Scientific, #H357), and the immunofluorescence was acquired after subtracting the
background intensities using the Nikon A1-Rsi confocal microscope (Nikon). Fluorescent detection of proteins in the nuclei and cytosolic regions was quantified
using the Imaris software (Bitplane) and its Cell module that use validated algorithms
to define the segmentation by permitting the recognition of selected protein
fluorescence in both nuclear and cytosolic regions. RPPA and western blot analyses. Methods
Tumor spec Tumor specimens. In compliance with all relevant ethical guidelines, patients
provided written informed consent to collect and use their tumor specimens for
research purposes using lab protocols LAB08-0151 or LAB04-0890, which are
approved by MDACC’s Institutional Review Board. The charts and electronic
medical records of patients with a confirmed diagnosis of DSRCT were included
for analysis and archived at the MDACC biospecimen bank or the collaborator PIs
laboratories. We identified 60 DSRCT patients treated at MDACC from 1990 to
2019 to generate a TMA. Also, we collected 16 DSRCT and 6 ES fresh frozen
tumors, all of them were profiled by RPPA. Specialist pathologists used clinical
information, immunohistochemistry, and cytogenic analysis for the EWSR1-WT1
or EWSR1-FLI1 fusions to confirm the DSRCT or ES diagnoses. In preparation for early-phase clinical trials now in develop-
ment, our work takes the first step to advance our mechanistic
understanding of AR signaling in DSRCT. As one of several
steroid and nuclear hormone superfamily receptors that include
the glucocorticoid receptor, mineralocorticoid receptor, proges-
terone and estrogen receptors, and the vitamin D receptor, AR
retains a conserved 66-amino acid DNA-binding domain able to
join two (5’-AGAACA-3’) hexameric half-sites arranged as an
inverted palindrome spaced 3-b.p. apart (IR3). Due to differences
in local steroid metabolism, ligand abundance, chromatin acces-
sibility, and cofactor occupancy, the DNA binding pattern of AR
varies significantly in PC compared to other tissues52. Interest-
ingly, among the pioneer factors that govern the lineage-specific RPPA and western blot analyses. The available snap-frozen DSRCT (n = 16) and
ES (n = 6) specimens collected during a core-needle biopsy or surgical debulking
procedures using clinical protocols approved by MDACC’s Institutional Review
Board and specimens of normal-appearing mesenteric tissue adjacent to DSRCT
obtained at the time of surgical debulking (n = 8) were used for the proteomic
analysis (Supplementary Table 1: Aggregate demographic information of DSRCT
and ES patients). Lysates were created, protein concentrations were determined,
and individual protein expression was measured using a well-validated RPPA and
WB technologies as previously described61–63. AR protein detection was performed
using the CST antibody (#5153). Additional details about RPPA and WB analyses
and normalized data are provided in the Supplementary Methods and Supple-
mental Data 4. 12 NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications Data availability y
All data that support the findings of this study are provided within the article,
Supplementary Information, and source data. All ChIP-seq data are available at GEO
accession number GSE151380, released publicly on September 1, 2022. RNA-seq data are
made publicly available in the European Genome-Phenome Archive: DSRCT at
EGAS00001004575; liposarcoma at EGAS00001002807; osteosarcoma at
EGAS00001003247; chondrosarcoma at EGAS00001004585; and prostate cancer through
Subudhi et al.83. RPPA DSRCT GEO (GSE108687) RNA-Seq DSRCT EGAS00001004575
[https://ega-archive.org/datasets/EGAD00001006394]. RNA-Seq LS data
EGAS00001002807. RNA-Seq OS data EGAS00001003247. RNA-Seq CS data
EGAS00001004585. RNA-Seq for PC: EGAS00001004050. RPPA data GEO
(GSE178406). AR-ChIP-seq GEO (accession #: GSE151380). H3K27Ac ChIP-seq GEO
(accession #: GSE151380). Additional RPPA GEO (accession #: GSE178406). WST1 cell proliferation assays. The cells were cultivated for 24 h to let them
adhere, before adding serially diluted concentrations of dihydrotestosterone (DHT,
0.0064-2000nM; Selleckchem), or AR-based drug treatments (Enzalutamide, AR-
ASO; 0.032–5 mM) for up to 48 or 72 h, respectively. WST1 was added, and cells
were incubated for an additional 2 h. Cell proliferation was measured at 450 nm in
a microplate reader (DTX880, Beckman Coulter). In both assays, the agonistic
effect of DHT or cytotoxic effect of the drugs was expressed as a percentage of cell
proliferation. IC50 values were calculated by the sigmoidal dose-response curve-fit
using Prism GraphPad 8.0. Immunohistochemistry and digital image analyses. Primary tumors from the
DSRCT xenograft and PDX1 group of mice, untreated and treated with control-
ASO or AR-ASO, were fixed in 10% formalin, embedded in paraffin, and then
sliced in 5-μm sections. The EZ-retriever microwave-based pretreatment and
antigen retrieval system (Biogenex, CA) was used for dewaxing, rehydration, and
antigen retrieval of these FFPE lung tissue sections. AR protein expression by IHC
was evaluated on Leica Bond MAX Autostainer by using primary antibody, rabbit
antibody anti-human AR (Cell Signaling Technologies, #5153). The primary
antibody was detected according to the manufacturer's protocol (Leica, #DS9800). Then, the slides were dehydrated in grade alcohols, cleared in xylene, cover slipped,
and imaged with Keyence Microscope (Keyence, Tokyo, Japan) at ×20 resolution. The digital images were processed and quantified using the Visiopharm software
version (2020.04) (Hoersholm, Denmark). An APP (Analysis Protocol Package)
was designed to quantify cell-based DAB staining in DSRCT preclinical samples
using traditional thresholding methods. References 1. Honore, C. et al. Abdominal desmoplastic small round cell tumor: multimodal
treatment combining chemotherapy, surgery, and radiotherapy is the best
option. Ann. Surg. Oncol. 22, 1073–1079. https://doi.org/10.1245/s10434-014-
4123-6 (2015). 2. Chang, F. Desmoplastic small round cell tumors: cytologic, histologic, and
immunohistochemical features. Arch. Pathol. Lab. Med. 130, 728–732 (2006). 3. Gerald, W. L., Rosai, J. & Ladanyi, M. Characterization of the genomic
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associated with t(11;22)(p13;q12): desmoplastic small round-cell tumor and its
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by pyflow-ChIP-seq71, a snakemake72 based ChIP-seq pipeline. Briefly, raw reads
were mapped by bowtie173 to hg19. Duplicated reads were removed, and only
uniquely mapped reads were retained. RPKM normalized bigwigs were generated
by deep tools74, and tracks were visualized with IGV75. Peaks were called using
macs1.476 with a p value of 1e–9 for H3K27ac and 1e–7 for AR. Heatmaps were
generated using R package EnrichedHeatmap. ChIP-seq peaks were annotated with
the nearest genes using ChIPseeker77. SEs were identified using ROSE78 based on
H3K27ac ChIP-seq data. 5. Lettieri, C. K., Garcia-Filion, P. & Hingorani, P. Incidence and outcomes of
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in specific DSRCT samples, enhancer peaks that overlap with 2.5 kb upstream and
2.5 kb downstream of any known TSSs were removed. The unique and shared peaks
within multiple groups were identified by Intervene79. The peaks were annotated with
ChIPseeker R package77, using addFlankGeneInfo function for enhancers. 9. Sawyer, J. R., Tryka, A. F. & Lewis, J. M. A novel reciprocal chromosome
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round-cell tumor. Am. J. Surg. Pathol. Data availability The algorithm was built around a three-
step approach: (1) pre-processing: HDAB-DAB and HDAB-hematoxylin features
were used to detect AR-positive cells (green masking) and negative cells (blue
masking), respectively, by setting pixel values. A median filter of size 5 × 5 was used
for the proper segmentation of cells. (2) Post processing: additional steps were
designed to enhance the performance of the APP. Change by shape excludes the
artifacts; merged cells were separated by separate labels step, and certain clear areas
in the cells were filled with fill holes step to fully mask the cells. (3) Output variable:
mean intensity of each cell and from each sample was extracted and exported into a
spreadsheet. Finally, the data were plotted on a Scatter Plot using GraphPad Prism
software, version 8 showing the AR Mean Intensity of each cell from all the
analyzed samples, and the AR Average mean intensity from each preclinical group
of treated mice. Code availability
Th
d
d The code used to generate the ChIP-seq data are available at https://github.com/
crazyhottommy/pyflow-ChIPseq. Received: 9 October 2020; Accepted: 13 May 2022; ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z or more regions of interest were randomly assessed for each microscopy image, and
representative images are shown. TMA preparation and immunohistochemistry analyses. Four‐micrometer
unstained slides were prepared from this TMA. Immunohistochemical studies were
performed using an autostainer (Bond‐Max; Leica Microsystems, Buffalo Grove, IL,
USA) with anti‐AR (1:30; clone AR441, Dako#M3562) antibody. This primary
antibody was detected using a Bond polymer refine detection kit according to the
manufacturer protocol (Leica, #DS9800). Then, the slides were dehydrated in grade
alcohols, cleared in xylene, and cover slipped. The TMA staining results were scored
on intensity (low, moderate, and high) as well as the percentage of tumoral labeling
(positive (>50%), low positive (10–50%), focal (1–10%), and negative (0–1%)). or more regions of interest were randomly assessed for each microscopy image, and
representative images are shown. Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z Male NOD (SCID)-IL-2Rgnull
mice 6 weeks in age (The Jackson Laboratory; Farmington, CT) were sub-
cutaneously injected with JN-DSRCT cells (5 × 106 cells/animal) or received PDX
explants (2 mm) to generate DSRCT xenograft and PDX mouse models. The
histologic and genetic analyses of DSRCT patients and PDX tumors are available in
Supplementary Fig. S12. All mice were maintained under barrier conditions and
treated using protocols approved by The University of Texas MDACC’s Institu-
tional Animal Care and Use Committee. Once their tumors reached a volume of
150 mm3, 5 mice per group received enzalutamide (25 mg/kg IP daily, 5 times per
week), or AR-ASOs (25 or 50 mg/kg subcutaneously daily, 5 times per week), or
control ASOs (50 mg/kg subcutaneously daily, 5 times per week), or a placebo 13 TURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications ARTICLE g
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the National Institutes of Health through Cancer Center Support Grant CA016672. The
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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
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Lamhamedi-Cherradi, Kunal Rai or Joseph A. Ludwig. 83. Subudhi, S. K. et al. Neoantigen responses, immune correlates, and favorable
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(2020). Peer review information Nature Communications thanks the anonymous reviewer(s) for
their contribution to the peer review of this work. Peer reviewer reports are available. Reprints and permission information is available at http://www.nature.com/reprints © The Author(s) 2022 Author contributions S.-E.L.-C., M.M., B.A.M., S.K., A.R.V., P.C., R.W.P., V.R., S.M., D.D.T., A.R.V., M.T.,
J.A.Lu., W.-L.W., and C.R.-.A. designed experiments, performed experiments, and ana-
lyzed data. D.R.I. helped identify and collect clinical samples for analysis. S.-E.L.-C.,
J.A.Lu., A.H.-J., and K.R. analyzed data and wrote the manuscript. P.A.F., M.T., A.R.M.,
N.C.D., R.R., J.A.Li., and A.H.-J. provided key scientific input and expertise. S.-E.L.-C., J.A.Lu., W. L.W., and C.R. .A. designed experiments, performed experiments, and ana
lyzed data. D.R.I. helped identify and collect clinical samples for analysis. S.-E.L.-C.,
J.A.Lu., A.H.-J., and K.R. analyzed data and wrote the manuscript. P.A.F., M.T., A.R.M.,
N.C.D., R.R., J.A.Li., and A.H.-J. provided key scientific input and expertise. S.-E.L.-C., © The Author(s) 2022 NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications 16 NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunicati
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Roles of XRCC2, RAD51B and RAD51D in RAD51-Independent SSA Recombination
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PLOS genetics
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cc-by
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To cite this version: Heïdi Serra, Olivier da Ines, Fabienne Degroote, Maria E. Gallego, Charles I. White. Roles of XRCC2,
RAD51B and RAD51D in RAD51-Independent SSA Recombination. PLoS Genetics, 2013, 9 (11),
pp.e1003971. 10.1371/journal.pgen.1003971. inserm-01907371 Introduction DNA double-strand breaks (DSB) are produced by ionizing
radiation, free radicals derived from metabolism, DNA cross-
linking reagents and during DNA replication [1,2]. DSB can lead
to mutations and rearrangements and/or loss of chromosomes,
causing tumorigenesis or cell death. DSB must be repaired to
maintain genome integrity, and this is carried out by end-joining
through non-homologous recombination or by homologous recom-
bination, which implicates DNA sequence homology of the
recombining molecules (for reviews, see [3,4]). The pathways that
utilize homology for repair can be distinguished by their dependence
or not on strand-invasion catalysed by the RAD51 recombinase (or
DMC1 in meiosis): gene conversion homologous recombination
(HR) is RAD51-dependent while single-strand annealing (SSA) is
RAD51-independent [3]. In addition to RAD51 and the meiosis-specific DMC1, a number
of RAD51 paralogue proteins have been described in a variety of
organisms. These share 20% to 30% homology with RAD51 and
presumably arose by gene duplication and evolved new functions
[5]. They clearly play key roles in DNA repair through HR, but
their exact functions are not fully understood (for reviews, see [6–8]). Two S. cerevisiae RAD51 paralogues, RAD55 and RAD57, form
a heterodimeric complex which associates with the RAD51 nucleo-
protein filament, stabilising it against disruption by the SRS2
antirecombinase [9]. Recent work has characterized novel yeast
RAD51 paralogues: Shu1, Shu2, Csm2 and Psy3, components of
the ‘‘suppresses sgs1 hydroxyurea sensitivity’’ (SHU or PCSS)
complex which also promotes RAD51 filament assembly and its
stability through counteracting the antirecombination activity of the
SRS2 and SGS1 helicases [10–17]. Fission yeast has homologues of
Shu1, Shu2 and Psy3 (Rlp1, Sws1 and Rdl1) and SWS1 and
SWSAP1 are members of a human SHU complex [11,12,18]. RAD51-dependent HR is an error-free DSB repair mechanism
involving the use of a homologous template for restoration of the
original sequence. It involves resection of the 59-ended DNA
strands at the DSB, generating 39 single-stranded DNA overhangs
that are bound by replication protein A (RPA). Assisted by
mediator proteins, RAD51 displaces RPA and forms a helical
nucleofilament on the exposed single-stranded DNA (ssDNA)
flanking the DSB. This nucleofilament performs the homology
search and catalyses invasion of the homologous template DNA,
following which the invading 39 ends are extended through DNA
synthesis. The joint recombination intermediate is resolved to Five RAD51 paralogues have been identified in animals and
plants: RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 (for
reviews, see [7,19,20]). Abstract The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living
organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and
the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination
pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence
or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present
here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the
RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-
induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence
of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the
individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple
knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2,
RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination. Citation: Serra H, Da Ines O, Degroote F, Gallego ME, White CI (2013) Roles of XRCC2, RAD51B and RAD51D in RAD51-Independent SSA Recombination. PLoS
Genet 9(11): e1003971. doi:10.1371/journal.pgen.1003971 Editor: Holger Puchta, Karlsruhe Institute of Technology, Germany ditor: Holger Puchta, Karlsruhe Institute of Technology, Germany Received June 26, 2013; Accepted October 7, 2013; Published November 21, 2013 Received June 26, 2013; Accepted October 7, 2013; Published November 21, 2013 Copyright: 2013 Serra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: HS was the recipient of a PhD fellowship financed by Institut National des Sciences Biologiques (INSB/CNRS) and the Conseil re´gional d’Auvergne
(PAVIRMA project). This work was financed by a European Union research grant (FP7-KBBE-2008-227190), the Centre National de la Recherche Scientifique, the
Universite´ Blaise Pascal, the Universite´ d’Auvergne, and the Institut National de la Sante´ et de la Recherche Me´dicale. 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. HAL Id: inserm-01907371
https://inserm.hal.science/inserm-01907371v1
Submitted on 29 Oct 2018 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. Abstract * E-mail: chwhite@univ-bpclermont.fr separate the recombining DNA molecules and thus restore
chromosome integrity (for a review, see [3]). Roles of XRCC2, RAD51B and RAD51D in RAD51-
Independent SSA Recombination Heı¨di Serra, Olivier Da Ines, Fabienne Degroote, Maria E. Gallego, Charles I. White*
Ge´ne´tique, Reproduction et De´veloppement, UMR CNRS 6293 - Clermont Universite´ - INSERM U1103, Aubie`re, France XRCC2 is required for SSA recombination Although XRCC2 is known to be involved in RAD51-
dependent homologous recombination in both vertebrates and in
plants [6,7,19,43,44], its potential role in RAD51-independent
SSA has not been tested. SSA recombination was monitored in xrcc2 mutant Arabidopsis
thaliana plants using the well-characterised DGU.US recombina-
tion reporter locus - consisting of an I-SceI restriction site flanked
by 39 and 59 truncated copies of the b-glucuronidase gene (GUS)
in direct orientation and with an overlap of 557 bp (Figure 1A;
[58]). Cleavage of the I-SceI site induces recombination between
the flanking GUS sequences and the resulting functional GUS
gene is scored histochemically as blue somatic spots. I-SceI
induced recombination at this tester locus has been shown not to
depend upon RAD51 [53]. rad51c and xrcc3 mutant plants are sterile due to recombination
defects [32,33]. We introduced the GUS recombination reporter locus into xrcc2
mutant
and
wild-type
(WT)
plants
through
crossing
and
transformed these DGU.US lines with an inducible I-SceI
expression cassette (Materials and Methods). Three independent
transformants (T2 lines) were selected for each genotype, each
with a single insertion site of the I-SceI cassette. Seeds of these
lines were plated onto medium containing hygromycin (in order to
select plants carrying the I-SceI cassette), in the presence or
absence of I-SceI expression inductor (b-estradiol), and numbers of
blue GUS+ spots counted after 14 days of growth (Figure 1). Induction of I-SceI expression by b-estradiol treatment in WT
plants
resulted
in
a
considerable
increase
of
numbers
of
recombinant blue spots/sectors (Figure 1B and C). In contrast,
expression of I-SceI had very little effect on numbers of blue spots
in xrcc2 mutant plants, with means of 5.9 spots per plant in the
presence of b-estradiol, and 4.1 in its absence. Repetition of these
analyses with two other independent I-SceI transformant lines
yielded similar results (Figure 1D). XRCC2 thus clearly plays an
important role in the SSA recombination pathway. Two-hybrid and co-immunoprecipitation studies have shown
that the five RAD51 paralogues form two major complexes:
RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) and RAD51C-
XRCC3 (CX3), as well as RAD51B-RAD51C (BC) and RAD51D-
XRCC2 (DX2) sub-complexes [5,7,8,34–40]. RAD51 paralogue
complexes act at both early and late stages of the recombinational
repair process, although their exact roles remain to be identified
[32,41–48]. XRCC2 is required for SSA recombination The early role of RAD51 paralogues in HR is to
promote formation and stabilization of RAD51 nucleoprotein
filament (reviewed by [6–8,19]), very probably through counter-
acting disruption of the filament by helicases [9–14]. Recent work
shows that the BCDX2 complex, and not the CX3 complex, is
responsible for RAD51 recruitment at DNA damage sites in human
cells [43]. After RAD51-mediated strand invasion, the RAD51
paralogues influence gene conversion tract length [42,47] and have
been linked to Holliday junction (HJ) resolvase activity [45,46]. In
addition, RAD51 paralogues can bind Y-shaped replication-like
intermediates and synthetic HJ, in accordance with a role for
RAD51 paralogues in repair during DNA replication and in
resolution of HR intermediary structures [49,50]. The histochemical GUS assay is an indirect measure of somatic
recombination and we thus carried out Southern analyses to
demonstrate directly that the decrease of number of GUS+ spots
in xrcc2 mutant plants is due to a failure of restoration of the GUS
gene. Southern analysis was carried out on SacI-digested genomic
DNA of WT and xrcc2 mutant plants (induced or not by b-
estradiol). In DGU.US lines, restoration of the GUS gene results in
deletion of the repeated sequence, including the inserted I-SceI
site. In DNA of WT plants, the reconstituted GUS gene is clearly
visible as a band at the expected size (2.5 kb) after induction of I-
SceI expression, but not in its absence (Figure 2, lanes 3 and 7). Treatment of the genomic DNA samples with I-SceI in vitro prior
to electrophoresis confirms that the 2.5 kb fragment has lost its I-
SceI site (Figure 2, lane 9), consistent with elimination of the I-SceI
restriction site during the restoration of the marker gene. No
restored GUS gene was detected in the xrcc2 mutant line (Figure 2,
lanes 8 and 10). This molecular analysis is thus fully consistent with
the results of the b-glucuronidase assay and confirms the
implication of the XRCC2 protein in the SSA recombination
pathway. The second main pathway using homology for repair, single-
strand annealing (SSA), promotes recombination between tan-
demly repeated DNA sequences flanking a DSB. SSA does not
involve
DNA-strand invasion and has been
shown to be
independent of RAD51 [51–54]. After bidirectional 59-39 resection
of the DSB ends, the exposed complementary sequences anneal. Subsequent removal of non-homologous 39-ended ssDNA tails,
filling-in of any single-strand gaps and ligation completes the
process. Author Summary The repair of DNA double-strand breaks by recombination
is key to the maintenance of genome integrity in all living
organisms. Recombination can however generate muta-
tions and chromosomal rearrangements, making the
regulation and the choice of specific pathways of great
importance. Through modulation of the activity of the
recombinase RAD51, the RAD51 paralogue proteins play
key roles in the regulation of recombination. Considerable
advances have been made in understanding of the RAD51
paralogue proteins and their roles in mediating RAD51-
mediated homologous recombination, however very little
is known of possible roles that they may have in other
recombination pathways. Working with the plant, Arabi-
dopsis thaliana, we show here major roles for three RAD51
paralogues in RAD51-independent single-strand annealing
recombination. Notwithstanding their clearly established
importance for RAD51-dependent homologous recombi-
nation, XRCC2, RAD51B and RAD51D thus also participate
in Single-Strand Annealing recombination. Introduction Animal cells defective in any of the RAD51
paralogues are hypersensitive to DNA cross-linking agents, such as
Cisplatin and Mitomycin C, and show spontaneous chromosomal
aberrations [21–27]. Mouse xrcc2, rad51b, rad51c and rad51d
mutants are embryonic lethal [28–31]. In contrast, all five RAD51
paralogues Arabidopsis mutants grow and develop normally and November 2013 | Volume 9 | Issue 11 | e1003971 1 PLOS Genetics | www.plosgenetics.org RAD51 Paralogues Act in SSA Recombination In this study, we describe an unexpected role in the SSA
pathway for Arabidopsis XRCC2, RAD51B and RAD51D,
highlighting for the first time a function of these three RAD51
paralogues in RAD51-independent SSA recombination. XRCC2 is required for SSA recombination The SSA recombination pathway thus leads to deletion of
the interstitial DNA sequence lying between the repeats and one of
the repeated homologous sequences (for reviews, see [3,55]). Little is known about possible involvement of the RAD51
paralogues in RAD51-independent SSA. Yeast Rad55 and Rad57
are not required for SSA in a plasmid assay [51] or spontaneous
direct repeat recombination [56,57] and a recent study has shown
that absence of Rad55, Csm2 or Psy3 result in increased SSA
recombination at a direct repeat chromosomal locus in yeast [15]. In Arabidopsis, RAD51, RAD51C and XRCC3 are not required
for SSA, although a mild reduction in the efficiency of SSA was
reported in the rad51c mutant [53]. We note also the presence of a 3.2 kb band in the SacI+I-SceI
digested DNA from WT plants (Figure 2, lane 9). That this I-SceI
resistant band is due to in planta rejoining of I-SceI breaks through November 2013 | Volume 9 | Issue 11 | e1003971 PLOS Genetics | www.plosgenetics.org 2 RAD51 Paralogues Act in SSA Recombination Figure 1. I-SceI induced DGU.US recombination depends upon XRCC2. (A) Schematic map of the recombination substrate DGU.US. (B) b-
glucuronidase assay of 14 day-old seedlings grown with or without induction of I-SceI by b-estradiol clearly shows reduced numbers of blue
recombinant GUS+ sectors in the xrcc2 mutant. (C) Quantification of recombination events confirms the role of XRCC2. Bars are mean values 6
standard errors. * Significant difference (p = 0.036, Mann-Whitney test). (D) Frequency distributions of recombinant spot numbers per plant of 3
independent WT and xrcc2 T2 lines grown in the presence of b-estradiol. doi:10.1371/journal.pgen.1003971.g001 Figure 1. I-SceI induced DGU.US recombination depends upon XRCC2. (A) Schematic map of the recombination substrate DGU.US. (B) b-
glucuronidase assay of 14 day-old seedlings grown with or without induction of I-SceI by b-estradiol clearly shows reduced numbers of blue
recombinant GUS+ sectors in the xrcc2 mutant. (C) Quantification of recombination events confirms the role of XRCC2. Bars are mean values 6
standard errors. * Significant difference (p = 0.036, Mann-Whitney test). (D) Frequency distributions of recombinant spot numbers per plant of 3
independent WT and xrcc2 T2 lines grown in the presence of b-estradiol. doi:10.1371/journal.pgen.1003971.g001 XRCC2 function in spontaneous DGU.US recombination
does not depend upon RAD51 activity XRCC2 function in spontaneous DGU.US recombination
does not depend upon RAD51 activity end-joining recombination was verified by PCR amplification and
DNA sequencing. XRCC2 is required for SSA recombination Approximately 10% of the sequences carried a
mutation at the I-SceI restriction site. DNA sequencing showed
that these result mostly from small deletions (Figure S1). As
previously described [59–63], these events can be ascribed to end-
joining exploiting the presence of microhomologies either side of
the I-SceI cleavage site. RAD51 Paralogues Act in SSA Recombination RAD51 Paralogues Act in SSA Recombination Figure 2. Molecular confirmation of recombination in WT, but
not xrcc2 mutant plants. Schematic representation of the GU.US
recombination tester locus (A) and Southern analysis (B) of DNA from
plants grown in the absence (lanes 1 to 6) or presence of b-estradiol
(lanes 7 to 10), digested with SacI (lanes 1,3,4,7,8) or SacI plus I-SceI
(lanes 2,5,6,9,10). The blot was hybridized with a GUS-specific probe as
indicated in panel (A). The recombined GUS gene has lost its I-SceI site
and is seen as a single 2.5 kb SacI fragment only in DNA from WT plants
grown in presence of b-estradiol (lanes 7 and 9). Col0: WT plants of
Columbia ecotype. doi:10.1371/journal.pgen.1003971.g002 break-induced replication [62,64,65]. We thus tested the RAD51-
dependence of
spontaneous recombination at
DGU.US
by
expressing the dominant-negative RAD51-GFP fusion protein
[66]. Plants were transformed with the RAD51-GFP fusion protein
construct and three T2 lines each with a single insertion (RAD51-
GFP plants) were selected and their rad51 mutant phenotype tested
by verification of sensitivity to the cross-linking agent, Mitomycin C
(MMC) (Figure S2). Wild-type plants are not sensitive to the MMC
dose used (2% sensitive plants), in contrast to the segregating
RAD51-GFP population, in which 76.9% are sensitive. PCR
genotyping confirmed that all of the MMC-sensitive and none of the
MMC-resistant T2 plants carry RAD51-GFP. Presence of RAD51-
GFP is thus perfectly correlated with MMC-sensitivity, confirming
the dominant-negative inhibition of RAD51 by the fusion protein
[66]. We then tested spontaneous DGU.US recombination in the
RAD51-GFP plants (Figure 4). No significant difference was
observed in numbers of GUS+ recombinant spots between control
and RAD51-GFP plants (Mann-Whitney test) clearly confirming
that spontaneous recombination of the DGU.US substrate does not
depend upon RAD51 activity. XRCC2, RAD51B and RAD51D have non-epistatic
functions in the SSA pathway XRCC2 is one of five RAD51 paralogue proteins, all of which
play important roles in recombination [7]. Given the function of
XRCC2 in the RAD51-independent SSA pathway presented
above, we also tested for evidence of roles of the other RAD51
paralogues, RAD51B and RAD51D, in this pathway. We thus
crossed the DGU.US recombination reporter locus into rad51b
and rad51d mutant plants and monitored spontaneous SSA
recombination at DGU.US in rad51b and rad51d mutants. Although less pronounced than the 15-fold reduction observed
in xrcc2 plants, numbers of spontaneous recombination events are
also reduced in rad51b and rad51d mutants (respectively 4.6-fold
and 3.4-fold; Figure 5; Table 2) clearly establishing roles for
RAD51B and RAD51D in the SSA pathway. Figure 2. Molecular confirmation of recombination in WT, but
not xrcc2 mutant plants. Schematic representation of the GU.US
recombination tester locus (A) and Southern analysis (B) of DNA from
plants grown in the absence (lanes 1 to 6) or presence of b-estradiol
(lanes 7 to 10), digested with SacI (lanes 1,3,4,7,8) or SacI plus I-SceI
(lanes 2,5,6,9,10). The blot was hybridized with a GUS-specific probe as
indicated in panel (A). The recombined GUS gene has lost its I-SceI site
and is seen as a single 2.5 kb SacI fragment only in DNA from WT plants
grown in presence of b-estradiol (lanes 7 and 9). Col0: WT plants of
Columbia ecotype. d i 10 1371/j
l
1003971 002 Epistasis relationships in SSA recombination between the three
RAD51 paralogue genes were tested in xrcc2 rad51b double and
xrcc2 rad51b rad51d triple mutants. Spontaneous SSA recombina-
tion was significantly less efficient in xrcc2 rad51b double mutants
that in the corresponding single mutants (p,0.02) (Figure 5). A
slight further reduction in numbers of blue spots per plant was
observed in the triple xrcc2 rad51b rad51d mutants with respect to
the double xrcc2 rad51b mutant, but the difference is not significant. yp
doi:10.1371/journal.pgen.1003971.g002 To confirm at the molecular level the results of the GUS assays,
we transformed rad51b, rad51d, xrcc2 rad51b and xrcc2 rad51b rad51d
mutant plants with the inducible I-SceI expression cassette. Southern analysis of recombination was carried out on b-estradiol
induced T2 plants. As expected, the 2.5 kb fragment of the
recombination product is only detected in the WT, confirming the
GUS assay data (Figure 6). XRCC2, RAD51B and RAD51D have non-epistatic
functions in the SSA pathway both), we monitored recombination in xrcc2 mutant and WT
plants with the same DGU.US locus (at the same location in
genome), but which do not carry I-SceI (Figure 3). This analysis
showed a reduction of number of recombinant spots in the absence
of the I-SceI cassette, for both WT and xrcc2 plants (from 13.24 to
5.46 and 4.10 to 0.36 spots per plant, respectively; Figures 1C and
3A), confirming the presence of some leakiness in expression of the
I-SceI inducible promoter in the absence of b-estradiol. In the
absence of the I-SceI cassette, mean numbers of blue spots per
plant were however still significantly (15-fold) reduced in xrcc2
mutants (0.36 ; s.e.m = 0.08) compared with WT controls (5.46 ;
s.e.m = 0.34 ; Figure 3). An independent repetition of this experi-
ment confirmed these results (Table 1). XRCC2 is thus clearly
involved in spontaneous recombination of the DGU.US substrate. Arabidopsis XRCC2, RAD51B and RAD51D thus play roles in
SSA recombination pathway and these roles are non-epistatic, at
least for XRCC2 and RAD51B. PLOS Genetics | www.plosgenetics.org XRCC2 function in spontaneous DGU.US recombination
does not depend upon RAD51 activity p
p
y
Although minor, the DGU.US recombination analyses shown
in Figure 1C also showed a difference in numbers of blue spots
between WT and xrcc2 plants in the absence of b-estradiol. To
check whether this is due to differences in spontaneous recombi-
nation rates or to leakiness of the inducible I-SceI cassette (or November 2013 | Volume 9 | Issue 11 | e1003971 PLOS Genetics | www.plosgenetics.org PLOS Genetics | www.plosgenetics.org 3 Discussion The roles of RAD51 paralogues in RAD51-dependent recom-
bination have been the subject of considerable interest in recent
years [6–8,19]. Little is known however of possible roles in
RAD51-independent SSA recombination. In Arabidopsis, no
effect was found on SSA in xrcc3 mutants and a barely statistically
significant reduction observed in rad51c plants [53]. We show here
the involvement of three RAD51 paralogues, XRCC2, RAD51B As mentioned above, I-SceI induced recombination at the
DGU.US locus has been shown to be RAD51-independent [53]. This has not however been confirmed for spontaneous recombi-
nation, for which different mechanisms can be envisaged - single-
strand annealing, intermolecular synthesis-dependent strand annealing, November 2013 | Volume 9 | Issue 11 | e1003971 PLOS Genetics | www.plosgenetics.org 4 RAD51 Paralogues Act in SSA Recombination Figure 3. Spontaneous DGU.US recombination is reduced in the xrcc2 mutant. A significant reduction in spontaneous recombination rate is
observed in xrcc2 mutant compared to WT plants. (A) Mean values 6 standard errors of the means. *** p,0.0001 (Mann-Whitney test). (B) Frequency
distributions. doi:10.1371/journal.pgen.1003971.g003 Figure 3. Spontaneous DGU.US recombination is reduced in the xrcc2 mutant. A significant reduction in spontaneous recombination rate is
observed in xrcc2 mutant compared to WT plants. (A) Mean values 6 standard errors of the means. *** p,0.0001 (Mann-Whitney test). (B) Frequency
distributions. doi:10.1371/journal.pgen.1003971.g003 against this interpretation. Data suggesting differing roles for
individual RAD51 paralogues, or sub-complexes, can be found in
a number of reports. Individual paralogue mutants in DT-40 cells
show non-epistatic phenotypes [67] and biochemical analyses
show specific roles for the sub-complexes [68–70]. In Arabidopsis,
absence of XRCC2 and RAD51B, but not RAD51D, increases
rates of meiotic crossing-over [44] and RAD51D appears to be the
only RAD51 paralogue to be essential for telomere integrity in
human cells [71]. A recent report shows opposing effects on cell-
cycle regulation of the inhibition of XRCC3 and RAD51C in
HeLa cells, with inhibition of XRCC3 eliciting checkpoint defects
and inhibition of RAD51C inducing G2/M cell cycle arrest [48]. and RAD51D, in RAD51-independent single-strand annealing in
Arabidopsis thaliana. XRCC2 plays a major role in this pathway with
a striking reduction of I-SceI induced recombination and a 15-fold
reduction in the number of spontaneous SSA events in its absence
(Figures 1 and 3). Spontaneous SSA is also clearly reduced in
rad51b and rad51d mutants (4.6-fold and 3.4-fold reduction
respectively; Figure 5; Table 2), although less strongly than in
xrcc2 mutants. Discussion The differing severity of the phenotypes of the three
mutants is suggestive of individual roles for these proteins, and this
is supported by epistasis analyses of double and triple mutant
plants (Figure 5, Table 2). An alternative to a direct role of these
proteins is that the presence of non-functional RAD51 nucleofila-
ments in these mutants which might block SSA. The lack of effect
on SSA of RAD51-GFP (which forms foci at DSBs and is
dominant-negative for GC/SDSA recombination) however argues g
y
[
]
What can the roles of XRCC2, RAD51B and RAD51D be in
the SSA pathway? The main steps of SSA are (1) bidirectional 59
to 39 resection of the DSB ends flanking a DSB, (2) annealing of
exposed complementary sequences, (3) excision of non-homolo-
gous 39-ended overhangs, (4) DNA synthesis and (5) ligation which
restores two continuous strands [72,73]. A role in the annealing
step is suggested by the capacity of the human BCDX2 complex to
catalyse annealing between single-strand DNAs in vitro [50]. This
study also showed a high affinity of the BCDX2 complex for
branched DNA structures, such as Y-shaped DNA, that result
from this annealing between tandem repeats during single-strand
annealing. Taken together, these results strongly suggest a role of
XRCC2, RAD51B and RAD51D in the annealing of the two
exposed repeat sequences on either side of the DSB. Table 1. Spontaneous DGU.US recombination in xrcc2
mutant and in wild-type plants. Experiment
n
N
m ± SEM
Ratio xrcc2/WT
1
WT
50
273
5.4660.34
xrcc2
50
18
0.3660.08
0.066
2
WT
50
310
6.2060.51
xrcc2
50
19
0.3860.09
0.061
Recombination in the mutants and WT were compared using non-parametric
statistical analysis (Mann–Whitney test). Differences between xrcc2 and WT are
highly significant (p,0.0001) in both cases. n, the number of plantlets screened;
N, the total number of blue spots (recombination events) ; m 6 SEM, the mean
number of recombination events per plant 6 standard error of the mean. doi:10.1371/journal.pgen.1003971.t001 Table 1. Spontaneous DGU.US recombination in xrcc2
mutant and in wild-type plants. Biochemical studies have identified two main complexes of the
five RAD51 paralogue proteins in animal and plant cells:
RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3
[5,7,8,34–40]. No self-assembly of individual RAD51 paralogues
have been detected. Discussion Analysis of epistasis relationships of RAD51
paralogues in chicken DT-40 cells show that rad51b and rad51d are November 2013 | Volume 9 | Issue 11 | e1003971 PLOS Genetics | www.plosgenetics.org 5 RAD51 Paralogues Act in SSA Recombination Figure 4. Spontaneous DGU.US recombination is RAD51-
independent. No significant effect on spontaneous recombination
rate was observed in three independent transformants carrying the
dominant-negative RAD51-GFP construct. Bars are mean values 6
standard errors. doi:10.1371/journal.pgen.1003971.g004
Figure 5. Individual and combined effects of xrcc2, rad51b and
rad51d on spontaneous DGU.US recombination. Significant
reductions in spontaneous recombination rate are observed in xrcc2,
rad51b and rad51d mutants, and the severities of the reductions differ
between these single mutants. A further significant reduction is seen in
xrcc2 rad51b mutant plants. The triple xrcc2 rad51b rad51d mutant
shows a further reduction, but this does not differ significantly from
that observed in the xrcc2 rad51b plants. Bars are mean values 6
standard errors. * 0.05,p,0.0001; *** p,0.0001 (Mann-Whitney test). doi:10.1371/journal.pgen.1003971.g005 Figure 4. Spontaneous DGU.US recombination is RAD51-
independent. No significant effect on spontaneous recombination
rate was observed in three independent transformants carrying the
dominant-negative RAD51-GFP construct. Bars are mean values 6
standard errors. doi:10.1371/journal.pgen.1003971.g004 Figure 5. Individual and combined effects of xrcc2, rad51b and
rad51d on spontaneous DGU.US recombination. Significant
reductions in spontaneous recombination rate are observed in xrcc2,
rad51b and rad51d mutants, and the severities of the reductions differ
between these single mutants. A further significant reduction is seen in
xrcc2 rad51b mutant plants. The triple xrcc2 rad51b rad51d mutant
shows a further reduction, but this does not differ significantly from
that observed in the xrcc2 rad51b plants. Bars are mean values 6
standard errors. * 0.05,p,0.0001; *** p,0.0001 (Mann-Whitney test). doi:10.1371/journal.pgen.1003971.g005 Figure 5. Individual and combined effects of xrcc2, rad51b and
rad51d on spontaneous DGU.US recombination. Significant
reductions in spontaneous recombination rate are observed in xrcc2,
rad51b and rad51d mutants, and the severities of the reductions differ
between these single mutants. A further significant reduction is seen in
xrcc2 rad51b mutant plants. The triple xrcc2 rad51b rad51d mutant
shows a further reduction, but this does not differ significantly from
that observed in the xrcc2 rad51b plants. Bars are mean values 6
standard errors. * 0.05,p,0.0001; *** p,0.0001 (Mann-Whitney test). doi:10.1371/journal.pgen.1003971.g005 Figure 4. Spontaneous DGU.US recombination is RAD51-
independent. No significant effect on spontaneous recombination
rate was observed in three independent transformants carrying the
dominant-negative RAD51-GFP construct. Discussion Bars are mean values 6
standard errors. doi:10.1371/journal.pgen.1003971.g004 doi:10.1371/journal.pgen.1003971.g004 reviews, [7,8]). Our data thus favour individual roles of XRCC2,
RAD51B and RAD51D in single-strand annealing recombination. reviews, [7,8]). Our data thus favour individual roles of XRCC2,
RAD51B and RAD51D in single-strand annealing recombination. epistatic while xrcc3 rad51d double mutant cells exhibit an additive
sensitivity to ionizing radiation [67], consistent with differential
actions of two major complexes in cellular response to DNA
damage. That the three RAD51 paralogues involved in SSA are
components of the BCDX2 complex suggests this complex is the
active species in SSA. However, the differing severity of the
phenotypes of the xrcc2, rad51b and rad51d (and rad51c; [53])
mutants argues against the implication of the BCDX2 complex as
such. The proposed structure of the complex also argues against
being the active form in SSA, with protein-protein interaction
studies showing that the four proteins are linked in the order:
RAD51B-RAD51C-RAD51D-XRCC2 [35]. Absence of RAD51D
should thus exclude XRCC2 from the complex, yet SSA in the xrcc2
mutants is significantly more affected than in rad51d (and similarly
for rad51b versus rad51c). This argument also applies to the
RAD51B-RAD51C and RAD51D-XRCC2 sub-complexes (for epistatic while xrcc3 rad51d double mutant cells exhibit an additive
sensitivity to ionizing radiation [67], consistent with differential
actions of two major complexes in cellular response to DNA
damage. That the three RAD51 paralogues involved in SSA are
components of the BCDX2 complex suggests this complex is the
active species in SSA. However, the differing severity of the
phenotypes of the xrcc2, rad51b and rad51d (and rad51c; [53])
mutants argues against the implication of the BCDX2 complex as
such. The proposed structure of the complex also argues against
being the active form in SSA, with protein-protein interaction
studies showing that the four proteins are linked in the order:
RAD51B-RAD51C-RAD51D-XRCC2 [35]. Absence of RAD51D
should thus exclude XRCC2 from the complex, yet SSA in the xrcc2
mutants is significantly more affected than in rad51d (and similarly
for rad51b versus rad51c). This argument also applies to the
RAD51B-RAD51C and RAD51D-XRCC2 sub-complexes (for The yeast RAD51 paralogues Rad55 and Rad57 are not
required in SSA recombination in a plasmid-based assay [51] and
a chromosomal assay shows that absence of Csm2, Psy3 (also
RAD51 paralogues) or Rad55 favours SSA with respect to gene
conversion recombination [15]. Discussion The description here of roles for
XRCC2, RAD51B and RAD51D in the RAD51-independent
SSA pathway thus highlights a difference in the roles of
Arabidopsis and yeast RAD51 paralogues in the SSA pathway. Such a difference is also seen in the roles of RAD51 paralogues in
meiotic recombination with psy3 mutants exhibiting a strong hypo-
recombination in yeast [12], while absence of XRCC2 or
RAD51B increases meiotic crossing-over in Arabidopsis [44]. In conclusion, we describe here an unexpected role in
recombination for the Arabidopsis RAD51 paralogues XRCC2,
RAD51B and RAD51D. The roles of these proteins are seen in November 2013 | Volume 9 | Issue 11 | e1003971 PLOS Genetics | www.plosgenetics.org 6 RAD51 Paralogues Act in SSA Recombination Table 2. Spontaneous DGU.US recombination in wild-type,
rad51b, rad51d, double and triple mutants. Experiment
n
N
m ± SEM
Ratio
mutant/WT
1
WT
50
273
5.4660.34
rad51b
50
63
1.2660.15
0.231
rad51d
50
81
1.6260.21
0.297
xrcc2, rad51b
50
8
0.1660.07
0.023
xrcc2, rad51b,
rad51d
50
6
0.1260.05
0.022
2
WT
50
310
6.2060.51
rad51b
50
62
1.2460.18
0.200
rad51d
50
92
1.8460.23
0.297
xrcc2, rad51b
50
6
0.1260.05
0.019
xrcc2, rad51b,
rad51d
50
1
0.0260.02
0.003
Recombination in the mutants and WT was compared using non-parametric
statistical analysis (Mann–Whitney test). Differences between each mutant and
corresponding WT are highly significant (p,0.0001). n, the number of plantlets
screened; N, the total number of blue spots (recombination events); m 6 SEM,
the mean number of recombination events per plant 6 standard error of the
mean. doi:10.1371/journal.pgen.1003971.t002 Table 2. Spontaneous DGU.US recombination in wild-type,
rad51b, rad51d, double and triple mutants. spontaneous and in DSB-induced recombination at a tandem
direct repeat recombination tester locus, both of which are
unaffected by the absence of RAD51. Notwithstanding their
clearly established importance for RAD51-dependent homologous
recombination, these proteins thus also participate in RAD51-
independent Single-Strand Annealing recombination. Plant material The Arabidopsis thaliana xrcc2, rad51b [74] and rad51d [44]
mutants used in this work have been previously described. A triple
xrcc2/xrcc2 rad51b/rad51b rad51d/rad51d mutant was crossed with
the recombination tester DGU.US-1 line [58] and single, double
and triple mutants homozygous for the DGU.US substrate were
identified in the F2. Wild-type control plants come from the same
crosses. The I-SceI coding sequence [75] was placed under control of b-
estradiol in the plasmid pMDC7 [76] by Gateway cloning. The
resulting vector was transferred into Agrobacterium tumefaciens, and
used to transform the plant lines utilising the floral dip method [77]. Recombination in the mutants and WT was compared using non-parametric
statistical analysis (Mann–Whitney test). Differences between each mutant and
corresponding WT are highly significant (p,0.0001). n, the number of plantlets
screened; N, the total number of blue spots (recombination events); m 6 SEM,
the mean number of recombination events per plant 6 standard error of the
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287: 20231–20239. 6. Krejci L, Altmannova V, Spirek M, Zhao X (2012) Homologous recombination
and its regulation. Nucleic acids research 40: 5795–5818. 7. Suwaki N, Klare K, Tarsounas M (2011) RAD51 paralogs: roles in DNA
damage signalling, recombinational repair and tumorigenesis. Seminars in cell &
developmental biology 22: 898–905. 18. Acknowledgments We thank Holger Puchta for providing the I-SceI plasmid and DGU.US
recombination tester. Members of the recombination group are thanked
for their help and discussions. Plant DNA extraction and Southern analysis DNA was prepared from seedlings as described previously [78]
and 1.5 mg digested with 100 units of SacI or 25 units of I-SceI in a
final volume of 200 ml for 15 h. Digested DNA samples were
isopropanol precipitated, resuspended in TE, and electrophoresed
in 0.8% agarose-TBE gel. Gel was blotted into a positively charged
nylon membrane (Hybond-XL, Amersham Biosciences), which
was hybridized in 0.5 M phosphate buffer, 7% w/v SDS, 1 mM
EDTA (pH 8) and 1% BSA at 65uC. The DNA probes to the
GUS gene (a PCR fragment amplified with 59-TGGATCCC-
CGGGATCATCTACTTCTG and 59-AGCCATGCACACTG-
ATACTCTTCACTCC) and the actin gene (a PCR fragment
amplified with 59-GGCTCCTCTTAACCCAAAGG and 59-TT-
ACCTGCTGGAATGTGCTG) were labelled with [a-32P]dCTP Growth conditions Surface-sterilized seeds were stratified at 4uC for 2 days and
grown in vitro on germination medium (0.8% w/v agar, 1% w/v
sucrose and half-strength Murashige & Skoog salts (M0255; Duchefa doi:10.1371/journal.pgen.1003971.t002 Figure 6. Molecular confirmation of recombination defects in rad51b, rad51d, xrcc2 rad51b and xrcc2 rad51b rad51d mutants. (A)
Southern analysis of DNA from rad51b and rad51d mutant plants grown in the presence of b-estradiol, digested with SacI (lanes 1,3,5,7) or SacI plus I-
SceI (lanes 2,4,6,8). (B) Southern analysis of DNA from xrcc2 rad51b and xrcc2 rad51b rad51d mutant plants grown in the presence of b-estradiol,
digested with SacI. The blots were hybridized with a GUS-specific probe. The recombined GUS gene has lost its I-SceI site and is seen as a 2.5 kb band
only in DNA from WT plants grown in presence of b-estradiol (A, lanes 3 and 4; B, lane 2). Col0: WT plants of Columbia ecotype. doi:10.1371/journal.pgen.1003971.g006 Figure 6. Molecular confirmation of recombination defects in rad51b, rad51d, xrcc2 rad51b and xrcc2 rad51b rad51d mutants. (A)
Southern analysis of DNA from rad51b and rad51d mutant plants grown in the presence of b-estradiol, digested with SacI (lanes 1,3,5,7) or SacI plus I-
SceI (lanes 2,4,6,8). (B) Southern analysis of DNA from xrcc2 rad51b and xrcc2 rad51b rad51d mutant plants grown in the presence of b-estradiol,
digested with SacI. The blots were hybridized with a GUS-specific probe. The recombined GUS gene has lost its I-SceI site and is seen as a 2.5 kb band
only in DNA from WT plants grown in presence of b-estradiol (A, lanes 3 and 4; B, lane 2). Col0: WT plants of Columbia ecotype. doi:10.1371/journal.pgen.1003971.g006 November 2013 | Volume 9 | Issue 11 | e1003971 PLOS Genetics | www.plosgenetics.org 7 RAD51 Paralogues Act in SSA Recombination Biochemie, Netherlands)) in a growth cabinet with a 16-h light/8-h
dark cycle, at 23uC with 45–60% relative humidity. using a random priming labelling kit (Megaprime DNA labelling
system, Amersham) according to the manufacturer’s instructions. Blots were washed with 0.5% SSC, 0.1% SDS solution at 65uC
and imaged with a PhosphoImager (Bio-Rad Personal FX). y
y
The growth medium was supplemented with 170 mM 17-b-
estradiol (E2758; Sigma-Aldrich) for induction of I-SceI expression. Mitomycin C treatment Figure S2
Sensitivity to Mitomycin C in T2 Rad51-GFP plants. WT and three independent Rad51-GFP T2 transformants were
tested for their sensitivity to the cross-linking agent MMC. The
dominant-negative effect of the RAD51-GFP allele is clearly
visible in the 3:1 segregating MMC hypersensitivity of the
plantlets. (A) photos of the plantlets and (B) quantitation of
sensitive versus resistant plants. (PDF) Figure S2
Sensitivity to Mitomycin C in T2 Rad51-GFP plants. WT and three independent Rad51-GFP T2 transformants were
tested for their sensitivity to the cross-linking agent MMC. The
dominant-negative effect of the RAD51-GFP allele is clearly
visible in the 3:1 segregating MMC hypersensitivity of the
plantlets. (A) photos of the plantlets and (B) quantitation of
sensitive versus resistant plants. Seeds were sown on plates containing fresh solid germination
medium supplemented with 40 mM Mitomycin C (M0503; Sigma-
Aldrich). The plates were then incubated for 20 days (23uC, 16-h
light). A plant with 3 or more true leaves is considered resistant. Author Contributions Conceived and designed the experiments: HS MEG CIW. Performed the
experiments: HS ODI FD MEG. Analyzed the data: HS ODI FD MEG
CIW. Contributed reagents/materials/analysis tools: MEG CIW. Wrote
the paper: HS CIW. Conceived and designed the experiments: HS MEG CIW. Performed the
experiments: HS ODI FD MEG. Analyzed the data: HS ODI FD MEG
CIW. Contributed reagents/materials/analysis tools: MEG CIW. Wrote
the paper: HS CIW. Supporting Information Fourteen-day old seedlings grown under standard conditions
(supplemented or not with 17-b-estradiol) were harvested and
incubated in staining buffer (0.2% Triton X-100, 50 mM sodium
phosphate buffer (pH 7.2), 2 mM X-Gluc (5-bromo-4-chloro-3-
indolyl-b-D-glucuronic acid; Biosynth), dissolved in N,N-dimethyl-
formamide). Plants were infiltrated under vacuum for 15 min and
incubated at 37uC overnight. The staining solution was then
replaced with 70% ethanol to remove leaf pigments, and the blue
spots were counted under a binocular microscope. Figure S1
I-SceI induced mutations in end-joining products of
DGU.US repair. The unmodified sequence surrounding the I-SceI
cut site of the DGU.US recombination tester locus is shown at the
top of the alignment, with the I-SceI restriction site boxed and the
cut-sites for each strand arrowed. Mutations are highlighted by
gray boxes and the size of deletions (bp) is indicated at right. Flanking microhomologies presumably involved in the end-joining
of I-SceI induced DSB are underlined. (PDF) RAD51 Paralogues Act in SSA Recombination Sigurdsson S, Van Komen S, Bussen W, Schild D, Albala JS, et al. (2001)
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Rad51 paralog Rad51B promotes homologous recombinational repair. Molecular and cellular biology 20: 6476–6482. PLOS Genetics | www.plosgenetics.org November 2013 | Volume 9 | Issue 11 | e1003971 November 2013 | Volume 9 | Issue 11 | e1003971 8 RAD51 Paralogues Act in SSA Recombination RAD51 Paralogues Act in SSA Recombination Yokoyama H, Sarai N, Kagawa W, Enomoto R, Shibata T, et al. (2004)
Preferential binding to branched DNA strands and strand-annealing activity of
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acids research 32: 2556–2565. 77. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-
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requirements for the single-strand annealing pathway of double-strand break
repair in Saccharomyces cerevisiae. Genetics 142: 693–704. November 2013 | Volume 9 | Issue 11 | e1003971 PLOS Genetics | www.plosgenetics.org 9
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THE DEVELOPMENT OF COVID-19 USING OUTBREAK THE SUSCEPTIBLE, INFECTED, AND RECOVERED (SIR) MODEL WITH VACCINATION
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| ERROR: type should be string, got "https://doi.org/10.30598/barekengvol17iss3pp1325-1340 \nSeptember 2023 Volume 17 Issue 3 Page 1325–1340 \nP-ISSN: 1978-7227 \n E-ISSN: 2615-3017 \n \nBAREKENG: Journal of Mathematics and Its Applications https://doi.org/10.30598/barekengvol17iss3pp1325-1340 \nSeptember 2023 Volume 17 Issue 3 Page 1325–1340 \nP-ISSN: 1978-7227 \n E-ISSN: 2615-3017 \n \nBAREKENG: Journal of Mathematics and Its Applications September 2023 Volume 17 Issue 3 Page 1325–1340 \nP-ISSN: 1978-7227 \n E-ISSN: 2615-3017 \nBAREKENG: Journal of Mathematics and Its Applications September 2023 Volume 17 Issue 3 Page 1325–1340 \nP-ISSN: 1978-7227 \n E-ISSN: 2615-3017 \nBAREKENG: Journal of Mathematics and Its Applications THE DEVELOPMENT OF COVID-19 USING OUTBREAK THE \nSUSCEPTIBLE, INFECTED AND RECOVERED (SIR) MODEL WITH \nVACCINATION Dorrah Azis 1*, La Zakaria2, Tiryono Ruby3, Muhammad Is’ad Arifaldi4 Dorrah Azis 1*, La Zakaria2, Tiryono Ruby3, Muhammad Is’ad Arifaldi4 1,2,3,4Mathematics Department, Faculty of Mathematic and Natural Sciences, University of Lampung \nSumantri Brojonegoro Street, Bandar Lampung, 35141, Indonesia \nCorresponding author’s e mail: * dorrah azis@fmipa unila ac id 4Mathematics Department, Faculty of Mathematic and Natural Sciences, University of Lampung \nSumantri Brojonegoro Street, Bandar Lampung, 35141, Indonesia , , , Mathematics Department, Faculty of Mathematic and Natural Sciences, University of Lampun\nSumantri Brojonegoro Street, Bandar Lampung, 35141, Indonesia \nCorresponding author’s e-mail: * dorrah.azis@fmipa.unila.ac.id Corresponding author’s e-mail: * dorrah.azis@fmipa.unila.ac.id ABSTRACT The COVID-19 pandemic in 2020 has caused severe problems in Indonesia. The COVID-19 \nvirus epidemic can be modeled using the Susceptible, Infected, and Recovered (SIR) model. This \nmodeling aims to look at the dynamics of COVID-19 to predict when disease-free and endemic \ndisease occurs and to find the basic reproduction number (𝑅0) for policy making in suppressing \nthe spread of COVID-19. In this article, we describe and solve a research result on the SIR \nmodel with an assumption. The assumption in the model is that there is vaccination for the \npopulation. There are live stages of research conducted. The first is creating the SIR model and \ndetermining the equilibrium points on disease-free and disease-endemic. The Second is getting \nthe basic reproduction number. The third is determining the stability around the equilibrium \npoints using the Routh-Hurwitz criteria. Fourth, create a diagram for the subpopulations state \nat a specific time using Wolfram Mathematica software. As an implementation of the model \ncreated, COVID-19 data at the Batanghari Community Health Center Inpatient UPTD was \nused. Finally, determine the model error percentage with MAPE. The SIR COVID-19 model \nwas made using eight parameters, namely 𝑁, 𝛼, 𝛽, 𝜏, µ, 𝜎, 𝛿, 𝛾, which are all positive. The results \nshowed that the disease-free and disease-endemic equilibrium points were locally \nasymptotically stable after being analyzed using the Routh-Hurwitz stability criteria. The model \ntrial using data from UPTD Puskesmas Batanghari obtained a stable condition for up to 100 \nmonths with a MAPE of 2.8%. From this study, obtained an 𝑅0 =\n𝛽𝜎\n𝛼+µ. This means that if you \nwant to reduce the rate of spread, then reduce the number of people who are easily infected (𝜎) \nand reduce contacts (𝛽), and increase the healing rate (𝛼). Article History: \nReceived: 06th January 2023 \nRevised: 9th July 2023 \nAccepted: 19th July 2023 Article History: \nReceived: 06th January 2023 \nRevised: 9th July 2023 \nAccepted: 19th July 2023 y\nSIR model; \nCOVID-19; \nBasic reproduction \nnumber; \nRouth-Hurwitz Criterion This article is an open access article distributed under the terms and conditions of the \nCreative Commons Attribution-ShareAlike 4.0 International License. How to cite this article: \nD. Azis, L. Zakaria, T. Ruby and M. I. Arifaldi, “THE DEVELOPMENT OF COVID-19 USING OUTBREAK THE SUSCEPTIBLE, INFECTED, \nAND RECOVERED (SIR) MODEL WITH VACCINATION,” BAREKENG: J. Math. &App., vol. 17, iss. 3, pp. 1325-1340, September, 2023. 1. INTRODUCTION For years, vaccines have been \nproven to reduce the incidence of infectious diseases through the mechanism of the human body's immunity \n[15]. The COVID-19 vaccine was developed to help the formation of individual body immunity so that the \nadministration of the COVID-19 vaccine is expected to accelerate the formation of group immunity (herd \nimmunity), which will have an impact on reducing the number of infected cases [16]. The vaccination policy \nhas an impact on reducing the number of COVID-19 cases that are still not under control in Indonesia [17]. This is reinforced by [18] which concluded that the severity of disease in individuals who had received the \nvaccine decreased, so it can be supposed that the vaccine effectively protects individuals from the SARS-\nCoV-2 variant. A disease can be modeled mathematically into an epidemiological model. One of the many types of \nmodeling available is the SIR model. The SIR model was first introduced in 1927 by Kermack and \nMcKendrick [19]. The SIR model determines the behavior of a pandemic and prediction [20]. This SIR \nmodel groups individuals in a population into three subpopulations, namely susceptible (groups of individuals \nwho are susceptible to being infected with a disease), infected (groups of individuals infected with disease), \nrecovered (groups of individuals who have recovered from the disease). SIR modeling is a model that is \nprepared with assumptions about a disease starting from the stage before being infected with a disease, being \ninfected, and until the individual is cured. Mathematical models of infectious diseases based on the classical \nSIR model are widely used to study the spread of a disease. These models show exciting results, especially \nin the early period of the pandemic [1]–[4], [21]–[28].The results of this model can be used as an illustration \nof how to suppress the spread of disease by looking at the effect of vaccination, ideal number of individuals \nwho must be vaccinated, and this model can predict within a certain time, the disease will become endemic. This requires field data to analyze the dynamics of the development of a disease. This modeling aims to \ndetermine the basic reproduction number (𝑅0), which plays a role in decision or policy-making for the \nauthorities in dealing with problems caused by the COVID-19 virus. 1. INTRODUCTION Mathematical models have played an essential role in explaining the dynamics of the disease. A \nmathematical model categorizes individuals into Susceptible, Infectious, and Recovered. In several very \nrecent research applied to the COVID-19 epidemic, researchers have developed and used SIR and SEIR-\nbased models with vaccination to overcome the limitations of the conventional SIR model. Mathematical \nmodeling specifically constructed to determine the development of disease outbreaks caused by COVID-19 \nhas not been widely carried out [1]–[9]. Handling the COVID-19 outbreak in Lampung Province involves various groups/communities of \nsociety, including academics. Even for Lampung province, dynamic models have not yet been found to solve \nthe problem of the COVID-19 outbreak in terms of the theory of the SIR model, which involves vaccination \nparameters. The research that has been carried out is to construct the SIR model to determine the development \nof the COVID-19 outbreak in Lampung Province, especially East Lampung Regency, by involving the \nparameters of vaccine administration to the community. Using data at the UPTD Puskesmas Batanghari \nDistrict, East Lampung Regency, the SIR model obtained was implemented to determine the trend of the \ngrowth of the COVID-19 outbreak in that place after the people were given the vaccine. In March 2020, WHO announced that the world was facing a pandemic called Corona Virus Infectious \nDisease 2019 or COVID-19 [10]. Until October 2020, the number of positive cases of. COVID-19 worldwide \nhas reached 37 million, with deaths reaching 1 million people [11]. The main medium of transmission of the \nSARS-Cov-2 virus is droplets that can be easily spread when humans interact directly with a certain distance. At the beginning of its spread, the average transmission power of the virus was still quite low, around 2.2 \n[12]. However, in its development, the SARS-Cov-2 virus underwent mutations so that several new virus \nvariants emerged with higher transmission capabilities, such as in England, South Africa, Brazil, and India \n[13]. The COVID-19 pandemic is developing so fast that many countries are not ready to adapt since the \nbeginning. WHO has advised focusing the handling of the pandemic on the health aspect by implementing \nregional isolation and banning activities that involve crowds. However, for some countries, this is not done \nbecause they doubt that the COVID-19 pandemic will last quite a long time [14]. One of the other efforts to \ndeal with the spread of COVID-19 is by implementing mass vaccination. How to cite this article: \nD. Azis, L. Zakaria, T. Ruby and M. I. Arifaldi, “THE DEVELOPMENT OF COVID-19 USING OUTBREAK THE SUSCEPTIBLE, INFECTED, \nAND RECOVERED (SIR) MODEL WITH VACCINATION,” BAREKENG: J. Math. &App., vol. 17, iss. 3, pp. 1325-1340, September, 2023. How to cite this article: \nD. Azis, L. Zakaria, T. Ruby and M. I. Arifaldi, “THE DEVELOPMENT OF COVID-19 USING OUTBREAK THE SUSCEPTIBLE, INFECTED, \nAND RECOVERED (SIR) MODEL WITH VACCINATION,” BAREKENG: J. Math. &App., vol. 17, iss. 3, pp. 1325-1340, September, 2023. Copyright © 2023 \nJournal homepage: https://ojs3.unpatti.ac.id/index.php/barekeng/ \nJournal e-mail: barekeng.math@yahoo.com; barekeng.journal@mail.unpatti.ac.id \nResearch Article • Open Access How to cite this article: \nD. Azis, L. Zakaria, T. Ruby and M. I. Arifaldi, “THE DEVELOPMENT OF COVID-19 USING OUTBREAK THE SUSCEPTIBLE, INFECTED, \nAND RECOVERED (SIR) MODEL WITH VACCINATION,” BAREKENG: J. Math. &App., vol. 17, iss. 3, pp. 1325-1340, September, 2023. 1325 326 \n \nAziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. 1326 1. INTRODUCTION In addition, this modeling is also to find \nout whether this disease can disappear or will remain (endemic) in an area and predict when this disease will \ndisappear or remain (endemic) in an area. BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 1327 2. RESEARCH METHODS This research is applied research conducted using secondary data obtained from the UPTD of the \nBatanghari District Inpatient Health Center related to cases of the spread of the COVID-19 disease that \noccurred throughout 2020-2021. The steps in this research are given in Figure 1: This research is applied research conducted using secondary data obtained from the UPTD of the \nBatanghari District Inpatient Health Center related to cases of the spread of the COVID-19 disease that \noccurred throughout 2020-2021. The steps in this research are given in Figure 1: Figure 1. 𝐒𝐭𝐞𝐩𝐬 of research diagram Figure 1. 𝐒𝐭𝐞𝐩𝐬 of research diagram 2.3 Next Generation Matrix Suppose there are 𝑛 infected classes and m uninfected classes. Furthermore, suppose that 𝑥 is an \ninfected sub-population and 𝑦 represents an uninfected (susceptible or cured) subpopulation, so that 𝑥̇ =\n𝜑𝑖(𝑥, 𝑦) −𝜔𝑖(𝑥, 𝑦), and 𝑦̇ = 𝑔𝑗(𝑥, 𝑦), where 𝑖= 1,2, … , 𝑛 and 𝑗= 1,2, … , 𝑚. 𝜑𝑖is the rate of secondary \ninfection in the infected class, and 𝜔𝑖 is the rate of disease progression, death, and recovery resulting in a \nreduced population of the infected class [32]. Next, Diekmann explain the next generation matrix 𝑲 is defined, which has the form 𝑲= 𝑭𝑽−1 \n(4) 𝑲= 𝑭𝑽−1 (4) where 𝑭 and 𝑽 are matrix of size 𝑛× 𝑛, which can also be written as follows: 𝑭= [\n𝜕𝜑𝑖\n𝜕𝑦𝑗] and 𝑽= [\n𝜕𝜔𝑖\n𝜕𝑦𝑗] \n(5) (5) (5) 2.1 SIR Model The SIR model divides the population into susceptible (S), infected (I), and recovered (R) \nsubpopulations. The number of susceptible, infected, and cured individuals at time t in a row can be written \nin the form of functions S(t), I(t), and R(t). The SIR model can be used to predict how a disease spreads, the total number of infected, the duration \nof the epidemic, and estimate various epidemiological parameters such as the number of reproductions. This \nmodel can determine how different public health interventions can affect epidemic outcomes [29]. Figure 2. 𝑺𝑰𝑹 model diagram \nS \n𝒓𝑺𝑰 \n𝜶𝑰 \nI \nR Figure 2. 𝑺𝑰𝑹 model diagram \nS \n𝒓𝑺𝑰 \n𝜶𝑰 \nI \nR Figure 2. 𝑺𝑰𝑹 model diagram 𝑆 = number of susceptible individuals in the population at the time \n𝐼 = number of infected individuals in the population at the time \n𝑅 = number of recovered individuals in the population at the time \n𝛼 = healing rate from infected to recovered \n𝑟 = rate of disease transmission from susceptible to infected \nThe SIR epidemic model is assumed as follows: 𝑆 = number of susceptible individuals in the population at the time 𝑟 = rate of disease transmission from susceptible to infected The SIR epidemic model is assumed as follows: 𝑑𝑆\n𝑑𝑡= −𝑟𝑆𝐼 \n(1) \n𝑑𝐼\n𝑑𝑡= 𝑟𝑆𝐼−𝛼𝐼 \n(2) \n𝑑𝑅\n𝑑𝑡= 𝛼𝐼 \n(3) 𝑑𝑆\n𝑑𝑡= −𝑟𝑆𝐼 \n(1) \n𝑑𝐼\n𝑑𝑡= 𝑟𝑆𝐼−𝛼𝐼 \n(2) \n𝑑𝑅\n𝑑𝑡= 𝛼𝐼 \n(3) 𝑑𝑆\n𝑑𝑡= −𝑟𝑆𝐼 \n𝑑𝐼\n𝑑𝑡= 𝑟𝑆𝐼−𝛼𝐼 \n𝑑𝑅\n𝑑𝑡= 𝛼𝐼 𝑑𝑆\n𝑑𝑡= −𝑟𝑆𝐼 \n𝑑𝐼\n𝑑𝑡= 𝑟𝑆𝐼−𝛼𝐼 \n𝑑𝑅\n𝑑𝑡= 𝛼𝐼 Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. 1328 Theorem Equilibrium Point [31]: 1. If all the real parts of the eigenvalues of the Jacobian matrix of a system of differential equations are \nnegative, then the equilibrium point of the system is stable. 1. If all the real parts of the eigenvalues of the Jacobian matrix of a system of differential equations are \nnegative, then the equilibrium point of the system is stable. 2. If one eigenvalue of the Jacobian matrix of a system of differential equations is positive, then the \nequilibrium point of the system is unstable. 2. If one eigenvalue of the Jacobian matrix of a system of differential equations is positive, then the \nequilibrium point of the system is unstable. 2.2 Equilibrium Point The equilibrium point is used to analyze the model. According to [19], the equilibrium point is the \nsolution of the system of differential equations which is independent of time. In [30], Meyer said the same \nthing too, the equilibrium condition is a condition where the system does not change over time. The equilibrium point is divided into 2, as follows: The equilibrium point is divided into 2, as follows: 1. The disease-free equilibrium point is the condition in which no individual is infected with the disease \ndiscussed in the population, so 𝐼 = 0. 1. The disease-free equilibrium point is the condition in which no individual is infected with the disease \ndiscussed in the population, so 𝐼 = 0. 2. The endemic equilibrium point is a condition where there are infected individuals in the population, so the \ncompartment at the endemic equilibrium point is 𝐼 ≠ 0. 2.4 Basic Reproduction Number (𝑹𝟎) The basic reproduction number 𝑅0 can be defined as the average number of infected individuals \ninfected by other infected individuals in a population. A basic reproductive number is a number that shows \nthe number of susceptible individuals who can suffer from diseases caused by one infected individual [33]. According [32], the basic reproduction number, which can be formulated as follows 𝑅0 = 𝜌(𝑲) = 𝜌(𝑭𝑽−𝟏) \n(6) (6) (6) The same thing regarding the basic reproduction number is denoted by 𝑅0 and is expressed by the \nfollowing equation [34]: 𝑅0 = 𝑓𝑎𝑐𝑡𝑜𝑟𝑠 𝑡ℎ𝑎𝑡 𝑐𝑎𝑢𝑠𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒\n𝑓𝑎𝑐𝑡𝑜𝑟𝑠 𝑡ℎ𝑎𝑡 𝑟𝑒𝑑𝑢𝑐𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒 \n(7) 𝑅0 = 𝑓𝑎𝑐𝑡𝑜𝑟𝑠 𝑡ℎ𝑎𝑡 𝑐𝑎𝑢𝑠𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒\n𝑓𝑎𝑐𝑡𝑜𝑟𝑠 𝑡ℎ𝑎𝑡 𝑟𝑒𝑑𝑢𝑐𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒 \n(7) (7) BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 1329 If 𝑅0 > 1, the each infected individual produces, on average, more than one new infection, and the disease \ncan invade the population. The disease will become an epidemic [35]. If 𝑅0 > 1, the each infected individual produces, on average, more than one new infection, and the disease \ncan invade the population. The disease will become an epidemic [35]. 2.5 Routh-Hurwitz Stability Criterion The necessary condition for the system to be said to be stable is if the coefficient of the characteristic \nequation is positive, while the sufficient condition is that each term of the first column of the matrix (6) is \npositive. 5. The necessary condition for the system to be said to be stable is if the coefficient of the characteristic \nequation is positive, while the sufficient condition is that each term of the first column of the matrix (6) is \npositive. Some of the conditions that will arise, namely: Some of the conditions that will arise, namely: the conditions that will arise, namely: If 𝑅0 < 1, then on average an infected individual produces \nless than one new infected individual over the course of its infectious period, and the infection \ncannot grow. The disease will disappear [35]. If 𝑅0 = 1, then the disease will persist [34]. BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 2.5 Routh-Hurwitz Stability Criterion The Routh-Hurwitz stability criterion is used to show a system's stability by taking into account the \ncoefficients of the characteristic equation without calculating the roots directly. If a polynomial equation is a \ncharacteristic equation, then this method can determine a system's stability. Thus, the procedures in the Routh-\nHurwitz criterion are [31]: 1. The 𝑛𝑡ℎ order polynomial equation is written in the form: 1. The 𝑛𝑡ℎ order polynomial equation is written in the form: 1. The 𝑛𝑡ℎ order polynomial equation is written in the form: det(𝜆𝐼−𝐴) = 𝑎𝑛𝜆𝑛+ 𝑎𝑛−1𝜆𝑛−1 + ⋯+ 𝑎1𝜆+ 𝑎0 \n(8) \n \nwhere the coefficients are real numbers and 𝑎𝑛≠0 det(𝜆𝐼−𝐴) = 𝑎𝑛𝜆𝑛+ 𝑎𝑛−1𝜆𝑛−1 + ⋯+ 𝑎1𝜆+ 𝑎0 \n(8) (8) where the coefficients are real numbers and 𝑎𝑛≠0. where the coefficients are real numbers and 𝑎𝑛≠0. 2. If there is a coefficient of 0 or negative, then there is one root or imaginary roots or has a positive real part \nwhich means the system is unstable. 2. If there is a coefficient of 0 or negative, then there is one root or imaginary roots or has a positive real part \nwhich means the system is unstable. 2. If there is a coefficient of 0 or negative, then there is one root or imaginary roots or has a positive real part \nwhich means the system is unstable. 3. If all coefficients are positive, then a matrix which is often called a Routh array, can be formed as follows |\n|\n𝜆𝑛\n𝑎𝑛\n𝑎𝑛−2\n𝑎𝑛−4\n⋯\n𝜆𝑛−1\n𝑎𝑛−1\n𝑎𝑛−3\n𝑎𝑛−5\n⋯\n𝜆𝑛−2\n𝑏1\n𝑏2\n𝑏3\n⋯\n⋮\n𝑐1\n𝑐2\n𝑐3\n⋯\n𝜆0\n⋮\n⋮\n⋮\n⋱\n|\n| \n(9) (9) The coefficients 𝑏1, 𝑏2, … , 𝑏𝑘 and 𝑐1, 𝑐2, … , 𝑐𝑘can be determined by the following formulas: 𝑏1 = −\n1\n𝑎𝑛−1 | 𝑎𝑛\n𝑎𝑛−2\n𝑎𝑛−1\n𝑎𝑛−3|, 𝑏2 = −\n1\n𝑎𝑛−3 |𝑎𝑛−2\n𝑎𝑛−4\n𝑎𝑛−3\n𝑎𝑛−5|,⋯ \n \n𝑐1 = −\n1\n𝑏1 |𝑎𝑛−1\n𝑎𝑛−3\n𝑏1\n𝑏2 |,𝑐2 = −\n1\n𝑏2 |𝑎𝑛−3\n𝑎𝑛−5\n𝑏2\n𝑏3 |,⋯ The scheme is continued until only zeroes appear (both to the right and down wards) [36]. The scheme is continued until only zeroes appear (both to the right and down wards) [36]. The scheme is continued until only zeroes appear (both to the right and down wards) [36]. 4. The number of unstable roots can be seen in the number of sign changes in the first matrix c 5. 2.6 Mean Absolute Percentage Error (MAPE) Mean Absolute Percentage Error is a method of finding the average absolute error value in the form of \na percentage in a comparison between actual data and existing projection or forecasting data. MAPE is \nformulated as follows: 𝑀𝐴𝑃𝐸= ∑|𝐴−𝑃|\n𝑛\n× 100% (10) 𝑀𝐴𝑃𝐸= ∑|𝐴−𝑃|\n𝑛\n× 100% (10) With With 𝐴: Actual data, 𝐴: Actual data, 𝑃: Forecasting data, 𝑃: Forecasting data, 𝑛: Total data. 𝑛: Total data. 𝑛: Total data. The percentage of MAPE is divided into four interpretation as follows [37]: Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. 1330 Table 1. MAPE Interpretation \nInterpretation of Typical MAPE Value’s \nMAPE \nInterpretation \n< 10 \nHighly Accurate Forecasting \n10 −20 \nGood Forecasting \n20 −50 \nReasonable Forecasting \n> 50 \nInaccurate Forecasting Table 1. MAPE Interpretation \nInterpretation of Typical MAPE Value’s \nMAPE \nInterpretation \n< 10 \nHighly Accurate Forecasting \n10 −20 \nGood Forecasting \n20 −50 \nReasonable Forecasting \n> 50 \nInaccurate Forecasting 3.1 SIR Model The assumptions in the SIR model for the COVID-19 disease are as follows: 1. Factors of birth and death are considered. Individuals born into the Susceptible (S) class because the \nindividual is assumed to be healthy but susceptible to COVID-19 disease. 1. Factors of birth and death are considered. Individuals born into the Susceptible (S) class because the \nindividual is assumed to be healthy but susceptible to COVID-19 disease. 2. The population birth and death rates in each compartment are assumed to be the same so that the total \npopulation is constant 2. The population birth and death rates in each compartment are assumed to be the same so that the total \npopulation is constant 3. Migration occurs in the population. No immigrants enter every S, I, and R class. 3. Migration occurs in the population. No immigrants enter every S, I, and R class. 4. COVID-19 disease can cause death (fatal). 5. Vaccinated individuals fall into class S. Vaccination efficacy is assumed as a percentage. WHO explained \nthat the performance of vaccines could be seen from three measurements, namely through the efficacy, \neffectiveness, and impact of the vaccine. One type of COVID-19 vaccine is Sinovac. The efficacy of the \nSinovac vaccine reaches 65.3% [38]. 5. Vaccinated individuals fall into class S. Vaccination efficacy is assumed as a percentage. WHO explained \nthat the performance of vaccines could be seen from three measurements, namely through the efficacy, \neffectiveness, and impact of the vaccine. One type of COVID-19 vaccine is Sinovac. The efficacy of the \nSinovac vaccine reaches 65.3% [38]. 6. COVID-19 disease can result in re-infection of individuals who have been infected before. However, some \nindividuals have recovered and formed antibodies to the COVID-19 virus. 6. COVID-19 disease can result in re-infection of individuals who have been infected before. However, some \nindividuals have recovered and formed antibodies to the COVID-19 virus. ased on the assumptions that have been made, the model parameters can be defined as follows: Figure 3. SIR model of COVID-19 disease with vaccination \n𝝉𝑰𝑹 \nS \n𝜷𝝈𝑰𝑺 \n𝜶𝑰 \nµ𝑰 \nµ𝑺 \nµ𝑹 \nµ𝑵 \nI \nR Figure 3. BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 1331 value is not far from 1 or very close. However, birth and death rates are equated with making this model easy \nto analyze. value is not far from 1 or very close. However, birth and death rates are equated with making this model easy \nto analyze. Then the mathematical model of the spread of the COVID-19 disease with vaccination was obtained \nin the form of a system of differential equations as follows: 𝑑𝑆\n𝑑𝑡= µ −𝛽𝜎𝐼𝑆−µ𝑆 \n(12) \n𝑑𝐼\n𝑑𝑡= 𝛽𝜎𝐼𝑆+ 𝜏𝐼𝑅−𝛼𝐼−µ𝐼 \n(13) \n𝑑𝑅\n𝑑𝑡= 𝛼𝐼−µ𝑅−𝜏𝐼𝑅 \n(14) 𝑑𝑆\n𝑑𝑡= µ −𝛽𝜎𝐼𝑆−µ𝑆 \n(12) \n𝑑𝐼\n𝑑𝑡= 𝛽𝜎𝐼𝑆+ 𝜏𝐼𝑅−𝛼𝐼−µ𝐼 \n(13) \n𝑑𝑅\n𝑑𝑡= 𝛼𝐼−µ𝑅−𝜏𝐼𝑅 \n(14) (12) (13) (14) where 𝑆(0) > 0, 𝐼(0) ≥0, 𝑅(0) > 0; \n \nµ, 𝛽, 𝜎, 𝛿, 𝛾, 𝜏, 𝛼> 0. where 𝑆(0) > 0, 𝐼(0) ≥0, 𝑅(0) > 0; \n \nµ, 𝛽, 𝜎, 𝛿, 𝛾, 𝜏, 𝛼> 0. where 𝑆(0) > 0, 𝐼(0) ≥0, 𝑅(0) > 0; \n \nµ, 𝛽, 𝜎, 𝛿, 𝛾, 𝜏, 𝛼> 0. 3.2.1 Disease-Free Equilibrium Point Disease-free population means that in the population, no one is sick, 𝐼= 0. This means that no \nindividual has been exposed to COVID-19 or no individual has been infected with COVID-19. Because no \nindividual has been exposed to COVID-19, the transmission rate (𝛽)and the infected group (𝐼)is worth 0. From the Equation (𝟏𝟐), substitute each equation containing the variables 𝛽and 𝐼 with a value of 0, it is \nobtained: quation (𝟏𝟐), substitute each equation containing the variables 𝛽and 𝐼 with a value of 0, it is µ −(0)𝜎(0)𝑆0 −µ𝑆0 = 0 \nµ −µ𝑆0 = 0 \n−µ𝑆0 = −µ \n𝑆0 = 1 (15) (15) Substituting the value 𝐼0 = 0 into Equation (14), obtained: 𝛼(0) −µ𝑅0 −𝜏(0)𝑅0 = 0 \n−µ𝑅0 = 0 \n𝑅0 = 0 𝛼(0) −µ𝑅0 −𝜏(0)𝑅0 = 0 \n−µ𝑅0 = 0 \n𝑅0 = 0 (16) (16) Then the disease-free equilibrium point is obtained as follows: Then the disease-free equilibrium point is obtained as follows: (𝑆0, 𝐼0, 𝑅0) = (1,0,0) \n(17) (17) 3.2 Equilibrium Point The equilibrium condition is a condition where the system does not change over time [30]. 3.2.1 Disease-Free Equilibrium Point The equilibrium condition is a condition where the system does not change over time [30]. The equilibrium condition is a condition where the system does not change over time [30]. 3.2.1 Disease-Free Equilibrium Point 3.1 SIR Model SIR model of COVID-19 disease with vaccination Information: 𝑁 : Total number of individuals in the population (%) µ : Expresses the birth rate in the S compartment and the death rate in each compartment (% 𝛽 : Expresses the infection rate in compartment S (%) 𝛽 : Expresses the infection rate in compartment S (%) \n𝛼 : Expresses the healing rate in compartment I (%) σ : Stating the total number of susceptible individuals (total individuals who are not vaccinated and already \n vaccinated but infected) (%) 𝜏: Expresses the rate of reinfection in compartment R (%) \nwith \n(\n) with \n𝜎= 1 −𝛿𝛾 (11) \n𝛿 : Vaccine efficiency (%) \n𝛾 : Number of individuals who have been vaccinated (%) 𝜎= 1 −𝛿𝛾 (11) \nbeen vaccinated (%) (11) 𝛾 : Number of individuals who have been vaccinated (%) 𝛾 : Number of individuals who have been vaccinated (%) Since the birth rate is considered equal to the number of deaths, the total population will be constant, \nso 𝑆+ 𝐼+ 𝑅= 1. If the number of births is not equal to the number of deaths, then N is not constant, but the BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 3.2.2. Disease Endemic Equilibrium Point Finally, \nwe have the COVID-19 endemic equilibrium point for is as follows: them into Equation (20), Equation (19) and Equation (3), the equilibrium point () will be obtained. Finally, \nwe have the COVID-19 endemic equilibrium point for is as follows: (𝑆, 𝐼, 𝑅) = (𝑆∗, 𝐼∗, 𝑅∗) \n(21) (21) 3.2.2. Disease Endemic Equilibrium Point Endemic disease means that in the population, there are always individuals who are infected with the \ndisease, meaning that there are individuals who are prone to be exposed to COVID-19, and there are \nindividuals who are infected with COVID-19. Because there are individuals who are exposed to COVID-19, \nthe transmission rate (𝛽) and the infected group (𝐼) are worth 𝛽> 0, 𝐼≠0. Let (𝑆∗, 𝐼∗, 𝑅∗) be an endemic \nequilibrium point. From the Equation (𝟏𝟐) for 𝜎> 0 and µ > 0, we can rewrite the equation as: 𝑆∗=\nµ\n𝐼∗ 𝛽𝜎+ µ \n(18) (18) Substituting Equation (18) into Equation (𝟏𝟑), and then solving it for 𝐼∗, we have: Substituting Equation (18) into Equation (𝟏𝟑), and then solving it for 𝐼∗, we have: 𝐼∗= 1\n2 ( −µ(𝛼−𝛽𝜎+ µ)\n𝛽𝜎(𝛼+ µ)\n+ √µ(4 𝑅∗ 𝛽𝜎𝜏(𝛼+ µ) + µ(𝛼−𝛽𝜎+ µ)2)\n𝛽2𝜎2(𝛼+ µ)2\n) \n(19) (19) Substituting Equation (19) into Equation (𝟏𝟒), and then solving it for 𝑅∗, we have: Substituting Equation (19) into Equation (𝟏𝟒), and then solving it for 𝑅∗, we have: 1332 \n \nAziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. 1332 \n \nAziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. 𝑅∗= 𝑎−𝑏 \n(20) \nWhere: \n𝑎=\n1\n2𝜏3 (𝛽𝜎𝜏µ + 𝛽𝜎µ2 −𝜏µ2 + 𝛼(2𝜏2 + 𝛽𝜎µ −𝜏µ) \n𝑏= 𝜏3√\n(𝛼2(2𝛽𝜎𝜏(2𝜏−µ)+𝛽2𝜎2µ+𝜏2µ)+µ(𝜏µ−𝛽𝜎(𝜏+µ))\n2+2𝛼µ(𝛽𝜎𝜏(𝜏−2µ)+𝜏2µ+𝛽2𝜎2(𝜏+µ)))\n(𝜏6µ) (20) (𝜏6µ) By selecting and setting parameter values that satisfy 𝜎> 0 µ > 0 ( (𝛼≥µ ((𝛽= 𝛼+µ\n𝜎 𝜏>\n0) (𝜏>\nµ(𝛼−𝛽𝜎+µ)\n𝛼\n 0 < 𝛽<\n𝛼+µ\n𝜎))) (0 < 𝛼< µ 0 < 𝛽≤\n𝛼+µ\n𝜎 𝜏>\nµ(𝛼−𝛽𝜎+µ)\n𝛼\n)) and substituting By selecting and setting parameter values that satisfy 𝜎> 0 µ > 0 ( (𝛼≥µ ((𝛽= 𝛼+µ\n𝜎 𝜏>\n0) (𝜏>\nµ(𝛼−𝛽𝜎+µ)\n𝛼\n 0 < 𝛽<\n𝛼+µ\n𝜎))) (0 < 𝛼< µ 0 < 𝛽≤\n𝛼+µ\n𝜎 𝜏>\nµ(𝛼−𝛽𝜎+µ)\n𝛼\n)) and substituting By selecting and setting parameter values that satisfy 𝜎> 0 µ > 0 ( (𝛼≥µ ((𝛽= 𝛼+µ\n𝜎 𝜏> 0) (𝜏>\nµ(𝛼−𝛽𝜎+µ)\n𝛼\n 0 < 𝛽<\n𝛼+µ\n𝜎))) (0 < 𝛼< µ 0 < 𝛽≤\n𝛼+µ\n𝜎 𝜏>\nµ(𝛼−𝛽𝜎+µ)\n𝛼\n)) an them into Equation (20), Equation (19) and Equation (3), the equilibrium point () will be obtained. 3.3 Basic Reproduction Number 1333 𝑅0 = 𝜌(\n𝛽𝜎\n(µ + 𝛼)\n0\n𝛼\n(µ + 𝛼)\n0\n) \n(26) (26) To get 𝑅0, the next step is to find the biggest eigenvalue of 𝐄𝐪𝐮𝐚𝐭𝐢𝐨𝐧 (𝟐𝟔) as follows: |𝑭∙𝑽−1 −𝜆𝑰| = ||(\n𝛽𝜎\n(µ + 𝛼)\n0\n𝛼\n(µ + 𝛼)\n0\n) −(𝜆\n0\n0\n𝜆)|| \n→|𝑭∙𝑽−1 −𝜆𝑰| = ||\n𝛽𝜎\n(µ + 𝛼) −𝜆\n0\n𝛼\n(µ + 𝛼)\n−𝜆\n|| \n→𝜆(𝜆−\n𝛽𝜎\n(µ + 𝛼)) = 0 \n𝜆1 = 0 and 𝜆2 =\n𝛽𝑂\nµ+𝛼 |𝑭∙𝑽−1 −𝜆𝑰| = ||(\n𝛽𝜎\n(µ + 𝛼)\n0\n𝛼\n(µ + 𝛼)\n0\n) −(𝜆\n0\n0\n𝜆)|| \n→|𝑭∙𝑽−1 −𝜆𝑰| = ||\n𝛽𝜎\n(µ + 𝛼) −𝜆\n0\n𝛼\n(µ + 𝛼)\n−𝜆\n|| \n→𝜆(𝜆−\n𝛽𝜎\n(µ + 𝛼)) = 0 \n𝜆1 = 0 and 𝜆2 =\n𝛽𝑂\nµ+𝛼 \n(27) (27) Because all variables are positive, so 𝜆2 > 𝜆1. Then obtained 𝑅0 as follows: Because all variables are positive, so 𝜆2 > 𝜆1. Then obtained 𝑅0 as follows: 𝑅0 =\n𝛽𝜎\nµ + 𝛼 , µ + 𝛼≠0 \n(28) 𝑅0 =\n𝛽𝜎\nµ + 𝛼 , µ + 𝛼≠0 (28) 3.3 Basic Reproduction Number The first step is to form a Jacobian matrix of the compartments containing infected individuals, namely \nI and R as follows: 𝑱(𝐼, 𝑅) = (\n𝑑(𝛽𝜎𝐼𝑆+ 𝜏𝐼𝑅−µ𝐼−𝛼𝐼)\n𝑑𝐼\n𝑑(𝛽𝜎𝐼𝑆+ 𝜏𝐼𝑅−µ𝐼−𝛼𝐼)\n𝑑𝑅\n𝑑(𝛼𝐼−𝜏𝐼𝑅−µ𝑅)\n𝑑𝐼\n𝑑(𝛼𝐼−𝜏𝐼𝑅−µ𝑅)\n𝑑𝑅\n) 𝑱(𝐼, 𝑅) = (𝛽𝜎𝑆+ 𝜏𝑅−µ −𝛼\n𝜏𝐼\n𝛼−𝜏𝑅\n−𝜏𝐼−µ) (22) (22) ext, substituting the disease-free equilibrium point (17) into the Jacobian matrix (22), we get: 𝑱(1,0,0) = (𝛽𝜎(1) + 𝜏(0) −µ −𝛼\n𝜏(0)\n𝛼−𝜏(0)\n−𝜏(0) −µ) \n𝑱(1,0,0) = (𝛽𝜎−µ −𝛼\n0\n𝛼\n−µ) \n(23) 𝑱(1,0,0) = (𝛽𝜎(1) + 𝜏(0) −µ −𝛼\n𝜏(0)\n𝛼−𝜏(0)\n−𝜏(0) −µ) \n𝑱(1,0,0) = (𝛽𝜎−µ −𝛼\n0\n𝛼\n−µ) \n(23) 𝑱(1,0,0) = (𝛽𝜎(1) + 𝜏(0) −µ −𝛼\n𝜏(0)\n𝛼−𝜏(0)\n−𝜏(0) −µ) \n𝑱(1,0,0) = (𝛽𝜎−µ −𝛼\n0\n𝛼\n−µ) 𝑱(1,0,0) = (𝛽𝜎−µ −𝛼\n0\n𝛼\n−µ) \n(23) (23) µ\nSince 𝑱= 𝑭−𝑽, by using manipulation, the Jacobian matrix (23) can be formed as follows: 𝑱(1,0,0) = (𝛽𝜎\n0\n𝛼\n0) −(µ + 𝛼\n0\n0\nµ) \n𝑭= (𝛽𝜎\n0\n𝛼\n0) and 𝑽= (µ + 𝛼\n0\n0\nµ) \n(24) (24) 𝛼\n0\nNext, looking for the inverse of the matrix V, we get: 𝛼\n0\n0\nµ\nNext, looking for the inverse of the matrix V, we get: ext, looking for the inverse of the matrix V, we get: 𝑽−1 =\n1\nµ(µ + 𝛼) (µ\n0\n0\nµ + 𝛼) \n𝑽−1 =\n(\n \n1\n(µ + 𝛼)\n0\n0\n1\nµ) 𝑽−1 =\n1\nµ(µ + 𝛼) (µ\n0\n0\nµ + 𝛼) \n𝑽−1 =\n(\n \n1\n(µ + 𝛼)\n0\n0\n1\nµ)\n \n(25) (25) (\nµ)\nThe next step is to find 𝑅0 = 𝜌(𝑭∙𝑽−1). Then obtained: (\nµ)\nThe next step is to find 𝑅0 = 𝜌(𝑭∙𝑽−1). Then obtained: (\nµ)\nThe next step is to find 𝑅0 = 𝜌(𝑭∙𝑽−1). Then obtained: (\nµ)\nThe next step is to find 𝑅0 = 𝜌(𝑭∙𝑽−1). Then obtained: 𝑅0 = 𝜌(𝐹∙𝑉−1) \n𝑅0 = 𝜌\n(\n \n (𝛽𝜎\n0\n𝛼\n0) ∙\n(\n \n1\n(µ + 𝛼)\n0\n0\n1\nµ)\n \n) BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 1333 BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 3.4.1 \nDisease-Free Equilibrium Point Stability Analysis By substituting the disease-free equilibrium point value in Equation (17) into the Jacobian matrix (32),\nwe get: \n𝛽\n0 we get: 𝐽0 = [\n−µ\n−𝛽𝜎\n0\n0\n𝛽𝜎−𝛼−µ\n0\n0\n𝛼\n−µ\n] \n(33) (33) Next, look for the eigenvalues of the matrix (33). So, the characteristic equation for the Jacobian matrix \n(33), which is analyzed by substituting the disease-free equilibrium point, can be written as follows: Next, look for the eigenvalues of the matrix (33). So, the characteristic equation for the Jacobian matrix \n(33), which is analyzed by substituting the disease-free equilibrium point, can be written as follows: 𝑑𝑒𝑡[\n𝜆+ µ\n−𝛽𝜎\n0\n0\n𝜆−𝛽𝜎+ 𝛼+ µ\n0\n0\n𝛼\n𝜆+ µ\n] = 0 \n (𝜆+ µ)2(𝛼−βσ + 𝜆+ µ) = 0 (34) 𝑑𝑒𝑡[\n𝜆+ µ\n−𝛽𝜎\n0\n0\n𝜆−𝛽𝜎+ 𝛼+ µ\n0\n0\n𝛼\n𝜆+ µ\n] = 0 (𝜆+ µ)2(𝛼−βσ + 𝜆+ µ) = 0 (34) (34) Then the eigenvalues obtained are 𝜆1 = −µ and 𝜆2 = βσ −𝛼−µ. It can be seen that the value of 𝜆1 \nis clearly negative, and 𝜆2 is not necessarily negative. For that, it will be proved that 𝜆2 is negative. It is \nknown that the condition for the disease-free equilibrium point is said to be stable if 𝑅0 < 1. From Equation \n(28) known value 𝑅0 = 𝛽𝜎\n𝛼+µ. Then obtained: Then the eigenvalues obtained are 𝜆1 = −µ and 𝜆2 = βσ −𝛼−µ. It can be seen that the value of 𝜆1 \nis clearly negative, and 𝜆2 is not necessarily negative. For that, it will be proved that 𝜆2 is negative. It is \nknown that the condition for the disease-free equilibrium point is said to be stable if 𝑅0 < 1. From Equation \n𝛽 (28) known value 𝑅0 = 𝛽𝜎\n𝛼+µ. Then obtained: 𝛽𝜎\n𝛼+ µ < 1 \n𝛽𝜎< 𝛼+ µ \n𝛽𝜎−𝛼−µ < 0 Because 𝜆2 = βσ −𝛼−µ . Then obtained: Because 𝜆2 = βσ −𝛼−µ . Then obtained: 𝜆2 < 0 𝜆2 < 0 Based on the Routh-Hurwitz criteria, every eigenvalue that exists is the same, namely negative. As a \nresult, there is no change in sign. It can be concluded that the disease-free equilibrium point is locally \nasymptotically stable. 3.4.1 \nDisease-Free Equilibrium Point Stability Analysis 3.4.1 \nDisease-Free Equilibrium Point Stability Analysis 3.4 Stability Analysis After obtaining the model equilibrium point, the next step is to perform stability analysis for each \nequilibrium point (𝑆0, 𝐼0, 𝑅0) and (𝑆1, 𝐼1, 𝑅1). As the first step in analyzing the equilibrium point, it is \nnecessary to linearize the system of differential Equation (12), Equation (13), and Equation (14). For \nexample: 𝑓(𝑆, 𝐼, 𝑅) = µ −𝛽𝜎𝐼𝑆−µ𝑆 \n(29) \n𝑔(𝑆, 𝐼, 𝑅) = 𝛽𝜎𝐼𝑆+ 𝜏𝐼𝑅−𝛼𝐼−µ𝐼 \n(30) \nℎ(𝑆, 𝐼, 𝑅) = 𝛼𝐼−µ𝑅−𝜏𝐼𝑅 \n(31) (29) \n(30) \n(31) (31) By linearizing Equation (𝟐𝟗), we get: 𝑑𝑓\n𝑑𝑆= 𝑑(µ −𝛽𝜎𝐼𝑆−µ𝑆)\n𝑑𝑆\n= −𝛽𝜎𝐼−µ \n𝑑𝑓\n𝑑𝐼= 𝑑(µ −𝛽𝜎𝐼𝑆−µ𝑆)\n𝑑𝐼\n= −𝐵𝜎𝑆 \n𝑑𝑓\n𝑑𝑅= 𝑑(µ −𝛽𝜎𝐼𝑆−µ𝑆)\n𝑑𝑅\n= 0 Then linearize Equation (𝟑𝟎). Then obtained: Then linearize Equation (𝟑𝟎). Then obtained: 𝑑𝑔\n𝑑𝑆= 𝑑(𝛽𝜎𝐼𝑆+ 𝜏𝐼𝑅−𝛼𝐼−µ𝐼)\n𝑑𝑆\n= 𝛽𝜎𝐼 \n𝑑𝑔\n𝑑𝐼= 𝑑(𝛽𝜎𝐼𝑆+ 𝜏𝐼𝑅−𝛼𝐼−µ𝐼)\n𝑑𝐼\n= 𝛽𝜎𝑆+ 𝜏𝑅−𝛼−µ \n𝑑𝑔\n𝑑𝑅= 𝑑(𝛽𝜎𝐼𝑆+ 𝜏𝐼𝑅−𝛼𝐼−µ𝐼)\n𝑑𝑅\n= 𝜏𝐼 Then linearize Equation (𝟑𝟏). Then obtained: Then linearize Equation (𝟑𝟏). Then obtained: 𝑑ℎ\n𝑑𝑆= 𝑑(𝛼𝐼−µ𝑅−𝜏𝐼𝑅)\n𝑑𝑆\n= 0 Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. 1334 𝑑ℎ\n𝑑𝐼= 𝑑(𝛼𝐼−µ𝑅−𝜏𝐼𝑅)\n𝑑𝐼\n= 𝛼−𝜏𝑅 \n𝑑ℎ\n𝑑𝑅= 𝑑(𝛼𝐼−µ𝑅−𝜏𝐼𝑅)\n𝑑𝑅\n= −µ −𝜏𝐼 The result of linearization will be the elements of the Jacobian matrix, which has the following general \nJacobian: The result of linearization will be the elements of the Jacobian matrix, which has the following general \nJacobian: Jacobian: 𝐽= [\n−𝛽𝜎𝐼−µ\n−𝛽𝜎𝑆\n0\n𝛽𝜎𝐼\n𝛽𝜎𝑆+ 𝜏𝑅−𝛼−µ\n𝜏𝐼\n0\n𝛼−𝜏𝑅\n−µ −𝜏𝐼\n] \n(32) (32) BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 1335 1335 𝑑𝑒𝑡[\n−𝛽𝜎𝐼1 −µ\n−𝛽𝜎𝑆1\n0\n𝛽𝜎𝐼1\n𝛽𝜎𝑆1 + 𝜏𝑅1 −𝛼−µ\n𝜏𝐼1\n0\n𝛼−𝜏𝑅1\n−µ −𝜏𝐼1\n] = 0 𝐼1𝜏(−𝛼+ 𝜏𝑅1)(𝜆+ 𝐼1𝛽𝜎+ µ) + (𝜆+ 𝜏𝐼1 + µ)(𝑆1𝐼1𝛽2𝜎2 + (𝜆+ 𝐼1𝛽𝜎+ µ)(𝛼+ 𝜆−𝑆1𝛽𝜎+\n0 𝐼1𝜏(−𝛼+ 𝜏𝑅1)(𝜆+ 𝐼1𝛽𝜎+ µ) + (𝜆+ 𝜏𝐼1 + µ)(𝑆1𝐼1𝛽2𝜎2 + (𝜆+ 𝐼1𝛽𝜎+ µ)(𝛼+ 𝜆−𝑆1𝛽𝜎+ 𝑅1𝜏+ µ)) \n= 0 𝜆3 + 𝜆2(𝛼−𝑆1𝛽𝜎+ 𝐼1𝛽𝜎+ 𝐼1𝜏+ 𝑅1𝜏+ 3µ) + 𝜆(2𝐼1𝛽𝜎µ \n−2𝑆1𝛽𝜎µ + 2𝐼1𝜏µ + 2𝑅1𝜏µ + 3µ2 + 𝐼1𝛼𝛽𝜎−𝑆1𝐼1𝛽𝜎𝜏 \n+𝐼1\n2𝛽𝜎𝜏+ 𝐼1𝑅1𝛽𝜎𝜏+ 2𝐼1𝑅1𝜏2 + 2𝛼µ) + 2𝐼1\n2𝑅1𝛽𝜎𝜏2 \n+𝐼1𝛼𝛽𝜎µ −𝑆1𝐼1𝛽𝜎𝜏µ + 𝐼1\n2𝛽𝜎𝜏µ + 𝐼1𝑅1𝛽𝜎𝜏µ \n+2𝐼1𝑅1𝜏2µ + 𝛼µ2 −𝑆1𝛽𝜎µ2 + 𝐼1𝛽𝜎µ2 + 𝐼1𝜏µ2 \n+𝑅1𝜏µ2 + µ3 = 0 Then obtained: Then obtained: 𝑎0 = 1 \n𝑎1 = 𝛼−𝑆1𝛽𝜎+ 𝐼1𝛽𝜎+ 𝐼1𝜏+ 𝑅1𝜏+ 3µ \n𝑎2 = 2𝐼1𝛽𝜎µ −2𝑆1𝛽𝜎µ + 2𝐼1𝜏µ + 2𝑅1𝜏µ + 3µ2 + 𝐼1𝛼𝛽𝜎 \n−𝑆1𝐼1𝛽𝜎𝜏+ 𝐼1\n2𝛽𝜎𝜏+ 𝐼1𝑅1𝛽𝜎𝜏+ 2𝐼1𝑅𝜏2 + 2𝛼µ \n𝑎3 = 2𝐼1\n2𝑅1𝛽𝜎𝜏2 + 𝐼1𝛼𝛽𝜎µ −𝑆1𝐼1𝛽𝜎𝜏µ + 𝐼1\n2𝛽𝜎𝜏µ \n+𝐼1𝑅1𝛽𝜎𝜏µ + 2𝐼1𝑅𝜏2µ + 𝛼µ2 −𝑆1𝛽𝜎µ2 \n+𝐼1𝛽𝜎µ2 + 𝐼1𝜏µ2 + 𝑅1𝜏µ2 + µ3 Forming an Array-Routh based on the above equation, we get: Forming an Array-Routh based on the above equation, we get: ||\n𝜆3\n𝛼0\n𝛼2\n𝜆2\n𝛼1\n𝛼3\n𝜆\n(𝛼1𝛼2 −𝛼0𝛼3\n𝛼1\n)\n0\n|| ||\n𝜆3\n𝛼0\n𝛼2\n𝜆2\n𝛼1\n𝛼3\n𝜆\n(𝛼1𝛼2 −𝛼0𝛼3\n𝛼1\n)\n0\n|| The next step is to analyze 𝛼0, 𝛼1, and (\n𝛼1𝛼2−𝛼0𝛼3\n𝛼1\n)There is no change in signs of a stable condition. The condition for a disease to be endemic is 𝑆, 𝐼, 𝑅> 0. Each case will create a different compartment value, \nmeaning that the difference between the compartments is absolute. Then obtained: Then obtained: \n𝛼1𝛼2 −𝛼0𝛼3 = 𝐼1𝛼2𝛽𝜎+ |𝐼1 −𝑆1|𝐼1𝛼𝛽2𝜎2 + |𝐼1 −𝑆1|𝐼1𝛼𝛽𝜎𝜏+ 2𝑅1𝛼𝛽𝜎𝜏+ 𝑆1\n2𝐼1𝛽2𝜎2𝜏\n+ |𝐼1 −2𝑆1|𝐼1\n2𝛽2𝜎2𝜏+ |𝐼1 −𝑆1|𝐼1𝑅1𝛽2𝜎2𝜏+ 2𝐼1𝑅1𝛼𝜏2 + |𝐼1 −𝑆1|𝐼1\n2𝛽𝜎𝜏2\n+ |𝐼1 −3𝑆1|𝑆1𝐼1𝑅1𝛽𝜎𝜏2 + 𝐼1𝑅1\n2𝛽𝜎𝜏2 + 2𝐼2𝑅1𝜏3 + 2𝐼1𝑅1\n2𝜏3 + 2𝛼2µ\n+ |6𝐼1 −4𝑆1|𝛼𝛽𝜎µ + 2𝑆1\n2𝛽2𝜎2µ + |2𝐼1 −4𝑆1|𝑆1𝐼1𝛽2𝜎2µ + 4𝐼1𝛼𝜏µ + 4𝑅1𝛼𝜏µ\n+ |6𝐼1 −6𝑆1|𝐼1𝛽𝜎𝜏µ + |6𝐼1 −4𝑆1|𝑅1𝛽𝜎𝜏µ + 2𝐼1\n2𝜏2µ + 8𝑅1𝐼1\n2𝑅1µ + 2𝑅1\n2𝜏2µ + 8𝛼µ2\n+ |8𝐼1 −8𝑆1|𝛽𝜎µ2 + 8𝐼1𝜏µ2 + 8𝑅1𝜏µ2 + 8µ3 + |\n𝛼1𝛼2 −𝛼0𝛼3 > 0 + |\n𝛼1𝛼2 −𝛼0𝛼3 > 0 𝛼1𝛼2 −𝛼0𝛼3 > 0 𝛼1 = 𝛼+ |𝐼1 −𝑆1|𝛽𝜎+ 𝐼1𝜏+ 𝑅1𝜏+ 3µ \n𝛼1 > 0 In this case, 𝛼1𝛼2 −𝛼0𝛼3 and 𝛼1 are clearly positive, so it can be concluded that 𝛼1𝛼2−𝛼0𝛼3\n𝛼1\n is positive. BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. Then all column one in Array Routh is positive. So, this equation is asymptotically stable. This means that \nthe disease will remain in a population. 3.4.2 \nDisease Endemic Equilibrium Point Stability Analysis Consider an endemic equilibrium Equation (21). Let the point (𝑆1, 𝐼1, 𝑅1) = (𝑆∗, 𝐼∗, 𝑅∗) = (𝑆, 𝐼, 𝑅). By substituting the disease-free equilibrium point value in Equation (21) into the Jacobian matrix (32), we \nget: 𝐽= [\n−𝛽𝜎𝐼1 −µ\n−𝛽𝜎𝑆1\n0\n𝛽𝜎𝐼1\n𝛽𝜎𝑆1 + 𝜏𝑅1 −𝛼−µ\n𝜏𝐼1\n0\n𝛼−𝜏𝑅1\n−µ −𝜏𝐼1\n] \n(35) (35) Next, look for the eigenvalues of the matrix (35). So the characteristic equation for the Jacobian matrix \n(35), which is analyzed by substituting the disease-free equilibrium point, can be written as follows: Next, look for the eigenvalues of the matrix (35). So the characteristic equation for the Jacobian matrix \n(35), which is analyzed by substituting the disease-free equilibrium point, can be written as follows: BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. Table 2. Parameter values in COVID-19 data at Health Center UPTD Batangh Table 2. Parameter values in COVID-19 data at Health Center UPTD Batanghari Table 2. Parameter values in COVID 19 data at Health Center UPTD Batanghari \nParameter \nValue Parameter \nN(Total Population) \n100% (24.977) \nµ(Death and Birth Rate) \n2% (Assumption) \n𝛽 (Transmission or \nInfection Rate) \n100% (Assumption) \nα(Recovery Rate) \n66% (138 of 206) \nγ(Total Vaccinated) \n65% (16.361 of 24.977) \nδ(Vaccine Efficacy) \n65% (Sinovac Standard) \n𝜏(Reinfection Rate) \n50% (Assumption) \nI (Infected Individual) \n0,82% (206 of 24.977) Parameter \nValue Parameter \nN(Total Population) \n100% (24.977) \nµ(Death and Birth Rate) \n2% (Assumption) \n𝛽 (Transmission or \nInfection Rate) \n100% (Assumption) \nα(Recovery Rate) \n66% (138 of 206) \nγ(Total Vaccinated) \n65% (16.361 of 24.977) \nδ(Vaccine Efficacy) \n65% (Sinovac Standard) \n𝜏(Reinfection Rate) \n50% (Assumption) \nI (Infected Individual) \n0,82% (206 of 24.977) With initial values 𝑆1: 0.9868, 𝐼1: 0.0082, and 𝑅1: 0.005. Subtitute Equation (36), Equation (37), and \nEquation (38) with existing parameter values. Then obtained: 𝑆2 = 𝑆1 + µ −𝛽(1 −𝛿𝛾)𝐼1𝑆1 −µ𝑆1 \n𝑆2 = 0.9868 + 2% −100% × (1 −65% × 70%) × 0.0082 × 0.9868 −2% × \n0.9868 = 𝑆1 + µ −𝛽(1 −𝛿𝛾)𝐼1𝑆1 −µ𝑆1 \n𝑆2 = 0.9868 + 2% −100% × (1 −65% × 70%) × 0.0082 × 0.9868 −2% × \n0.9868 3.5 Model Application This simulation aims to test the balance points that have been formed following the Routh-Hurwitz \ncriteria. This simulation uses the following equation: This simulation aims to test the balance points that have been formed following the Routh-Hurwitz \ncriteria. This simulation uses the following equation: Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. 1336 𝑆𝑛+1 = 𝑆𝑛+ µ𝑁−𝛽𝜎𝐼𝑛𝑆𝑛−µ𝑆𝑛 \n(36) \n𝐼𝑛+1 = 𝐼𝑛+ 𝛽𝜎𝐼𝑛𝑆𝑛+ 𝜏𝐼𝑛𝑅𝑛−𝛼𝐼𝑛−µ𝐼𝑛 \n(37) \n𝑅𝑛+1 = 𝑅𝑛+ 𝛼𝐼𝑛−µ𝑅𝑛−𝜏𝐼𝑛𝑅𝑛 \n(38) (36) \n(37) \n(38) f this model uses data obtained from the UPTD Puskesmas Batanghari, as follows: lication of this model uses data obtained from the UPTD Puskesmas Batanghari, as follows: ACKNOWLEDGEMENT All authors would like to thank the academic community at the University of Lampung and LPPM at \nthe University of Lampung for supporting and funding the research and publication of this article. 4. CONCLUSIONS 4. CONCLUSIONS Our research results can be explained as follow: h results can be explained as follow: Our research results can be explained as follow: 1. The COVID-19 SIR Model, made locally asymptotically stable at the balance point of disease-free \nand endemic disease, means that this disease can disappear and remain in an area. 1. The COVID-19 SIR Model, made locally asymptotically stable at the balance point of disease-free \nand endemic disease, means that this disease can disappear and remain in an area. 2. The effect of the vaccination given is to inhibit the spike in infection. We can pay attention to the \nbasic reproduction number A in handling this case. 2. The effect of the vaccination given is to inhibit the spike in infection. We can pay attention to the \nbasic reproduction number A in handling this case. 3. Based on the application of the model to the data at the Batanghari Health Center UPTD, we obtained \nthat the dynamics of the COVID-19 development monitored to be conducive, and the condition was \nstable for the next 100 months, with a Mean Absolute Percentage Error (MAPE) percentage of 2.8% \nwhich it's Highly Accurate Forecasting 3.6 MAPE To find out the errors that exist in the forecasting process, the data in the Batanghari Health Center \nUPTD in months 1, 2, 3, 4, and 5 in 2022 with forecasting results with the SIR COVID-19 model in months \n1, 2, 3, 4, and 5 in 2022. Table 3. Comparison of Data for Months 1, 2, 3, 4, and 5 in 2022 Table 3. Comparison of Data for Months 1, 2, 3, 4, and 5 in 2022 \nMonths \nModel SIR \nCOVID-19 \n(𝑨) \nHealth \nCenter \n(𝑷) \n𝜟|𝑨−𝑷| \n1 \n12.239 \n10 \n2.239 \n2 \n10.965 \n11 \n0.035 \n3 \n9.796 \n7 \n2.796 \n4 \n8.761 \n8 \n0.761 \n5 \n7.837 \n9 \n1.163 \nTotal \n49.598 \n45 \n6.994 \n \nThen it can be obtained that the average absolute error or Mean Absolute Error (MAPE) is Then it can be obtained that the average absolute error or Mean Absolute Error (MAPE) is 𝑀𝐴𝐸= 𝛥|𝐴−𝑃|\n𝑛\n= 6,994\n5\n= 1.3988 \n𝑀𝐴𝑃𝐸= 1.3988\n49.598 = 0.028202 = 2.8% 𝑀𝐴𝐸= 𝛥|𝐴−𝑃|\n𝑛\n= 6,994\n5\n= 1.3988 \n𝑀𝐴𝑃𝐸= 1.3988\n49.598 = 0.028202 = 2.8% BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. decisions or policies implemented by the government and Health Center are correct. However, they must \ncontinue to apply health protocols properly so that there is no spike in unwanted infections. decisions or policies implemented by the government and Health Center are correct. However, they must \ncontinue to apply health protocols properly so that there is no spike in unwanted infections. [5] \nA. Mortellaro and P. Ricciardi-Castagnoli, “From vaccine practice to vaccine science: The contribution of human immunology \nto the prevention of infectious disease,” Immunol. Cell Biol., vol. 89, no. 3, pp. 332–339, 2011, doi: 10.1038/icb.2010.152. [2] \nA. Ajbar, R. T. Alqahtani, and B. Mourad, “Dynamics of an SIR-Based COVID-19 Model With Linear Incidence Rate, \nNonlinear Removal Rate, and Public Awareness,” Front. Phys., vol. 9, no. 634251, pp. 1–13, 2021, doi: \nhttps://doi.org/10.3389/fphy.2021.634251. ,\n,\n, pp\n,\n,\ng\nP. Lan, Z. Liao, Y. Zhang, and S. Liu, “TW-SIR: time-window based SIR for COVID-19 forecasts.,” Sci. Rep., vol\n, 2020, doi: https://doi.org/10.1038/s41598-020-80007-8. [1] \nE. Callaway, “The race for coronavirus vaccines: a graphical guide,” Nature, vol. 580, no. 7805, pp. 576–577, 2020, doi: \ndoi:10.1038/d41586-020-01221-y. [4] \nZ. Liao, P. Lan, Z. Liao, Y. Zhang, and S. Liu, “TW-SIR: time-window based SIR for COVID-19 forecasts.\n10, no. 1, 2020, doi: https://doi.org/10.1038/s41598-020-80007-8. 𝑺𝟐= 𝟎, 𝟗𝟕𝟗𝟒𝟗𝟖𝟐𝟎𝟒 𝑺𝟐= 𝟎, 𝟗𝟕𝟗𝟒𝟗𝟖𝟐𝟎𝟒 𝐼2 = 𝐼1 + 𝛽(1 −𝛿𝛾)𝐼1𝑆1 + 𝜏𝐼1𝑅1 −𝛼𝐼1 −µ𝐼1 \n𝐼2 = 0.000132495 + 100% × (1 −65% × 70%) × 0.000132495 × 0.999847505 + 10% × \n0.000132495 × 0.00002 −20% × 0.000132495 \n− 2% × 0.000132495 𝐼2 = 𝐼1 + 𝛽(1 −𝛿𝛾)𝐼1𝑆1 + 𝜏𝐼1𝑅1 −𝛼𝐼1 −µ𝐼1 \n𝐼2 = 0.000132495 + 100% × (1 −65% × 70%) × 0.000132495 × 0.999847505 + 10% × \n0.000132495 × 0.00002 −20% × 0.000132495 \n− 2% × 0.000132495 𝑅2 = 𝑅1 + 𝛼𝐼1 −µ𝑅1 −𝜏𝐼1𝑅1 \n𝑅2 = 0.00002 + 20% × 0.000132495 −2% × 0.00002 −10% × 0.000132495 × 0.00002 \n𝑹𝟐= 𝟎. 𝟎𝟎𝟎𝟎𝟒𝟔 2\n𝑅1 + 𝛼𝐼1\nµ𝑅1\n𝜏𝐼1𝑅1 \n2 = 0.00002 + 20% × 0.000132495 −2% × 0.00002 −10% × 0.000132495 × 0.00002 \n𝟐= 𝟎. 𝟎𝟎𝟎𝟎𝟒𝟔 Continued until the 100𝑡ℎ iteration, so that the graph is obtained as follows: a) \n(b) \n(c) \n \nFigure 4. Graph of the SIR Model for Covid 19 Data at Batanghari Health Center. (a) Population conditions \nfor individuals who are healthy but susceptible to COVID-19, (b) Population conditions for individuals infected \nwith COVID-19, and (c) Population conditions for individuals who have been given the COVID-19 vaccine. Based on the resulting graph, it can be seen that the dynamics of the development of COVID-19 are \nmonitored to be conducive, and the condition is stable for the next 100 months. It can be concluded that the Figure 4. Graph of the SIR Model for Covid 19 Data at Batanghari Health Center. (a) Population conditions \nfor individuals who are healthy but susceptible to COVID-19, (b) Population conditions for individuals infected \nwith COVID-19, and (c) Population conditions for individuals who have been given the COVID-19 vaccine. Based on the resulting graph, it can be seen that the dynamics of the development of COVID-19 are \nmonitored to be conducive, and the condition is stable for the next 100 months. It can be concluded that the Based on the resulting graph, it can be seen that the dynamics of the development of COVID-19 are \nmonitored to be conducive, and the condition is stable for the next 100 months. It can be concluded that the BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 1337 [3] \nB. Wacker and J. Schlüter, “Time-continuous and time-discrete SIR models revisited: theory and applications,” Adv. Differ. \nEquations, vol. 2020, no. 556, pp. 1–44, 2020, doi: doi.org/10.1186/s13662-020-02995-1. \n[4] \nZ. Liao, P. Lan, Z. Liao, Y. Zhang, and S. Liu, “TW-SIR: time-window based SIR for COVID-19 forecasts.,” Sci. Rep., vol. \n10, no. 1, 2020, doi: https://doi.org/10.1038/s41598-020-80007-8. q\n,\n,\n, pp\n,\n,\ng\nZ. Liao, P. Lan, Z. Liao, Y. Zhang, and S. Liu, “TW-SIR: time-window based SIR for COVID-19 forecasts.,” Sci. R\n10\n1 2020 d i htt\n//d i\n/10 1038/ 41598 020 80007 8 [3] \nB. Wacker and J. Schlüter, Time continuous and time discrete SIR models revisited: theory and applicatio\nEquations, vol. 2020, no. 556, pp. 1–44, 2020, doi: doi.org/10.1186/s13662-020-02995-1. \n[4] \nZ. Liao, P. Lan, Z. Liao, Y. Zhang, and S. Liu, “TW-SIR: time-window based SIR for COVID-19 forecasts.\n10 no 1 2020 doi: https://doi org/10 1038/s41598-020-80007-8 p\ng\np y\n[3] \nB. Wacker and J. Schlüter, “Time-continuous and time-discrete SIR models revisited: theory and applications,” Adv. Differ. \nEquations, vol. 2020, no. 556, pp. 1–44, 2020, doi: doi.org/10.1186/s13662-020-02995-1. \n[4]\nZ Liao P Lan Z Liao Y Zhang and S Liu “TW SIR: time window based SIR for COVID 19 forecasts ” Sci Rep vol REFERENCES [1] \nE. Callaway, “The race for coronavirus vaccines: a graphical guide,” Nature, vol. 580, no. 7805, pp. 576–577, 2020, doi: \ndoi:10.1038/d41586-020-01221-y. y\n[2] \nA. Ajbar, R. T. Alqahtani, and B. Mourad, “Dynamics of an SIR-Based COVID-19 Model With Linear Incidence Rate, \nNonlinear Removal Rate, and Public Awareness,” Front. Phys., vol. 9, no. 634251, pp. 1–13, 2021, doi: \nhttps://doi.org/10.3389/fphy.2021.634251. [3] \nB. Wacker and J. Schlüter, “Time-continuous and time-discrete SIR models revisited: theory and applications,” Adv. Differ. Equations, vol. 2020, no. 556, pp. 1–44, 2020, doi: doi.org/10.1186/s13662-020-02995-1. [4] \nZ. Liao, P. Lan, Z. Liao, Y. Zhang, and S. Liu, “TW-SIR: time-window based SIR for COVID-19 forecasts.,” Sci. Rep., vol. 10, no. 1, 2020, doi: https://doi.org/10.1038/s41598-020-80007-8. [5] \nA. Mortellaro and P. Ricciardi-Castagnoli, “From vaccine practice to vaccine science: The contribution of human immunology \nto the prevention of infectious disease,” Immunol. Cell Biol., vol. 89, no. 3, pp. 332–339, 2011, doi: 10.1038/icb.2010.152. Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. 1338 [6] \nP. Thapa, “Predicating COVID19 Epidemic in Nepal Using the SIR Model,” Stud. Syst. Decis. Control, vol. 358, no. September, \npp. 229–237, 2021, doi: 10.1007/978-3-030-69744-0_14. pp\n[7] \nO. Diekmann, J. A. P. Heesterbeek, and J. A. J. Metz, “On the definition and the computation of the basic reproduction ratio \nR0 in models for infectious diseases in heterogeneous populations,” J. Math. Biol., vol. 28, no. 4, pp. 365–382, 1990, doi: \n10.1007/BF00178324. [8] \nW. F. Putra, “Analisis Efikasi dan Efektivitas Vaksin COVID-19 terhadap Varian SARS-CoV-2: Sebuah Tinjauan Literatur,” \nJ. Kedokt. Meditek, vol. 28, no. 1, pp. 107–119, 2022, doi: 10.36452/jkdoktmeditek.v28i1.2243. [9] \nC. van Oosterhout, N. Hall, H. Ly, and K. M. Tyler, “COVID-19 evolution during the pandemic–Implications of new SARS-\nCoV-2 variants on disease control and public health policies,” Virulence, vol. 12, no. 1, pp. 507–508, 2021, doi: \n10.1080/21505594.2021.1877066. [10] J. Li et al., “Epidemiology of COVID-19 : A Systematic Review and Meta-analysis of Clinical Epidemiology of COVID-19 : \nA systematic review and meta-analysis of clinical characteristics, risk factors , and outcomes,” Med. Virol., pp. 1–10, 2020, \ndoi: 10.1002/jmv.26424. [11] WHO, \n“Coronavirus \ndisease \n( \nCOVID-19 \n),” \n2020. [Online]. Available: \nhttps://www.who.int/docs/default-\nsource/coronaviruse/situation-reports/20201012-weekly-epi-update-9.pdf p\ny p\np\np\n2] J. Sun et al., “COVID-19 : Epidemiology , Evolution , and Cross-Disciplinary Perspectives,” no. January, 2020. [\n]\n,\np\ngy ,\n,\np\ny\np\n,\ny,\n[13] C. Van Oosterhout, N. Hall, H. Ly, and K. M. REFERENCES Tyler, “COVID-19 evolution during the pandemic – Implications of new SARS-\nCoV-2 variants on disease control and public health policies,” Virulence, vol. 12, no. 1, pp. 507–508, 2021, doi: \n10.1080/21505594.2021.1877066. [14] S. Setiati and M. K. Azwar, “Dilemma of Prioritising Health and the Economy During COVID-19 Pandemic in Indonesia,” \nActa Med Indones, vol. 52, no. 3, pp. 196–198, 2020. [15] A. Mortellaro and P. Ricciardi-Castagnoli, “From vaccine practice to vaccine science : the contribution of human immunology \nto the prevention of infectious disease,” Immunol. Cell Biol., vol. 89, pp. 332–339, 2011, doi: 10.1038/icb.2010.152. [16] WHO “T\nJ\nb L\nkd\nd\nh d i\nit ” 2021\nh i t/i d\ni /\n/\nl\ni\n/\n/\nl\nkd\nd [15] A. Mortellaro and P. Ricciardi-Castagnoli, “From vaccine practice to vaccine science : the contribution of human immunology \nto the prevention of infectious disease,” Immunol. Cell Biol., vol. 89, pp. 332–339, 2011, doi: 10.1038/icb.2010.152. [16] WHO, “Tanya Jawab : Lockdown dan herd immunity,” 2021. who.int/indonesia/news/novel-coronavirus/qa/qa-lockdown-and-\nherd-immunity (accessed Jun. 10, 2020). y (\n,\n)\n[17] N. M. Nasir, I. S. Joyosemito, B. Boerman, and I. Ismaniah, “Kebijakan Vaksinasi COVID-19 : Pendekatan Pemodelan \nMatematika Dinamis Pada Efektivitas Dan Dampak Vaksin Di Indonesia,” J. Abdimas UBJ, vol. 4, no. 2, pp. 191–204, 2021. [18] W. F. Putra, “Analisis Efikasi dan Efektivitas Vaksin COVID-19 terhadap Varian SARS-CoV-2 : Sebuah Tinjauan Literatur \nAnalysis the Efficacy and Effectivity of COVID-19 Vaccines to the SARS-CoV-2 Variants : A Literature Review,” Meditek, \nvol. 28, no. 1, pp. 107–119, 2022, doi: https://doi.org/10.36452/jkdoktmeditek.v28i1.2243. pp\np\ng\nj\n[19] W. O. Kermarck and A. G. McKendrick, “A Contribution to the Mathematical Theory o f Epidemics.,” in The Royal Society \nLondon A, Royal Society, 1927, pp. 700–721. doi: 10.1098/rspa.1927.0118. [20] P. Thapa, “Predicating COVID19 epidemic in Nepal using the SIR model,” Artif. Intell. COVID-19., 2021, doi: 10.1007/978-\n3-030-69744-0_14. [21] M. A. Shereen, S. Khan, A. Kazmi, N. Bashir, and R. Siddique, “COVID-19 infection: origin, transmission, an\nof human coronaviruses.,” J. Adv. Res., vol. 24, no. 2020, pp. 91–98, 2020, doi: 10.1016/j.jare.2020.03.005. [22] Y. F. Lin et al., “Spread and Impact of COVID-19 in China: A Systematic Review and Synthesis of Predictions From \nTransmission-Dynamic Models. Frontiers in Medicine, 7. doi:10.3389/fmed.2020.00321,” Front. Med., vol. 7, no. 321, pp. 1–\n11, 2020, doi: doi:10.3389/fmed.2020.00321. [23] Q. Griette and P. REFERENCES Magal, “Clarifying predictions for COVID-19 from testing data: The example of New York State.,” Infect. Dis. Model., vol. 6, pp. 273–283, 2021, doi: https://doi.org/10.1016/j.idm.2020.12.011. pp\np\ng\nj\n[24] Z. Liu, P. Magal, and G. Webb, “Predicting the number of reported and unreported cases for the COVID- 19 epidemics in \nChina, South Korea, Italy, France, Germany and United Kingdom,” J. Theor. Biol. 509, 110501., vol. 509, no. 110501, 2021, \ndoi: doi:10.1016/j.jtbi.2020.110501. j j\n[25] A. J. Kucharski, T. W. Russell, J. Diamond, C., Liu, Y., Edmunds, S. Funk, and R. M. Eggo, “Early dynamics of transmission \nand control of COVID-19: a mathematical modelling study.,” Lancet Infect. Dis., vol. 20, pp. 1–7, 2020, doi: \nhttps://doi.org/10.1016/S1473-3099(20)30144-4. p\ng\n(\n)\n[26] E. S. Kurkina and E. M. Koltsova, “Mathematical Modeling of the Propagation of COVID-19 Pandemic Waves in the World.,” \nComput. Math. Model., vol. 32, no. 2, pp. 147–170, 2021, doi: doi:10.1007/s10598-021-09523-0. pp\n[27] A. Lobo et al., “COVID-19 epidemic in Brazil: where we at?,” Int. J. Infect. Dis., vol. 97, pp. 382–385, 2020, doi: \ndoi:10.1016/j.ijid.2020.06.044. [28] K. Roosa et al., “Real-time forecasts of the COVID-19 epidemic in China from February 5th to February 24th, 2020.,” Infect. Dis. Model., vol. 5, no. 2020, pp. 256–263, 2020, doi: 10.1016/j.idm.2020.02.002. [29] H. W. Hethcote, “The mathematics of infectious diseases,” SIAM Rev., vol. 42, no. 4, pp. 599–653, 2000, [On\nhttp://www.siam.org/journals/sirev/42-4/37190.html p\ng j\n[30] K. R. Meyer, “Normal forms for the general equilibrium,” Funkc. Ekvacioj, vol. 27, pp. 261–271, 1984. p\ng j\n] K. R. Meyer, “Normal forms for the general equilibrium [31] G. J. Olsder and J. W. van der Woude, Mathematical Systems Theory, Second. Delft, The Netherland: Delft \n1997. [32] O. Diekmann, J. A. P. Heesterbeek, and J. A. J. Metz, “On the definition and the computation of the basic reproduction ratio \nRo in models for infectious diseases in heterogeneous populations,” J. Math. Biol, vol. 28, pp. 365–382, 1990. [33] A. Faruk, “Model Epidemik Tuberkulosis Seir dengan Terapi pada Individu Terinfeksi,” J. Penelit. Sains, vol. 18, no. 3, pp. 99–104, 2016, doi: 10.56064/jps.v18i3.16. jp\n[34] J. Giesecke, Modern Infectious Disease Epidemiology, Third. CRC Press Taylor & Francis Group, 2017. [35] P. Van Den Driessche and J. Watmough, “Reproduction numbers and sub-threshold endemic equilibria for compartmental \nmodels \nof \ndisease \ntransmission,” \nMath. Biosci. 180, \nvol. 180, \npp. 29–48, \n2002, \n[Online]. Available: \nhttp://www.math.unb.ca/?watmough. p\ng\n[36] F. R. Gantmacher, The theory of matrices. REFERENCES Chelsea Publishing Company, 1959. p\ng\n[36] F. R. Gantmacher, The theory of matrices. Chelsea Publishing Company, 1959. ] F. R. Gantmacher, The theory of matrices. Chelsea Publi ewis, Demand Forecasting and Inventory Control: A computer aided learning approach, vol. 2. Woodhead Publishing\n7. [37] C. D. Lewis, Demand Forecasting and Inventory Control: A computer aided learning approach, vol. 2. Woodhead Publishing \nLtd, 1997. [38] WHO “Evaluation of COVID-19 vaccine effectiveness ” 2021 https://www who int/publications/i/item/WHO-2019-nCoV- Ltd, 1997. [38] WHO “Evaluation of COVID-19 vaccine effectiveness ” 2021 https://www who int/publications/i/item/WHO-2019-nCoV- Ltd, 1997. [38] WHO, “Evaluation of COVID-19 vaccine effectiveness,” 2021. https://www.who.int/publications/i/item/WHO-2019-nCoV- Evaluation of COVID-19 vaccine effectiveness,” 2021. https://www.who.int/publications/i/item/WHO-2019-nCoV [38] WHO, “Evaluation of COVID-19 vaccine effectiveness,” 2021. https://www.who.int/publications/i/item/WH BAREKENG: J. Math. & App., vol. 17(3), pp. 1325- 1340, September, 2023. 1339 \n \nvaccine_effectiveness- measurement-2021 1339 Aziz, et. al. THE DEVELOPMENT OF COVID-19 USING OUTBREAK …. 1340"
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Lupus Anticoagulant Antibody
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Definitions
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Qeios · Definition, February 7, 2020
Ope n Pe e r Re v ie w on Qe ios
Lupus Anticoagulant Antibody
National Cancer Institute
Source
National Cancer Institute. Lupus Anticoagulant Antibody. NCI T hesaurus. Code C70620.
Antibodies directed against plasma proteins (such as beta-2-glycoprotein I, prothrombin,
or annexin V) bound to anionic phospholipids. T he anticoagulant blocks in vitro assembly
of the prothrombinase complex, resulting in prolongation of in vitro clotting assays such
as the activated partial thromboplastin time, the Russell viper venom time, and others.
Paradoxical to these changes which suggest impaired coagulation, patients with the
lupus anticoagulant have an increase in thrombotic events.
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Achieving high spatial resolution in a large field-of-view using lensless x-ray imaging
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Applied physics letters
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RESEARCH ARTICLE | SEPTEMBER 21 2021
Achieving high spatial resolution in a large field-of-view
using lensless x-ray imaging RESEARCH ARTICLE | SEPTEMBER 21 2021
Achieving high spatial resolution in a large field-of-view
using lensless x-ray imaging RESEARCH ARTICLE | SEPTEMBER 21 2021 Achieving high spatial resolution in a large field-of-view
using lensless x-ray imaging Yi Jiang
; Junjing Deng
; Yudong Yao; Jeffrey A. Klug
; Sheikh Mashrafi; Christian Roehrig;
Curt Preissner; Fabricio S. Marin; Zhonghou Cai; Barry Lai; Stefan Vogt Appl. Phys. Lett. 119, 124101 (2021)
https://doi.org/10.1063/5.0067197
CHORUS Articles You May Be Interested In
Hard X‐Ray Scanning Microscopy with Coherent Diffraction Contrast
AIP Conference Proceedings (September 2011)
Hard x-ray scanning microscopy with coherent radiation: Beyond the resolution of conventional x-ray
microscopes
Appl. Phys. Lett. (June 2012)
The Imaging and Coherence Beamline I13 at Diamond
AIP Conference Proceedings (September 2011) Articles You May Be Interested In Hard X‐Ray Scanning Microscopy with Coherent Diffraction Contrast AIP Conference Proceedings (September 2011) Appl. Phys. Lett. (June 2012) 24 October 2024 05:18:07 ABSTRACT X-ray ptychography, a powerful scanning lensless imaging technique, has become attractive for nondestructively imaging internal structures
at nanoscale. Stage positioning overhead in conventional step-scan ptychography is one of the limiting factors on the imaging throughput. In
this work, we demonstrate the use of advanced fly scan ptychography to achieve high-resolution ptychograms of modern integrated circuits
on a large field-of-view at millimeter scale. By completely removing stage overheads between scan points, the imaging time for millimeter-
size sample can be significantly reduced. Furthermore, we implement the orthogonal probe relaxation technique to overcome the variation of
illumination across the large scan area as well as local vibrations. The capability of x-ray ptychography shown here is broadly applicable for
various studies, which requires both high spatial resolution and large scan area. 24 October 2024 05:18:07 V
C 2021 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://
creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0067197 experimentally demonstrated in a step-scan ptychography to achieve
hundreds-of-micrometers FOV.9 However, the positioning overhead
of 150 ms still contributed about 43% of the total scan time in this
demonstration. An even larger beam (>50 lm) was used in near-field
ptychography,2,10 which enables efficient large FOV imaging at the
cost of degraded spatial resolution. The development of high-precision
stages continues to decrease the positioning overhead, which, however,
is still determined by the mobile mass and stage motion range in the
experiment (e.g., 50–80 ms reported in this Ref. 11). A further signifi-
cant decrease in overhead to 5 ms has been reported in a proof-of-con-
cept demonstration through a hybrid motion of sample and optics.11
To pursue negligible positioning overhead under any circumstances,
several groups12–14 have developed a so-called fly scan scheme in
which objects move at a constant velocity during single exposure, thus
reducing the idle time to detector’s readout time that can be a few
microseconds for numerous pixel-array-detectors. The problem is tra-
ditionally solved with the mixed-states ptychography algorithm15 that
describes the fly scan diffraction pattern as an incoherent summation
of diffraction intensities generated from multiple orthogonal probe
modes. The number of probe modes required in fly scan ptychography
increases with the step size,13,16 resulting in an increase in the recon-
struction difficulty. AFFILIATIONS Advanced Photon Source, Argonne National Laboratory, Lemont, Illinois 60439, USA a)Author to whom correspondence should be addressed: junjingdeng@anl.gov Achieving high spatial resolution in a large
field-of-view using lensless x-ray imaging Cite as: Appl. Phys. Lett. 119, 124101 (2021); doi: 10.1063/5.0067197
Submitted: 14 August 2021 . Accepted: 5 September 2021 . Published Online: 21 September 2021 Cite as: Appl. Phys. Lett. 119, 124101 (2021); doi: 10.1063/5.0067197
Submitted: 14 August 2021 . Accepted: 5 September 2021 . Published Online: 21 September 2021 Yi Jiang,
Junjing Deng,a)
Yudong Yao, Jeffrey A. Klug,
Sheikh Mashrafi, Christian Roehrig, Curt Preissner,
Fabricio S. Marin, Zhonghou Cai, Barry Lai, and Stefan Vogt Applied Physics Letters ARTICLE scitation.org/journal/apl ABSTRACT Therefore, the step size in fly scan ptychography is
generally limited at a given spatial resolution,12 and a small step size is Imaging large objects with millimeter or even centimeter size at
nanoscale provides unprecedented information and has been increas-
ingly demanded in a wide range of applications from semiconductors
characterization1,2 to brain research.3 Among most popular micros-
copy techniques, x-ray ptychography has a unique advantage in
achieving this goal, thanks to its ability to reach spatial resolution
beyond the probe-forming lens. By utilizing redundant information
from scanning diffraction patterns, ptychography provides projected
electron density of the specimen by directly reconstructing the
complex-valued transmission function,4,5 with the state-of-the-art
reaching below 10 nm spatial resolution in x-ray regime.6,7 Moreover,
the scanning nature allows ptychography to bypass the “isolated
object” requirement in conventional coherent diffraction imaging8
and able to image extended sample. Conventional ptychography is implemented in a step-scan mode
in which the scanning microscope acquires diffraction patterns in a
move-settle-acquire sequence. The time spent on “move-settle” pro-
cess, called overhead, is not used for acquiring diffraction patterns,
which makes scan inefficient. To efficiently achieve large field-of-view
(FOV) ptychographic imaging in step-scan mode, a large beam (e.g., a
few micrometers) with a big scan step size and a long exposure time
can be used to reduce scan points and, thus, reduce the percentage of
the scan overhead in the scan time. Such a scheme has been Appl. Phys. Lett. 119, 124101 (2021); doi: 10.1063/5.0067197
V
C Author(s) 2021 119, 124101-1 119, 124101-1 Applied Physics Letters scitation.org/journal/apl ARTICLE usually preferred for high-throughput ptychography17 at the cost of
more data points. Such restriction can be mitigated with the develop-
ment of the arbitrary-path fly scan technique18 that allows the use of
larger fly scan steps. Nevertheless, with the reconstruction quality close
to the conventional step-scan scheme using advanced computation
methods, the fly scan technique can achieve higher photon efficiency13
and will greatly improve ptychography throughput with the fourth-
generation photon source upgrade that will increase the photon flux
by several orders of magnitude. with mixed-states model, scan positions refinement, and probe varia-
tion correction. The tiles are then stitched together using the a cross
correlation based method21 during post-processing. A single recon-
struction of 500 iterations and an update group size (the number of
diffraction patterns that are processed in parallel) of 143 took about
40 min on a Nvidia Tesla V100 GPU. ABSTRACT The
results demonstrate fly scan ptychography’s potential for high-
throughput experiments that characterize large objects at nanoscale. Detailed descriptions about the Velociprobe instrument, includ-
ing granite air-bearing stages and the high-bandwidth interferometric
system, was reported in our instrumentation paper.19 The instrument
was initially designed to acquire diffraction patterns by moving the
light-weight focusing zone plate instead of the sample stage, thus pro-
viding more accurate and stable measurement at high scan speed. Although the scan area of a single zone-plate scan is generally limited
(<10 10 lm2), large FOV imaging can be achieved via image stitch-
ing during or post-reconstruction. 24 October 2024 05:18:07 g
g
p
Modern integrated circuits (ICs) assemble a myriad of complex
features at many length scales inside a chip to achieve certain function-
alities. Therefore, they are ideal samples for testing the capability of x-
ray ptychography. We first show a large FOV ptychographic imaging
of a TSMC chip fabricated with 16-nm technology. The entire chip of
2.5 2.5mm2 was backside thinned down to a Si substrate thickness
of about 130 lm. The sample was then mounted on a customized
invar sample holder. The orthogonal alignment of the sample plane
relative to x-rays was achieved by adjusting the sample tilt around the
horizontal and vertical axis through designed flexure structures in the
holder. An incident x-ray beam with a photon energy of 8.8 keV was
spectral-filtered by a Si h111i double crystal monochromator and then
focused by a Fresnel zone plate (FZP) with a 180 lm diameter and
50 nm outermost zone width, which produces a circular-shaped illu-
mination with a diameter of 150 nm on the sample that was
62 mm behind the zone plate. The measurement consisted of 676
(26 26) tile scans with a 6 6 lm2 FOV. Each tile was raster
scanned in a snake pattern19 with a 50-nm step size in both translation
directions. The diffraction patterns were continuously recorded at 250
frames/s by the Eiger X 500K detector, which was placed 1.92 m
downstream from the sample. Each tile scan only took 58 s, and a total
of 9.7 106 diffraction patterns were acquired in about 11.5 h. g
To have the flexibility of switching zone plates for different appli-
cations, the beam stop on the Velociprobe stays at a fixed location as
the zone plate scans the sample. ABSTRACT As shown in Fig. 1(a), the
FOV of the final phase image (with contrast inverted) is about
131 131 lm2. The dark regions at four corners are solder bumps
that absorb most of x-rays and, hence, were not reconstructed. Figures
1(b)–1(d) show the zoom-in areas indicated by the boxes with a FOV
of 50 50, 20 20, and 3.3 3.3 lm2, respectively. Figure 1(d) shows
distinct circuitry features that are projected from different IC layers
with a total thickness of about 4.5 lm. In addition to the 720 nm-size
metal blocks from top layers, the tungsten source/drain connections to
the bottom oxide diffusion layer (OD) and the gate vias to the poly-
crystalline layer (PO) are also clearly resolved in high contrast with a
width size of 40 nm. The inset in Fig. 1(d) (labeled by “GDS”) is
the stacked phase image calculated from chip design files on the
indicated brown-box region, displaying comparable contrast and
consistent features as the ptychographic reconstruction. For exam-
ple, the 50 nm-thick OD connections (magenta, only two are
highlighted in the GDS image) with 90 nm spacing and 40 nm-thick
PO vias (yellow) can be correspondingly located on the ptycho-
graphic image. The two green arrows demonstrate that the transis-
tor gates on the second lowest layer can also be clearly identified
with 16 nm linewidth and 90 nm pitch. The smallest features in this
chip are silicon FinFET fins with 10 nm width and 16 nm thickness,
which are very challenging to be resolved. Surprisingly, some weak-
contrast structures (cyan) with a spacing of about 50 nm can be
seen on some locations in Fig. 1(d), which is consistent with the fin
pitch of 48 nm in the chip design file. Recently, the Advanced Photon Source (APS) at Argonne
National Laboratory developed a dedicated fly scan ptychography
instrument, namely, the Velociprobe, and demonstrated sub-10 nm
resolution imaging over a 3 3 lm2 area at a 3000 Hz frame rate (the
maximum continuous frame rate allowed by the Dectris Eiger X 500K
detector) using a double-multilayer monochromator (DMM).19 Here,
we show the instrument’s capability for high-speed and large FOV
(millimeter scale) fly scan ptychography. With advanced sample stage
and reconstruction techniques, areas beyond 1 mm2 can be scanned in
only a few hours, meanwhile achieving 37 nm spatial resolution. ABSTRACT This causes the incident probe to
slightly vary during a single scan and results in a circular background
in the conventional ptychographic reconstruction. This issue can be
worked around by integrating the beam stop in the hard x-ray FZP.11
However, the scan range of FZP will be then limited by the diameter
difference between the beam stop and the order sorting aperture
(OSA). We found that such problem can also be alleviated in the
reconstruction using the orthogonal probe relaxation (OPR) tech-
nique,22 whose model allows different probe functions at different
scan positions. All the reconstructed probes are constrained via a trun-
cated singular value decomposition, and the number of orthonormal
modes that is kept to represent all probes can be used as an additional
reconstruction parameter. Figure 2 demonstrates the effect of OPR
technique on the zone-plate scan data on the Velociprobe. A test
pattern that was acquired with spiral fly scan was reconstructed with
the traditional mixed-states ePIE23 algorithm with position correc-
tion.20 As shown in Fig. 2(a), the reconstructed phase suffers from
strong circular variation and streaking artifacts that are caused by inac-
curate position refinement [Fig. 2(b)]. When the OPR method is
incorporated into reconstruction, the quality is greatly improved, as
shown in both reconstructed phase and the refined scan positions
[Figs. 2(c) and 2(d)]. Moreover, the OPR method is also effective in
reducing artifacts from probe variation during large FOV measure-
ments, which will be discussed in later section. For ptychographic reconstruction, 512 512 pixels from each
raw diffraction pattern were cropped and then binned to 256 256
pixels, resulting in a 7-nm pixel size in the reconstruction. Limited by
computer’s memory, each tile scan was reconstructed independently
using the least-square maximum likelihood (LSQ-ML) algorithm20 119, 124101-2 Appl. Phys. Lett. 119, 124101 (2021); doi: 10.1063/5.0067197
V
C Author(s) 2021 119, 124101-2 119, 124101-2 Applied Physics Letters scitation.org/journal/apl ARTICLE FIG. 1. Large area ptychography of an integrated circuit by tile scanning. (a) Phase image of 131 131 lm2 integrated circuit with 16 nm technology, reconstructed from
26 26 tiles (6 6 lm2 per tile), which were scanned in fly raster-scan mode using an 8.8 keV x-ray beam. The reconstructions have about 500 megapixels with a pixel size
of 7 nm. ABSTRACT The tiles that cover solder bumps at four corners cannot be reconstructed correctly due to little scattering signals and, hence, were not included in the final stitched
image. (b) A zoomed region of 50 50 lm2 indicated by a blue box in (a), while the red boxes in (a) and (b) outline an area of 10 10 lm2 shown in (c). (d) A 3.3 3.3 lm2
region from the denoted green box showing the circuitry details from the projected 21 layers. The inset in (d) is the stacked phase of the 21 circuitry layers calculated from the
chip design files at 8.8 keV, displaying the same chip area indicated by the solid brown square on the reconstruction. The gate (PO) and source/drain (OD) connections can be
clearly visualized in the ptychographic image. The green arrows point to two transistor gate lines that locate between OD lines, with a separation of 90 nm. The thin silicon fin
structures (cyan) fabricated with 16 nm FinFET technology can also be seen on some locations. FIG 1 Large area ptychography of an integrated circuit by tile scanning (a) Phase image of 131 131 lm2 integrated circuit with 16 nm technology reconstructed from 24 October 2024 05:18:07 24 October 2024 05:18:07 FIG. 1. Large area ptychography of an integrated circuit by tile scanning. (a) Phase image of 131 131 lm2 integrated circuit with 16 nm technology, reconstructed from
26 26 tiles (6 6 lm2 per tile), which were scanned in fly raster-scan mode using an 8.8 keV x-ray beam. The reconstructions have about 500 megapixels with a pixel size
of 7 nm. The tiles that cover solder bumps at four corners cannot be reconstructed correctly due to little scattering signals and, hence, were not included in the final stitched
image. (b) A zoomed region of 50 50 lm2 indicated by a blue box in (a), while the red boxes in (a) and (b) outline an area of 10 10 lm2 shown in (c). (d) A 3.3 3.3 lm2
region from the denoted green box showing the circuitry details from the projected 21 layers. The inset in (d) is the stacked phase of the 21 circuitry layers calculated from the
chip design files at 8.8 keV, displaying the same chip area indicated by the solid brown square on the reconstruction. ABSTRACT The gate (PO) and source/drain (OD) connections can be
clearly visualized in the ptychographic image. The green arrows point to two transistor gate lines that locate between OD lines, with a separation of 90 nm. The thin silicon fin
structures (cyan) fabricated with 16 nm FinFET technology can also be seen on some locations. Next, we demonstrate enhanced imaging throughput of the
Velociprobe instrument after recent modifications that increase
sample stage’s travel range up to 35 and 25 mm in horizontal and
vertical direction, respectively.24 An fly scan ptychography with
1 1 mm2 FOV was carried out on a new IC sample with a total
thickness of about 50 lm and solder bumps removed. The focusing
optics and x-ray energy same to the previous example were used to
produce an incident beam with a diameter of 1.5 lm on the sample,
which was placed about 600 lm downstream of the zone plate focus-
ing position. Instead of moving the FZP, the sample was continu-
ously moved in a snake pattern in ptychography scan. The entire
area was measured by 5 10 tile scans that have 205 105 lm2
FOV on each tile and a 5-lm overlap in between. The scan step sizes are 100 and 500 nm in the horizontal and vertical directions, respec-
tively. The data were acquired at a 2000 Hz frame rate using the
Dectris Eiger X 500 K detector, and a total of 24 106 diffraction
patterns were collected in 3.3 h. Each tile was reconstructed using
the LSQ-ML algorithm with 9 mixed-states probe modes, scan posi-
tion refinement, and probe variation correction. For low-resolution
(56 nm) reconstructions, we run 50 iterations with a group size of
1050 for each tile scan, which took approximately 1 h on a Nvidia
Tesla V100 GPU. Figure 3(a) shows the phase image of the entire reconstruction of
1 mm2 FOV, which was obtained by stitching all sub-images in post-
processing. A single tile reconstruction is shown in Fig. 3(b), which
contains large features such as 10-lm wide metal lines or the solder Appl. Phys. Lett. 119, 124101 (2021); doi: 10.1063/5.0067197
V
C Author(s) 2021 Appl. Phys. Lett. 119, 124101 (2021); doi: 10.1063/5.0067197 119, 124101-3 119, 124101-3 Applied Physics Letters scitation.org/journal/apl ARTICLE FIG. 2. Influence of the orthogonal probe relaxation (OPR) technique on zone-plate scan data acquired on the Velocipede. ABSTRACT (a) ePIE reconstruction of a test pattern measured
in a spiral fly scan trajectory. (b) Refined scan position that corresponds to (a). (c) ePIE reconstruction with the OPR method that account for beam variation. (d) Refined scan
position that corresponds to (c). 24 October 2024 05:18:07 24 October 2024 05:18:07 FIG. 2. Influence of the orthogonal probe relaxation (OPR) technique on zone-plate scan data acquired on the Velocipede. (a) ePIE reconstruction of a test pattern measured
in a spiral fly scan trajectory. (b) Refined scan position that corresponds to (a). (c) ePIE reconstruction with the OPR method that account for beam variation. (d) Refined scan
position that corresponds to (c). advanced LSQ-ML algorithm that is implemented in the PtychoShelves
package,26 which not only allows for multiple OPR modes but also
imposes additional constraints to the object and probes evolution to
further regularize the reconstruction. As the number of OPR modes
increases, reconstruction artifacts become greatly reduced [Figs. 4(c)
and 4(d)] and eventually become identical to an artifact-free recon-
struction from a small FOV data [Fig. 4(a)]. pad with a 90-lm diameter. The zoom-in detail of this large-FOV
reconstruction can be found in the supplementary material Movie 1. Figures 3(c) and 3(d) further show high-resolution reconstructions (7-
nm pixel size) of a smaller region highlighted in Fig. 3(b). Although
the data were acquired at a high frame rate and has limited photon
flux (5 108 ph/s), small features with 40 nm width can still be
resolved in the ptychographic reconstruction, as indicated in the line
profile in Fig. 3(e). The imaging reproducibility of two ptychographic
scans (20 10 lm2 area acquired with the same scan parameters) was
evaluated using Fourier ring correlation (FRC).25 The FRC gives a spa-
tial resolution of 37.1 nm with 1/2-bit threshold, which is consistent
with the line profile analysis. Therefore, with fly scan ptychography,
high-resolution imaging of a whole IC can be achieved within a rea-
sonable amount of time. pad with a 90-lm diameter. The zoom-in detail of this large-FOV
reconstruction can be found in the supplementary material Movie 1. Figures 3(c) and 3(d) further show high-resolution reconstructions (7-
nm pixel size) of a smaller region highlighted in Fig. 3(b). ABSTRACT Although
the data were acquired at a high frame rate and has limited photon
flux (5 108 ph/s), small features with 40 nm width can still be
resolved in the ptychographic reconstruction, as indicated in the line
profile in Fig. 3(e). The imaging reproducibility of two ptychographic
scans (20 10 lm2 area acquired with the same scan parameters) was
evaluated using Fourier ring correlation (FRC).25 The FRC gives a spa-
tial resolution of 37.1 nm with 1/2-bit threshold, which is consistent
with the line profile analysis. Therefore, with fly scan ptychography,
high-resolution imaging of a whole IC can be achieved within a rea-
sonable amount of time. In conclusion, this Letter reports two large FOV x-ray fly scan
ptychography experiments conducted on the Velociprobe instrument
at the APS, demonstrating the possibility of efficient imaging of objects
beyond 1 mm in size at tens of nanometer spatial resolution. We have
achieved 6.2 103 resolution elements per second (a method used in
Ref. 9 to evaluate ptychography throughput) with about 10 nm resolu-
tion in the first IC sample, while we have achieved 7.5 104 resolution
elements (35 nm) per second in the second sample. Even though only
IC samples were chosen for the demonstration here, this imaging
capability has broad research applications, such as brain imaging that
requires high resolution to resolve synaptic connections and high
imaging throughput to map neuronal circuitry composing of billions As scan area increases, the reconstruction quality often degrades
due to beam variation over a long scan time or local vibrations from
the focusing optics. Figure 4 shows that the phase image from a single
tile scan suffers from distracting streaky artifacts when only a single
OPR mode is used. To overcome the problem, we used the more Appl. Phys. Lett. 119, 124101 (2021); doi: 10.1063/5.0067197
V
C Author(s) 2021 119, 124101-4 119, 124101-4 119, 124101-4 Applied Physics Letters scitation.org/journal/apl FIG. 3. Fly scan ptychography reconstruction of a 16-nm technology IC. The data were acquired at a 2000 Hz frame rate. (a) Phase image of 1 mm 1 mm reconstruction of
the sample. (b) Reconstructed phase on a single tile of 205 105 lm2 FOV. The pixel size is 56 nm. (c) A high-resolution reconstruction of a smaller region highlighted in (b). The pixel size is 7 nm. (d) A zoom-in of the region highlighted in (c). ABSTRACT It further shows the small features that can be reconstructed from the data. (e) A line profile across several
vertical lines in (d). Features that are 80-nm apart are well resolved in the image, and the full-width half maximum of a single line is estimated to be 35 nm. FIG. 3. Fly scan ptychography reconstruction of a 16-nm technology IC. The data were acquired at a 2000 Hz frame rate. (a) Phase image of 1 mm 1 mm reconstruction of
the sample. (b) Reconstructed phase on a single tile of 205 105 lm2 FOV. The pixel size is 56 nm. (c) A high-resolution reconstruction of a smaller region highlighted in (b). The pixel size is 7 nm. (d) A zoom-in of the region highlighted in (c). It further shows the small features that can be reconstructed from the data. (e) A line profile across several
vertical lines in (d). Features that are 80-nm apart are well resolved in the image, and the full-width half maximum of a single line is estimated to be 35 nm. challenging goal toward 1 MHz regime hopefully aided by on-chip
digital data compression techniques to improve frame rates.30,31
Photon-counting detectors, such as the one used in this study, may
suffer from count rate saturation with upgraded sources; thus,
charge-integrating or hybrid counting/integrating detectors will
become favored in this case. Meanwhile, continuous developments of
instrumentation and methodology can relax requirements of such
detectors. For instance, position smearing caused by a slow detector
in fly scanning can be alleviated by the idea of “pseudo step scanning”
using hybrid continuous scanning,11 while the use of a highly diverg-
ing beam17 and/or semi-transparent beam stop32 can reduce demands
on detector dynamic range. On the other hand, with tremendous data
volume provided by high-speed scan, the reconstruction process is
becoming the major bottleneck in practice. Therefore, future work
will also focus on improving the reconstruction speed as well as
implementing advanced algorithms in high-performance computing
environments. of neurons. The high-resolution imaging with fast data acquisition
speed can also facilitate a 3D high-resolution characterization of bulk
materials and electronic devices via either computed tomography or
laminography.2 The current imaging speed is not limited by the instru-
ment’s scan speed but rather by the x-ray flux because the achieved
resolution is fundamentally limited by the x-ray dose on the sample. ABSTRACT As many synchrotron x-ray sources, including the APS, are being
upgraded in the next decade to provide at least two orders of magni-
tude increase in coherent flux, we expect that the imaging speed will
increase correspondingly, and fly scan ptychography will gain more
interest in materials science. The increasing speed of fly scan ptychog-
raphy at next-generation sources also requires a sustained effort in
developing fast detectors that can keep up with scanning rates and
withstand such high flux densities. Some detectors developed for x-ray
free-electron lasers are already achievable at 5–10 MHz but only for a
limited number of frames in burst mode.27 Detectors with a continu-
ous frame rate of tens of kHz are currently being developed,28,29 with a 24 October 2024 05:18:07 24 October 2024 05:18:07 FIG. 4. Influence of the orthogonal probe relaxation (OPR) technique on large FOV ptychographic reconstruction of an integrated circuit. (a) Phase image cropped from a small
FOV (14 105 lm2) reconstruction using 1 OPR mode. (b)–(d) Cropped reconstruction from a full tile scan (205 105 lm2) using 1, 3, and 5 OPR modes, respectively. The
streaky artifacts due to beam variations are reduced as more OPR modes are used in reconstruction. FIG. 4. Influence of the orthogonal probe relaxation (OPR) technique on large FOV ptychographic reconstruction of an integrated circuit. (a) Phase image cropped from a small
FOV (14 105 lm2) reconstruction using 1 OPR mode. (b)–(d) Cropped reconstruction from a full tile scan (205 105 lm2) using 1, 3, and 5 OPR modes, respectively. The
streaky artifacts due to beam variations are reduced as more OPR modes are used in reconstruction. 119, 124101-5 Appl. Phys. Lett. 119, 124101 (2021); doi: 10.1063/5.0067197
V
C Author(s) 2021 119, 124101-5 Applied Physics Letters scitation.org/journal/apl ARTICLE See the supplementary material for a movie showing the ptycho-
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Sandberg and Nina Weisse-Bernstein from Los Alamos National
Laboratory for preparing the second chip in this study. The
Velociprobe was supported by Argonne LDRD 2015-153-N0. This
research used resources of the Advanced Photon Source, U.S. Department of Energy (DOE) Office of Science User Facilities
operated for the DOE Office of Science by Argonne National
Laboratory under Contract No. DE-AC02-06CH11357. This work
was partially supported by the Office of the Director of National
Intelligence (ODNI), Intelligence Advanced Research Projects
Activity (IARPA) via Contract No. D2019-1903270004. The views
and conclusions contained herein are those of the authors and
should not be interpreted as necessarily representing the official
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C Author(s) 2021 Appl. Phys. Lett. 119, 124101 (2021); doi: 10.1063/5.0067197 V
C Author(s) 2021
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English
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The bottom–up approach to defining life: deciphering the functional organization of biological cells via multi-objective representation of biological complexity from molecules to cells
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Frontiers in physiology
| 2,013
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cc-by
| 9,611
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The bottom–up approach to defining life: deciphering
the functional organization of biological cells via
multi-objective representation of biological
complexity from molecules to cells Sathish Periyasamy 1,2,3*, Alex Gray 1 and Peter Kille 2 Sathish Periyasamy 1,2,3*, Alex Gray 1 and Peter Kille 2
1 Cardiff School of Computer Science and Informatics, Cardiff University, Cardiff, UK
2 Organisms and Environment Division, Cardiff School of Biosciences, Cardiff University, Cardiff, UK
3 Department of Bioinformatics, King Abdullah International Medical Research Center, National Guard Health Affairs, Riyadh, Saudi Arabia Sathish Periyasamy 1,2,3*, Alex Gray 1 and Peter Kille 2
1 Cardiff School of Computer Science and Informatics, Cardiff University, Cardiff, UK
2 Organisms and Environment Division, Cardiff School of Biosciences, Cardiff University, Cardiff, UK
3 Department of Bioinformatics, King Abdullah International Medical Research Center, National Guard Health Affairs, Riyadh, Saudi Arabia Sathish Periyasamy 1,2,3*, Alex Gray 1 and Peter Kille 2 Sathish Periyasamy 1,2,3*, Alex Gray 1 and Peter Kille 2
1 Cardiff School of Computer Science and Informatics, Cardiff University, Cardiff, UK
2 Organisms and Environment Division, Cardiff School of Biosciences, Cardiff University, Cardiff, UK
3 Department of Bioinformatics, King Abdullah International Medical Research Center, National Guard Health Affairs, Riyadh, Saudi Arabia 1 Cardiff School of Computer Science and Informatics, Cardiff University, Cardiff, UK
2 Organisms and Environment Division, Cardiff School of Biosciences, Cardiff University, Cardiff, UK
3 Department of Bioinformatics, King Abdullah International Medical Research Center, National Guard Hea In silico representation of cellular systems needs to represent the adaptive dynamics
of biological cells, recognizing a cell’s multi-objective topology formed by temporally
cohesive intracellular structures. The design of these models needs to address the
hierarchical and concurrent nature of cellular functions and incorporate the ability to
self-organize in response to transitions between healthy and pathological phases, and
adapt accordingly. The functions of biological systems are constantly progressing, due to
the ever changing demands of their environment. Biological systems meet these demands
by pursuing objectives, aided by their constituents, giving rise to biological functions. A biological cell is organized into an objective/task hierarchy. These objective hierarchy
corresponds to the nested nature of temporally cohesive structures and representing
them will facilitate in studying pleiotropy and polygeny by modeling causalities propagating
across multiple interconnected intracellular processes. Although biological adaptations
occur in physiological, developmental and reproductive timescales, the paper is focused on
adaptations that occur within physiological timescales, where the biomolecular activities
contributing to functional organization, play a key role in cellular physiology. HYPOTHESIS AND THEORY ARTICLE
bli h d 18 D
b
2013 published: 18 December 2013
doi: 10.3389/fphys.2013.00369 The bottom–up approach to defining life: deciphering
the functional organization of biological cells via
multi-objective representation of biological
complexity from molecules to cells The bottom–up approach to defining life: deciphering
the functional organization of biological cells via
multi-objective representation of biological
complexity from molecules to cells The paper
proposes a multi-scale and multi-objective modeling approach from the bottom–up by
representing temporally cohesive structures for multi-tasking of intracellular processes. Further the paper characterizes the properties and constraints that are consequential to
the adaptive dynamics in biological cells. Reviewed by: Reviewed by:
Osbaldo Resendis-Antonio,
Universidad Nacional Autónoma de
México, Mexico
David L. Robertson, University of
Manchester, UK Reviewed by:
Osbaldo Resendis-Antonio,
Universidad Nacional Autónoma de
México, Mexico David L. Robertson, University of
Manchester, UK *Correspondence: *Correspondence:
Sathish Periyasamy, King Abdullah
International Medical Research
Center, National Guard Health
Affairs, Bioinformatics, Khashm Al
Ann, MC 1515’, PO Box 22490,
Riyadh 11426, Saudi Arabia
e-mail: periyasamysa@ngha.med.sa;
sathish.periyasamy@gmail.com Keywords: multi-objective, cohesive structures, temporal modeling, cellular physiology, adaptation Edited by: Jean-Marc Schwartz, University of
Manchester, UK Jean-Marc Schwartz, University of
Manchester, UK HYPOTHESIS AND THEORY ARTICLE
published: 18 December 2013 Keywords: multi-objective, cohesive structures, temporal modeling, cellular physiology, adaptation INTRODUCTION Hence, the paper states how
diverse biomolecular activities are groped based on identifying
the functionally cohesive structures and measuring the perfor-
mance of these structures to assess their functions. Further, the
concept of Pareto Optimal Frontier is used to assess the level of
organization among numerous conflicting biochemical activities. Biological adaptations occur within physiological, develop-
mental and reproductive timescales. Although this research is
focused on physiological timescales, it is useful to understand how
biological systems are organized to adapt across these timescales,
i.e., how information regarding performance between biolog-
ical systems and the environment are exchanged across these
scales. Biological systems dynamically adapt to multiple objectives
concurrently. This is a process facilitated by their constituents
forming spatial and temporal cohesive structures. The objectives
and cohesive structures of biological systems are constantly adapt-
ing due to the ever changing demands of their environment. These objectives are imposed by the environment, which consists
of physical, chemical and biological elements of the individual
biological systems. Biological systems are driven toward meeting
the imposed objectives aided by their constituents, giving rise to
biological processes which are perceived as biological functions. Biological tasks emerge through the formation and development
of spatially and temporally cohesive structures when pursuing
these objectives. Due to the nested nature of cohesive struc-
tures, the biochemical tasks appear to be concurrent and mutually
dependent, which leads to the manifestation of pleiotropy and
polygeny in cells. Pleiotropy is defined as one gene giving rise
to two or more unrelated phenotypic traits (Stearns, 2010) and
polygeny is defined as a phenotypic trait controlled by more than
one gene. These objectives, on which the selective pressure is
imposed, are eventually organized into a spatially and temporally
cohesive hierarchy forming the biological organization strata,
where the amount of time required in pursuing the objectives
increases, when moving up the hierarchy (Schnell et al., 2007;
Noble, 2008; Dada and Mendes, 2011). The cellular activities are
hierarchically organized into various basic tasks, which merge
to form the complex and greater tasks of the cell, thus reflect-
ing the nested nature of intracellular cohesive structures. Hence,
managing competition and cooperation of these structures will
require coordination via hierarchical regulation (Westerhoff and
Palsson, 2004) that includes transcriptional, post-transcriptional,
translational and post translational regulators. The paper also evaluates the properties and constraints that are
consequential to the adaptive dynamics in biological cells. INTRODUCTION perception at their system level, that one outcome is qualitatively
better than another at this level, but cannot determine whether
this will be true at higher levels and thus cannot determine, if
it will lead to an absolute fitness specific to the requirements. Moreover, these mechanisms don’t inherently know what is the
optimum solution, or even if one exist (Eberhart and Shi, 2007). Although each of the objectives will not have an optimal solution,
the solutions observed will ultimately satisfy the requirements
in a sustained biological equilibrium. However, challenges to
this equilibrium, which exceed the capacity of a specific system
to compensate, will create a pathological process, resulting in
the multi-objective re-organization manifested as biological
adaptation. Further, pathological processes have become an
integral part of biological adaptation due to failure in achieving
objectives caused by unanticipated constraints. Moreover there
will be multiple biological solutions, which represent different
“trade-offs” among the objectives and constraints, associated
with the biological system. The preferred solution will vary
depending on changing requirements (i.e., criteria) exerted by Biomolecules
give
rise
to
living
entities
by
self-
ordering/organizing into coordinated biochemical activities,
whose ultimate outcome is the production of life (Abel, 2011). The multi-dimensional problem that needs to be resolved,
involves balancing the myriad of biological activities at various
levels of biological organization to result in a viable living system. However, a suitable resolution must exist within the “organi-
zational space” defined by the constraints of each constituent
biomolecule and their activities. At the cellular level, solutions
to the adaptive requirement emerge from the simultaneous
adaptation of multiple and mostly conflicting objectives formed
by competition amongst temporally cohesive structures (i.e.,
functional units). This is subjected to various control mech-
anisms (forms of feedback and reinforcement mechanisms
which facilitate self-organization and selection, respectively),
which act as regulators in space and time. These regulators can
exchange information directly via feedbacks and indirectly via
reinforcement mechanisms. These control mechanisms have a December 2013 | Volume 4 | Article 369 | 1 www.frontiersin.org www.frontiersin.org The bottom–up approach to defining life Periyasamy et al. the biological system’s dynamic environment. Biological cells are
mostly driven toward meeting the objectives which are temporal
in nature and strive to explore and exploit solutions with respect
to time within the bounds of the spatial constraints. theoretical frameworks through computational models to study
complex biological phenomena. INTRODUCTION Further
the paper describes the multi-objective nature of biological
systems, the constraints involved in pursuing these objectives,
and the hierarchical nature of biological systems by simpli-
fying cellular complexity via the construction/deconstruction
of basic objectives/tasks into mutually dependent complex
global tasks. To model uncertainty, concurrency, self-organization
and emergence in intracellular biochemical activities, a suit-
able modeling formalism will have to be utilized. The col-
lective dynamics approach aided by multi-objective topology,
has the ability to represent concurrency and functional hier-
archy. The model captures the diverse activities of functional
products that occur concurrently in space and time, and avoid
the combinatorial explosion inherent in network representa-
tions (Takahashi et al., 2005; Felix and Wagner, 2006; Kitano,
2010). Multi-objective topology provides a concurrent and hier-
archical view of biological systems, whereas network topol-
ogy provides a sequential and horizontal view of biological
systems. Frontiers in Physiology | Systems Biology THE CHARACTERISTICS AND PROPERTIES OF BIOLOGICAL
C Since systems biology addresses the
missing links between molecules and physiology (Bruggeman
and Westerhoff, 2007), it has to integrate experimentation and December 2013 | Volume 4 | Article 369 | 2 Frontiers in Physiology | Systems Biology The bottom–up approach to defining life Periyasamy et al. the biological organization strata, in terms of perceivable and spa-
tially cohesive entities (molecules, organelles, cells, organs, and
individuals), their applicability in modularizing the intracellular
activities of functional products into functional units and cellu-
lar processes is doubtful. Intracellular functions that lack spatial
boundaries are temporal phenomena, which emerge from the
causally linked temporal cohesive structures. A logical approach
to simplify cellular processes, is by constructing/deconstructing
these processes into objectives/tasks, on which the selective pres-
sure is imposed. Further, the modularity is concealed, due to
mutual dependency amongst the higher level tasks. The effects
of mutual dependency amongst the objectives/tasks, which occur
due to the presence of degenerate and redundant factors, and
the convergence and divergence of causal effects of biomolecu-
lar activities, adds to the complexity of modularizing biochemical
activities. Mutual dependencies complicate the process of iden-
tifying the degree of orthogonality (i.e., independence), which
facilitates the modularization from molecular resolution to cel-
lular resolution via deconstruction of objectives into basic and
molecular tasks required to pursue them. The emergence of global
cellular behavior is a result of functional products, which are
specialized to pursue their intended tasks. Further, acts of coop-
eration, competition and coordination emerge from the collective
dynamics of functional products. These actions are not mutu-
ally exclusive, rather they contribute concurrently to the pursuit
of various collective tasks of the cell and higher multi-cellular
organizations. The criteria used to modularize the interactions
among functional products, are based on performance/fitness
interactions, which emerge out of competition and cooperation
among functional products. This is the mechanism by which evo-
lution formed and evolved collaborative groups (Axelrod and
Hamilton, 1981; Axelrod, 2006; Nowak, 2006) containing one
or more species of functional product. These functional prod-
ucts within a group cooperate with each other for a common
objective/task. In physiological timescales competitive and coop-
erative adaptation among various biomolecular species is ubiqui-
tous amongst their activities. While inverse performance/fitness
interaction exists between the competing biomolecular species,
positive performance/fitness interaction will exist among cooper-
ating biomolecular species. THE CHARACTERISTICS AND PROPERTIES OF BIOLOGICAL
C Direct and inverse fitness interactions
can reveal the organization of the objective hierarchy in order
to construct/deconstruct the tasks between molecular resolution
and cellular resolution. Further this relationship is appropriate
to model the impact amongst various species of biomolecule’s
activity on the intracellular and cellular level processes, as a
whole. Gene Space
Protein Space
Gene 1
Gene 2
Gene 3
Gene 4
Protein 1
Protein 4
Protein 3
Protein 2
Complex
Metabolite A
Metabolite D
Metabolite C
Metabolite B
Metabolite Space
Resource Flow
Physical interaction
Influence
FIGURE 1 | The autocatalytic cycles traverse across gene space to
metabolite space. Biomolecular activities occurring within the gene,
transcript, protein and metabolite space contribute to the organization of a
biological cell. These activities form various causalities (i.e., causal links
amongst events), which form the organizational closure of the cell. Gene Space
Protein Space
Gene 1
Gene 2
Gene 3
Gene 4
Protein 1
Protein 4
Protein 3
Protein 2
Complex
Metabolite A
Metabolite D
Metabolite C
Metabolite B
Metabolite Space Metabolite Space Metabolite B Metabolite A Resource Flow
Physical interaction
Influence FIGURE 1 | The autocatalytic cycles traverse across gene space to
metabolite space. Biomolecular activities occurring within the gene,
transcript, protein and metabolite space contribute to the organization of a
biological cell. These activities form various causalities (i.e., causal links
amongst events), which form the organizational closure of the cell. systems are organizationally closed, they are thermodynamically
open systems that exist far from thermodynamic equilibrium by
exchanging matter and energy with their environment (Bachmair
et al., 1986; Van Regenmortel, 2007; Yafremava et al., 2013). For example, at the organizational level various resources (e.g.,
metabolites) are consumed and produced by various enzyme
mediated reactions, and if this is visualized by comparing every
resource against every reaction in a matrix, the complex depen-
dencies between enzyme mediated reactions at the thermody-
namic level can be observed. As a physical system the laws
of thermodynamics direct cellular metabolism (Wolfe, 2001;
Alberty, 2010), as a chemical system competition, cooperation
and coordination stabilizes cellular metabolism and as living sys-
tem adaptability, robustness and efficacy ensure persistence of the
system. THE CHARACTERISTICS AND PROPERTIES OF BIOLOGICAL
C Modeling and simulating the multi-level dynamics of biologi-
cal systems are one of the most complex endeavors in systems
biology studies, due to the fact that biological processes con-
sist of multi-level spatial and temporal scales (Bassingthwaighte
et al., 2006; Schnell et al., 2007; Noble, 2008). Living systems
are the most complex systems known in nature, which is due to
the multiple levels of constraints associated with them. Living
systems are constrained by physical laws, like non-living sys-
tems and also have additional levels of constraints associated
with complex biological processes (Abel, 2011). These two levels
constitute the fundamental and organizational principles, which
are required to model the complexity of biological cells from
the bottom up (Kitano, 2007). When considering the relation-
ship between individual biomolecules and the cells to which
they contribute, we can identify their resemblance to complex,
dynamic, self-organizing, adaptive, concurrent, robust, reactive
(Efroni et al., 2003) and proactive systems (Michener et al., 2001). Some typical properties of complex systems include dynamism,
emergent behavior, non-linearity, multi-stability, nested organi-
zation, feedbacks (i.e., horizontal and vertical) and scale free-
ness (Dubitzky, 2006). Biomolecular activities occurring within
the gene, transcript, protein and metabolite space contribute
to the organization of a biological cell. These activities form
various causalities (i.e., causal links amongst events), which
form the organizational closure of the cell (Shapiro, 2007) (see
Figure 1). This closure is different from thermodynamic clo-
sure, which is observed in isolated systems. Although biological The paper evaluates the mechanisms of biological adaptation
and specifies two categories of goals/objectives, which define these
tasks and drive the adaptive process. The aim is to elicit require-
ments for in silico representations of the adaptive dynamics
from molecules to cell. An appropriate systems biology approach
(Bruggeman and Westerhoff, 2007) will have to be adapted to
model the self-organization of biomolecular activities in order
to study the emergence of intracellular functional organization. Since it requires a mechanism based explanation, it has to be
mechanistically modeled using a bottom–up approach which
integrates molecular level information. Modeling at the level
of molecular resolution will require representing the molecu-
lar properties together with the spatial and temporal constraints
of the cellular environment. TASK FORMATION AND INTEGRATION OF FUNCTIONAL
UNITS IN CELLS Modularization is a way of simplifying complex systems into
a set of simple systems using functional abstractions, which
constitute the functional units. To this end various criteria for
simplifying the complex biochemical activities of life have been
proposed, using modularity to encapsulate biological complex-
ity. One such modularity is based on the cellular component
and biological processes, which is not compatible in represent-
ing the evolution of different functional products and gene
families, as functional products are “co-opted” for new tasks
beyond what is believed to be their original role (Hodgkin, 1998). Moreover, the long standing question is to what extent the con-
cept of modularity introduced for engineered systems, provides
realistic and useful abstractions for systems organized by bio-
logical adaptation from physiological to reproductive timescales
(Szallasi et al., 2006). Although modularity can be observed in Temporal networks are formed due to convergence and diver-
gence of causalities. The interaction between a common tran-
scription factor and various cis regulatory sites, is an indication
of divergence in causality. The presence of divergence points in
biochemical networks is an indication of competition for a com-
mon substrate and this leads to conflicts among higher level
cellular tasks/objectives. Shared resources are a major cause of
conflicts in intracellular organization. A basic task or a cooper-
ative module (functional unit) in biochemical activities is defined
as a group of one or more species of functional product col-
laborating for a common objective. These modules will have December 2013 | Volume 4 | Article 369 | 3 www.frontiersin.org The bottom–up approach to defining life Periyasamy et al. the characteristic, that every functional product’s performance
will have a beneficial effect on the other and the whole group’s
performance. The absence of any one member species of the
group will have no value for the existence of the remaining
member species of the group (all or nothing phenomena). In
molecular complexes the participating biomolecular species form
cooperative groups. In the context of metabolic networks, this
is a pathway which exists between two junction points. This
will be the basic functional unit, from which higher levels of
functional units are assembled. The fitness, at the functional
product level, is a function of its efficiency and stability. Efficiency
depends on the product’s affinity for interaction, and the time
and energy requirements for its activity. TASK FORMATION AND INTEGRATION OF FUNCTIONAL
UNITS IN CELLS An improved perfor-
mance for one competing unit implies a decreased performance
for another unit. Hence this has an inhibitory effect on other
competing functional units. Further, biomolecules are forced to
sacrifice their own efficiency for the betterment of cellular orga-
nization. This inverse performance between two levels can only
occur in the presence of conflicting objectives, and the conflicting
units will impose immense selection pressure on their regulatory
mechanism. FIGURE 2 | The outcome of the law of sufficiency is diversity in
solutions, where the fittest solutions converge into an attractor basin. The diversity in outcomes could give rise to the diverse solutions observed
in nature. If perfection is the norm, there will be no room for deviation or
defects in the outcomes, eventually leading to uniformity in solutions. FIGURE 2 | The outcome of the law of sufficiency is diversity in
solutions, where the fittest solutions converge into an attractor basin. The diversity in outcomes could give rise to the diverse solutions observed
in nature. If perfection is the norm, there will be no room for deviation or
defects in the outcomes, eventually leading to uniformity in solutions. behavior results from the collective organization of biomolecules
and their interactions in space and time. Each biomolecule is
simply reacting in a determinate way to stimuli and in-turn
responding by stimulating other biomolecules to regulate activ-
ities amongst them. The uncertainty of when, where and what
inter molecular interactions may occur is a stochastic process. However, molecular interactions that lead to meaningful out-
come (i.e., either positive or negative for a cell) are relatively
deterministic and this is determined by the affinity of the molec-
ular domains to other molecules. For example DNA or protein
sequences will determine the potential interactions that can hap-
pen in their functional forms. However, when and where these
potential interactions may happen is a stochastic process. Various
activities are required to provide system wide responses to per-
turbations. However these activities have limitations, and have to
be regulated in terms of when, where and what activities should
occur to provide timely responses to perturbations in a con-
strained environment. As a result, various stages of regulation
have evolved in anticipation of perturbations, which facilitate
transformation of the activities of functional products, which
are merely reactive to a collectively proactive organization. CELLULAR LEVEL PROPERTIES The cellular features reflect on the intracellular goals and objec-
tives that are manifested at the biological and chemical and
physical levels. Adaptability, robustness and efficacy of biological
systems govern the process at the biological level, and competi-
tion, cooperation and coordination among functional products
govern processes at the chemical level. Further, thermodynamics,
special and temporal constrains govern processes at the physical
level. These three levels which comprise the physical, chemical
and biological processes operate concurrently by influencing each
other during functional organization. TASK FORMATION AND INTEGRATION OF FUNCTIONAL
UNITS IN CELLS The
presence of higher stages of regulation such as translational and
post-translational regulation, facilitate the anticipation of recur-
ring perturbations, which also improves the performance of
the cell. CELLULAR LEVEL PROPERTIES
h
ll l
f
fl
h Frontiers in Physiology | Systems Biology December 2013 | Volume 4 | Article 369 | 4 ADAPTABILITY In the context of biological adaptation which
spans from physiological to reproductive timescales, function is
defined as the progression along some causality, to the goal or
successful outcome (Dusenbery, 1992). Some of the factors that
contribute to robustness are redundancy and degeneracy, plastic-
ity and concurrency. Degeneracy (Edelman and Gally, 2001; Felix
and Wagner, 2006) is the ability of different solutions to per-
form the same function, such as the enzyme’s performance can be
maintained by altering its processing time or abundance. In con-
trast Redundancy occurs, when the same function is performed
by identical solutions. Also redundancy refers to the degree of
replica. One of the outcomes of degeneracy is the pleiotropic
(Hodgkin, 1998; Lobo, 2008) and polygenic nature of the func-
tional products, where they positively and negatively influence
multiple cellular functions, concurrently. The term functional
product is currently more favored, than the term gene product,
due to changing views of genes (Gerstein et al., 2007). Although
degeneracy provides flexibility (many options) for the cell to
arrive at a solution (i.e., possibly accelerate adaptation), it adds
to the complexity in recognizing the contributions and compen-
satory adjustments made by different options to the solution. Plasticity is the ability of the system to readily adapt to new, differ-
ent, or changing requirements (Garnier et al., 2007). Concurrency
manifests in the existence of redundant and specialized biological
entities, such as diverse biomolecular species and cell types. The
effects of robustness are sensitiveness (fluctuation of performance
to perturbations) and adaptability. Robustness facilitates adapt-
ability by accumulating variations whilst maintaining a functional
phenotype(Wagner, 2008), such as silent or neutral mutations in
the genome. Response
Behaviour
Passive
Reactive
Proactive
Probabilistic
Deterministic
Biomolecule
Biological cell
FIGURE 3 | The nature of biomolecule and the biological cell. From the
reactive and deterministic nature of biomolecular activities, the complex,
non-deterministic and proactive cellular behavior will emerge. Biomolecule Behaviour FIGURE 3 | The nature of biomolecule and the biological cell. From the
reactive and deterministic nature of biomolecular activities, the complex,
non-deterministic and proactive cellular behavior will emerge. FIGURE 3 | The nature of biomolecule and the biological cell. From the
reactive and deterministic nature of biomolecular activities, the complex,
non-deterministic and proactive cellular behavior will emerge. to the complex global tasks of the cell and tracing causalities
via causal links amongst biomolecular activities. ADAPTABILITY Adaptation is a learning process which is associated with
Intelligence. The most appropriate definition for intelligence, that
covers all computational intelligence approaches, is “the capa-
bility of a system to adapt its behavior to meet its goals in a
range of environment” (Fogel, 2006). The ability to learn or
adapt is one of the hallmarks of intelligent systems. This can
also be witnessed in biological cells, where cellular intelligence
emerges as an organizational/system level property. The mecha-
nism that drives this intelligent behavior is reinforcement adap-
tation, which is ubiquitous to biological systems. Reinforcement
adaptation is facilitated via a critic, which follows a general prin-
ciple that serves to guide the adaptive process. These phenom-
ena is observed in cooperative binding (Stefan and Le Novère,
2013) of biomolecules and transcriptional memory (Flintoft,
2007) where the level of response varies which prior stimulus. Biological systems can be assumed to follow the law of suffi-
ciency, which states that if a solution is good enough, fast enough,
and cheap enough, it is sufficient (Eberhart and Shi, 2007). Hence the suitability of a solution (i.e., fitness) is not an abso-
lute measure, rather it is a relative measure (i.e., how good the
solution is relative to other solutions). Figure 2 shows the out-
come of the law of sufficiency. The proactive nature of cellular From a reductionist perspective, the organizational prop-
erties evident at the cellular level such as efficacy, robust-
ness and adaptability, cannot be perceived by characterizing
the biomolecules. In the context of reductionism, the cell is
perceived tangibly as its constituent biomolecules migrating,
physically interacting and causing the density of biomolec-
ular populations to fluctuate in space and time. However,
this perception is misleading, since the cell is a collective of
autonomous biomolecules exhibiting cohesiveness only at a holis-
tic level. Hence, observing individual biomolecular activities
will not provide vital insights about cellular level properties. Moreover the intra-organizational performance of a cell can-
not be directly observed or empirically measured, because this
requires analysis of the performances of biomolecular species
via their activities, analysing the contributions of basic tasks December 2013 | Volume 4 | Article 369 | 4 Frontiers in Physiology | Systems Biology The bottom–up approach to defining life Periyasamy et al. biomolecular activities and the availability of resources, which the
cell cannot produce. EFFICACY Efficacy refers to the effectiveness in responding to the
intracellular objectives. The intracellular organization has the
ability to efficiently adapt within the bounds of biological equi-
librium and gracefully degrade its performance, when func-
tional/performance requirements, perturbation or uncertainty
levels demand more than the capacity of robustness. Hence, not
only does the biological cell, which is constrained by its genome,
maintain performance within the capacity of its robustness, but
it also has the ability to reconfigure the responsiveness at the
genome level to meet the performance demands of the dynami-
cally changing capacity of robustness. The fitness/effectiveness of
solution in the internal cellular organization is constantly being
evaluated and it is the measure of performance with respect to
an objective. That is how well an intended task is being ful-
filled. Although every functional product has a purpose (intended
activity), ultimately their contribution to the overall performance
of the cellular organization, which in turn contributes to the
reproductive success, is essential to understanding of their impact
from the bottom up. These functional products will have pos-
itive contributions to sustaining biological equilibrium, when
their activities are performed when required. However, when
their activities are silenced or performed when not required, it
can have a negative contribution to sustaining biological equi-
librium. This is due to the fact that biomolecular activities are ADAPTABILITY At an orga-
nizational level, cellular behavior can only be probabilistically
determined, since causalities occur due to concurrent biomolecu-
lar activities. Figure 3 shows the deterministic and reactive nature
of biomolecules giving rise to a cellular organization, which is
probabilistic and proactive in nature. The deterministic nature
of biomolecular behavior can produce coordinated behavior
amongst biomolecules, causing reproducible or rhythmic intra-
cellular organizational behavior, in the face of perturbation and
uncertainty. ROBUSTNESS Robustness is an organizational/system level property (Kitano,
2004, 2010), which is defined as “the ability to maintain per-
formance in the face of perturbation and uncertainty” (Stelling
et al., 2004). The difficulty of comprehending how robustness
is accomplished at the cellular or molecular level is due to its
intimate link with the complexity of cellular systems (Stelling
et al., 2006). However, articles about robustness at these lev-
els have just begun to emerge (Hartman et al., 2001; Felix and
Wagner, 2006; Wagner, 2008; Yafremava et al., 2013). An impor-
tant realization is, that robustness is concerned with preserving
the functions of a system rather than system states. This distin-
guishes robustness from stability or homeostasis (Kitano, 2007). Homeostasis is a process, that preserves the state of the system
rather than its function. Robustness determines the boundaries
(see Figure 4) of the multi-dimensional problem (i.e., pertur-
bation and uncertainty) and the function (i.e., performance)
space, in which biological equilibrium can exist (Periyasamy et al.,
2009). Perturbation defines the extrinsic (environmental) stimu-
lus and intrinsic (programmed) stimulus. Uncertainty defines the
stochastic nature of the constraints, such as the intervals between December 2013 | Volume 4 | Article 369 | 5 www.frontiersin.org The bottom–up approach to defining life Periyasamy et al. boundaries in 2-dimensional problem space. (B) Performance—The existence
of Pareto optimal frontier (The region of high fitness) boundaries in
2-dimensional function space. FIGURE 4 | The formation of robustness and the associated biological
equilibrium. (A) Perturbation and Uncertainty- The existence of the normal
system phases (biological equilibrium within the bounds of robustness)
boundaries in 2-dimensional problem space. (B) Performance—The existence
of Pareto optimal frontier (The region of high fitness) boundaries in
2-dimensional function space. boundaries in 2-dimensional problem space. (B) Performance—The existence
of Pareto optimal frontier (The region of high fitness) boundaries in
2-dimensional function space. boundaries in 2-dimensional problem space. (B) Performance—The existence
of Pareto optimal frontier (The region of high fitness) boundaries in
2-dimensional function space. FIGURE 4 | The formation of robustness and the associated biological
equilibrium. (A) Perturbation and Uncertainty- The existence of the normal
system phases (biological equilibrium within the bounds of robustness) depends on the predictability of biomolecular activates, which
have to significantly dominate uncertain activities. Due to the
uncertain nature of the cellular environment, cellular physiology
is driven by the most probable molecular activities that occur,
based on the constraints in their local environment. CONSTRAINTS WITHIN CELLS The distinction between objectives and constraints in biological
cells is that when the constraints are subjected to selection pres-
sure they eventually become one of the focuses of the system and
will transform into a goal/objective which will have to be pursued
by the system for its persistence. Biological cells mostly pursue
objectives which are temporal in nature and strive to explore
and exploit solutions with respect to time within the bounds of
the spatial and thermodynamic constraints. Further, uncertainty
(Pearson, 2008; Johnston, 2012; Viney and Reece, 2013) adds to
the constraints in meeting the objectives of the cell. The stability of functional products also plays a major role
in the self-organizing process of the cell, because it deter-
mines the functional ability of these molecules. The main
factors, which affect the stability of molecules, are temper-
ature, pH and vulnerability to destruction (Plotkin, 2011). Proteins are the molecular machines of the cell and they have
evolved to be the major contributors to the organizational
dynamics of the cell. Proteins exist in various stages of the
lifecycle (Belle et al., 2006) and differ noticeably in their half-
lives (Bachmair et al., 1986) (see Table 1), which reflects on
their stability. While some are destructed very rapidly (typi-
cally enzymes), others are very stable (mechanical proteins). In Proteins, the half-life is determined to a large extent by
its amino-terminal residue, which acts as a signal for stabil-
ity and has been retained over the course of evolution. There
is a complex interplay between protein degradation, its reg-
ulation and other determinants of protein metabolism (Saric
and Goldberg, 2006). The cellular organization has adopted this ROBUSTNESS Constraints
reduce uncertainty by guiding the system. The main constraints
for molecular activities include the cost of the activity in terms
of time and energy (i.e., enzyme turnover cycle), the spacetime
interval amongst the activities (i.e., invariant interval between
the two activities/events, which takes into account both spa-
tial separation and their temporal separation), and the stability
and availability of reactants (biomolecules) to participate in the
activity. The uncertainty involved in spacetime intervals amongst
activities, depends on the probability at which respective reac-
tants meet. Biomolecules utilize three kinds of diffusion search
spaces. These are one dimensional (along DNA), two dimen-
sional (within the membrane) and three dimensional (in the
cytosol) to find their counterparts which initiate the activi-
ties. However, the cost of biomolecular activities has been the
major constraint (limiting factor) in cellular physiology, since
the amount of time required for various biomolecular activi-
ties, significantly dominates the time requirements for diffusion
mediated encounters. directional/vectorial in terms of their causality (cause and effect),
which contributes to the transformation of the cellular organi-
zation’s equilibrium state, either toward or away from equilib-
rium, depending on the phase of the cell. Hence the purpose
of the functional product in the context of its higher organiza-
tion (cell) depends on the circumstance, in which the activities
are performed. In a normal system, various feedback mecha-
nisms formed by regulatory switches, which span from tran-
scriptional to post translational level, ensure the activities occur
in appropriate circumstance to ensure effectiveness of cellular
functions. CONSTRAINTS WITHIN CELLS
h d
b
b Frontiers in Physiology | Systems Biology HIGHLY DESTABILIZING Various regulatory switches have evolved to self-organize the
cellular environment. While some switches utilize little or no
energy (e.g., binding of signaling molecules), others require
chemical modifications using high energy bonds (e.g., chemi-
cal modifications mainly by phosphate groups and other groups
such as acetyl, methyl and adenyl). Activities of functional prod-
ucts are orchestrated via various regulatory mechanisms which
range from transcriptional regulation (genetic level), through
post-transcriptional regulation, translational regulation (tran-
script level) and post-translational regulation (protein level). While transcriptional regulation provides slow and globalized
cellular responses, post-translational regulation provides rapid
and localized cellular responses. Transcriptional response is the
most time and energy consuming process, since genetic infor-
mation has to be transcribed and mostly translated to produce
a functional product. In contrast, post-translational response is
the least time and energy consuming process, since the functional
product is simply switched between an active and inactive state. Further transcriptional regulating is relatively centralized and sta-
tionary, while the remaining regulatory mechanisms are mobile
and provide rapid, distributed and localized regulation within
the cellular environment. Regulations facilitate in the timing of
functional products’ activities. Appropriate timing of activities is
essential, because its impact depends on the phenotypic state of
the cell. While some are destructed very rapidly (typically enzymes), others are very sta-
ble (mechanical proteins). In Proteins the half-life is determined to a large extent
by its amino-terminal residue, which acts as a signal for stability and has been
retained over the course of evolution. susceptibility of biomolecular degradation as non-specific nega-
tive feedbacks, which contribute to the internal organization of
the cell. THE IMPACT OF TIME AND ENERGY The role of energy in biological adaptation that spans from
physiological to reproductive timescales has been emphasized
in “thermoeconomics,” as the productivity, efficiency and prof-
itability of various mechanisms for capturing and utilizing
available energy to build biomass and do work (Corning,
2002). In metabolism there is a net energy gain in catabolic
activities, and a net energy loss in anabolic activities. In bio-
chemical systems, the energy released by catabolism is uti-
lized to drive the synthesis of Adenosine Tri Phosphate (ATP)
(known as the currency of energy), which in turn is used
for anabolism. Since ATP is released to a common pool and
used as a currency the cells have the flexibility to utilize it
for any activity that requires it. To facilitate this enzymes play
a crucial role in metabolism, because they drive biologically
desirable but thermodynamically unfavorable reactions by cou-
pling them to favorable ones. The self-organization processes in
cells are non-spontaneous, because energy is required to pro-
duce various functional products to maintain order in the cells
(see Figure 5). www.frontiersin.org UNCERTAINTY WITHIN CELLS FIGURE 5 | The role of metabolism in cellular homeostasis. Various
d
f bi l
i
l
hi h h
d
i
i FIGURE 5 | The role of metabolism in cellular homeostasis. Various
steady states of biological systems, which have emerged to maintain
biological equilibrium far from thermodynamic equilibrium, attract
non-spontaneous processes to increase order, whereas thermodynamic
equilibrium attracts spontaneous processes to decrease order. The
trajectory between these two biochemical system phases is controlled by
metabolism, where anabolism dominated by non-spontaneous processes,
and catabolism dominated by spontaneous processes, are coupled mostly
using ATP as a shared medium. FIGURE 5 | The role of metabolism in cellular homeostasis. Various
steady states of biological systems, which have emerged to maintain
biological equilibrium far from thermodynamic equilibrium, attract
non-spontaneous processes to increase order, whereas thermodynamic
equilibrium attracts spontaneous processes to decrease order. The
trajectory between these two biochemical system phases is controlled by
metabolism, where anabolism dominated by non-spontaneous processes,
and catabolism dominated by spontaneous processes, are coupled mostly
using ATP as a shared medium. process (Prigogine, 1984; Kauffman, 1993). If the metabolic
phase of a biological system reaches thermodynamic equilib-
rium, it will no longer be considered as a living system. The
frequency of reproduction of cells will depend on the amount
of energy utilized for reproduction. Energy utilized for other
mundane activities of the cell can reduce the frequency of
reproduction. UNCERTAINTY WITHIN CELLS The process of biological adaptation which spans from phys-
iological to reproductive timescales involves self-organization
and selection, which contributes to the optimization of bio-
logical systems. These two mechanisms, which are facilitated
by feedback and reinforcement mechanisms, should occur with
acceptable fidelity to ensure persistent behavior in biological sys-
tems. The cell’s ability to organize implies, that it has the ability
to optimize cellular activities under various perturbations and
uncertainty. The existence of uncertainty in the cellular envi-
ronment, for which the genome has no control, is due to the
presence of faulty activities, unpredictability of causal activities
inherent due to concurrency, and the downstream amplifica-
tion of activities. For example, during the course of evolution
an error frequency of about 10−4 per amino acid residue, has
been selected to produce the greatest number of functional pro-
teins in the shortest time (Stryer, 1988). The ability to organize December 2013 | Volume 4 | Article 369 | 6 Frontiers in Physiology | Systems Biology The bottom–up approach to defining life Periyasamy et al. Table 1 | Half-lives of cytosolic proteins which depend on the nature
of their amino-terminal residue (Adapted from Stryer (1988). Amino-terminal residue
Half-life
STABILIZING
Methionine
Glycine
Alanine
>20 h
Serine
Threonine
Valine
DESTABILIZING
Isoleucine
∼30 min
Glutamate
∼30 min
Tyrosin
∼30 min
Glutamine
∼10 min
Proline
∼7 min
HIGHLY DESTABILIZING
Leucine
∼3 min
Phenylalanine
∼3 min
Aspartate
∼3 min
Lysin
Arginine
∼2 min FIGURE 5 | The role of metabolism in cellular homeostasis. Various
steady states of biological systems, which have emerged to maintain
biological equilibrium far from thermodynamic equilibrium, attract
non-spontaneous processes to increase order, whereas thermodynamic
equilibrium attracts spontaneous processes to decrease order. The
trajectory between these two biochemical system phases is controlled by
metabolism, where anabolism dominated by non-spontaneous processes,
and catabolism dominated by spontaneous processes, are coupled mostly
using ATP as a shared medium. Table 1 | Half-lives of cytosolic proteins which depend on the nature
of their amino-terminal residue (Adapted from Stryer (1988). Amino-terminal residue
Half-life
STABILIZING
Methionine
Glycine
Alanine
>20 h
Serine
Threonine
Valine
DESTABILIZING
Isoleucine
∼30 min
Glutamate
∼30 min
Tyrosin
∼30 min
Glutamine
∼10 min
Proline
∼7 min
HIGHLY DESTABILIZING
Leucine
∼3 min
Phenylalanine
∼3 min
Aspartate
∼3 min
Lysin
Arginine
∼2 min Table 1 | Half-lives of cytosolic proteins which depend on the nature
of their amino-terminal residue (Adapted from Stryer (1988). CONCLUSIONS AND PROSPECTS This paper has defined adaptive dynamics of biological cells by
utilizing the multi-objective topology which deviates from the
conventional network topology based description of intracellu-
lar dynamics. Further, it has exemplified biological complexity
from molecules to cell by deciphering the functional organi-
zation of biological cells via multi-objective representation of
the intracellular adaptive dynamics. The paper has characterized
the crucial factors involved in biological adaptation occurring
in physiological time scales such as adaptability, robustness and
efficacy in the context of multi-objective topology which pro-
vides a hierarchical and concurrent view of the intracellular
dynamics. An appropriate systems biology approach will have
to be adopted to model self-ordering and self-organization of
biomolecular activities in order to study the emergence of intra-
cellular functional organization. One of the challenges is that
the organizational behavior of the cell is not something that can
be directly observed or empirically measured. Instead it needs a
group of actors to represent the functional products, represent a
set of cellular resources utilized by these functional products, cap-
ture the results of functional products’ activities and a method
to evaluate these results (Schut, 2007). This approach provides a
novel paradigm which may be harnessed in the development of
improved in silico representations of cells. The major generic chal-
lenge lying at the interface of biology and informatics is that of
generating a computational representation of a cell. We propose
that these models should represent the self-organizing nature
of competing concurrent processes organized into hierarchy;
from those activities performed by each biomolecular species, to
the tasks delivered by gene families contribute to overall cellu-
lar behavior. Models thus developed should enable simulation )
Cellular level functions are constrained by lower level func-
tions, many of which are in conflict, so various regulatory mecha-
nisms facilitate in managing these conflicts. The higher level tasks
enforce adaptive requirements for lower level tasks. Measuring
the performances of the functions within the hierarchy would
facilitate understanding of the functional organization of the
cell. Multi-objective topology provides a concurrent and hierar-
chical view of intracellular dynamics. A typical multi-objective
optimization scenario will generate a set of dominant solutions,
which forms the Pareto optimal frontier (the efficient frontier)
(Coello Coello, 2006). Optimization uses a controlled trial and
error process, where the cellular system is steered along a path
of increasing organization. MULTI-LEVEL BIOLOGICAL ORGANIZATION Hence there is a gradual tran-
sition from functions being independent at the molecular level
to mutual dependency of functions at the cellular level. The
objectives between the levels of the hierarchy are semantically
different. The tasks/objectives range from being physical to chem-
ical and biological, when traversing from molecular resolution to
cellular resolution. At molecular resolution, the tasks are phys-
ical. At the biomolecular species level, a task is represented by
its ensemble activity. At the cooperative level where basic func-
tional units emerge, the objectives are involved in completing
chemical tasks. However, at the cellular level objectives have
characteristics that are fundamental to living systems. That is
efficient use of energy, timely responses to perturbation, persis-
tence and other biological characteristics. Further these system
level tasks/objectives are not communicated directly to the con-
stituent biomolecules, rather they are self-maintained in a con-
current manner. Nature is inherently concurrent (Corrado, 2009)
and biological systems are no exceptions. Since cellular func-
tions are not maintained centrally, cells have adopted a unique
strategy to continuously realize their objectives by eliminating
obsolete information from their organization. The propensity of
biomolecular degradation by means of random or regulated pro-
cesses and collective autocatalysis provides an ideal reinforcement
adaptive mechanism for a cell. The process of biomolecular degra-
dation can eliminate obsolete biomolecular activities and so keep
cellular activities up to date, and recycle resources to maintain
cellular activities in a resource constrained and dynamic environ-
ment. These mechanisms are ubiquitous cellular processes and
are pivotal for intracellular adaptive dynamics (Periyasamy et al.,
2008). For example, aerobic and anaerobic respirations are dynamic
solutions, which have emerged to fulfill the task of liberating
energy in the presence and absence of oxygen, respectively. In
presence of oxygen, biomolecular activities pertaining to aerobic
respiration will dominate, and in absence of oxygen, biomolecu-
lar activities pertaining to anaerobic respiration tend to dominate. Hence, these two solutions, although they appear redundant
with respect to a cellular objective of releasing energy, are really
complementary (i.e., degenerate) with respect to the problem
of oxygen content (Rosenfeld, 2002). These adaptive strategies,
which are the result of collaborative efforts of biomolecules, pro-
vide complimentary solutions for cells. The adaptive mechanisms
of biological systems are destined to select appropriate anatomical
or physiological solutions (Regenmortel, 2004). Multi-level interactions deal with associating molecular level
activities to cellular level processes. MULTI-LEVEL BIOLOGICAL ORGANIZATION A biological cell is organized into an objective/task hierarchy,
which contains various cohesive levels. These tasks range from
the level of molecular species, where they are atomic and inde-
pendent of one another, to basic tasks and finally the cellular
level, where the tasks become global, mutually dependent and
biological. When more than one biomolecular species is involved
in the formation of a basic task, mutual dependency will exist This specificity has constrained and guided self-organization
in biochemical systems. The constant energy flux (energy dis-
sipation) between spontaneous and non-spontaneous processes
provides instability, which is required for the self-organization December 2013 | Volume 4 | Article 369 | 7 www.frontiersin.org The bottom–up approach to defining life Periyasamy et al. amongst the biomolecular species. Hence there is a gradual tran-
sition from functions being independent at the molecular level
to mutual dependency of functions at the cellular level. The
objectives between the levels of the hierarchy are semantically
different. The tasks/objectives range from being physical to chem-
ical and biological, when traversing from molecular resolution to
cellular resolution. At molecular resolution, the tasks are phys-
ical. At the biomolecular species level, a task is represented by
its ensemble activity. At the cooperative level where basic func-
tional units emerge, the objectives are involved in completing
chemical tasks. However, at the cellular level objectives have
characteristics that are fundamental to living systems. That is
efficient use of energy, timely responses to perturbation, persis-
tence and other biological characteristics. Further these system
level tasks/objectives are not communicated directly to the con-
stituent biomolecules, rather they are self-maintained in a con-
current manner. Nature is inherently concurrent (Corrado, 2009)
and biological systems are no exceptions. Since cellular func-
tions are not maintained centrally, cells have adopted a unique
strategy to continuously realize their objectives by eliminating
obsolete information from their organization. The propensity of
biomolecular degradation by means of random or regulated pro-
cesses and collective autocatalysis provides an ideal reinforcement
adaptive mechanism for a cell. The process of biomolecular degra-
dation can eliminate obsolete biomolecular activities and so keep
cellular activities up to date, and recycle resources to maintain
cellular activities in a resource constrained and dynamic environ-
ment. These mechanisms are ubiquitous cellular processes and
are pivotal for intracellular adaptive dynamics (Periyasamy et al.,
2008). amongst the biomolecular species. MULTI-LEVEL BIOLOGICAL ORGANIZATION These include representing
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robustness and adaptability from molecular resolution to cellu-
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10.1038/npg.els.0001363 This article was submitted to Systems Biology, a section of the journal Frontiers in
Physiology. Yafremava, L. S., Wielgos, M., Thomas, S., Nasir, A., Wang, M., Mittenthal, J. December 2013 | Volume 4 | Article 369 | 10 Frontiers in Physiology | Systems Biology REFERENCES E.,
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helps explain domains of life. Front. Genet. 4:16. doi: 10.3389/fgene.2013.00016 Copyright © 2013 Periyasamy, Gray and Kille. This is an open-access arti-
cle distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in other forums is permitted, pro-
vided the original author(s) or licensor are credited and that the original pub-
lication 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. 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. 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. December 2013 | Volume 4 | Article 369 | 10 Frontiers in Physiology | Systems Biology Frontiers in Physiology | Systems Biology
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English
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Comparison of Relative Waist Circumference between Asian Indian and US Adults
|
Journal of obesity
| 2,014
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cc-by
| 6,004
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Hindawi Publishing Corporation
Journal of Obesity
Volume 2014, Article ID 461956, 10 pages
http://dx.doi.org/10.1155/2014/461956 Hindawi Publishing Corporation
Journal of Obesity
Volume 2014, Article ID 461956, 10 pages
http://dx.doi.org/10.1155/2014/461956 Hindawi Publishing Corporation
Journal of Obesity
Volume 2014, Article ID 461956, 10 pages
http://dx.doi.org/10.1155/2014/461956 1 STOP Diabetes Foundation, Inc., Brampton, ON, Canada L6S 0C9 2 Division of Epidemiology & Community Health, University of Minnesota, 1300 South Second Street,
Suite 300, Minneapolis, MN 55454, USA p
3 Madras Diabetes Research Foundation and Dr. Mohan’s Diabetes Specialties Centre, Chennai, Tamil Nadu 600 086, India
4Columbia University New York NY 10032 USA 3 Madras Diabetes Research Foundation and Dr. Mohan’s Diabetes Specialties Centre, Chennai, Tamil Nadu 600 086, India
4Columbia University, New York, NY 10032, USA
5 5 University of Minnesota, Minneapolis, MN 55454, USA Correspondence should be addressed to Mark A. Pereira; pereira@epi.umn.edu Correspondence should be addressed to Mark A. Pereira; pereira@epi.umn.edu Received 27 February 2014; Revised 29 May 2014; Accepted 15 June 2014; Published 21 September 2014 Academic Editor: B. J. Ammori Copyright © 2014 Harpreet S. Bajaj et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. Background. Relative to Europeans, Asian Indians have higher rates of type 2 diabetes and cardiovascular disease. Whether
differences in body composition may underlie these population differences remains unclear. Methods. We compared directly
measured anthropometric data from the Chennai Urban Rural Epidemiology Study (CURES) survey of southern Indians (I) with
those from three US ethnic groups (C: Caucasians, A: African Americans, and M: Mexican Americans) from NHANES III (Third
National Health and Nutrition Examination Survey). A total of 15,733 subjects from CURES and 5,975 from NHANES III met
inclusion criteria (age 20–39, no known diabetes). Results. Asian Indian men and women had substantially lower body mass index,
waist circumference, hip circumference, waist-to-hip ratio, and body surface area relative to US groups (𝑃values <0.0001). In
contrast, the mean (±se) waist-weight ratio was significantly higher (𝑃< 0.001) in I (men 1.35 ± 0.002 and women 1.45 ± 0.002)
than in all the US groups (1.09, 1.21, and 1.14 in A, M, and C men; 1.23, 1.33, and 1.26 in A, M, and C women (se ranged from 0.005 to
0.006)). Conclusions. Compared to the US, the waist-weight ratio is significantly higher in men and women from Chennai, India. These results support the hypothesis that Southeast Asian Indians are particularly predisposed toward central adiposity. Harpreet S. Bajaj,1 Mark A. Pereira,2 Rajit Mohan Anjana,3 Raj Deepa,3
Viswanathan Mohan,3 Noel T. Mueller,4 Gundu H. R. Rao,5 and Myron D. Gross5 1 STOP Diabetes Foundation, Inc., Brampton, ON, Canada L6S 0C9
2 1. Introduction populations by setting lower thresholds of BMI to identify
those who may be at high risk [20]. However, some have
suggested that BMI has a relatively weak association with
chronic disease prevalence [21, 22] and with visceral abdom-
inal fat [23] in AI. As alternative measures, studies in various
populations, including AI [9, 22–24], US [25], and elsewhere
[26], suggest that either waist circumference (WC) alone [23,
25] or waist-hip ratio (WHR) [9, 22, 24, 26] may be a better
single anthropometric marker of chronic disease risk, as
compared to BMI, because they may more specifically reflect
abdominal body fatness. However, WC alone does not reflect
lean body mass, which is known to be protective [27], and
fails to allow comparisons between subjects and populations
due to confounding by body size and weight. Evidence that The number of people with type 2 diabetes in India is highest
in the world and is predicted to increase 150% by year 2025,
when the projected 69.9 million cases will comprise almost
a quarter of the world’s diabetic population [1]. The Asian
Indian (AI) phenotype, which refers to certain unique clinical
and biochemical characteristics such as greater abdominal
obesity despite lower body mass index, has been well docu-
mented and shown to make AI more prone to diabetes [2–14]
and coronary artery disease [15–19] than Caucasians (C) of
European ancestry.h The World Health Organization has addressed this para-
dox of low obesity and high chronic disease risk in Asian 2 2 Journal of Obesity Journal of Obesity high WHR may contribute to the high incidence of diabetes
in AI is equivocal [19, 28–30].l high WHR may contribute to the high incidence of diabetes
in AI is equivocal [19, 28–30].l high WHR may contribute to the high incidence of diabetes
in AI is equivocal [19, 28–30].l 0.5 kg with a calibrated scale, allowing light clothing. Waist
was measured to the nearest cm with a tape measure at the
smallest horizontal girth between the costal margins and
the iliac crest at minimal respiration. Hip was taken as the
greatest circumference at the level of greater trochanters (the
widest portion of the hip) on both sides. It was measured to
the nearest cm with a tape measure. In order to compare the
sex-specific prevalence of overweight and obesity among the
four ethnic groups, we used the currently accepted definitions
based on BMI and WC cutoffs [33]. 1. Introduction These include lower
cutoffs for AI recommended by WHO for BMI [25] and IDF
for WC [34]. To address the potential influence on chronic disease risk
of high abdominal fatness relative to total body fatness in
AI, we propose waist-to-weight ratio (WWR), as a single
continuous index, to distinguish differences between cultures
in the propensity to store fat in the visceral depots, con-
sistent with differences in insulin resistance, diabetes, and
cardiovascular disease (CVD). Therefore, we hypothesized
that the WWR will be higher in Indian men and women
than in US men and women. This hypothesis was tested
by comparing anthropometric characteristics between young
adults of the National Health and Nutrition Examination
Survey (NHANES III) [27] to young adults of the Chennai
Urban Rural Epidemiology Study (CURES) [31]. 2.3. Exclusion Criteria and Final Sample Sizes. In order to
minimize the likelihood of bias due to age-cohort effects
and the potential impact of clinical or subclinical illness
on anthropometry, we implemented the following exclusion
criteria: (1) missing or aberrant values for anthropometric
variables (excluded 7,824 from NHANES III and 446 from
CURES), (2) missing race/ethnicity or race/ethnicity other
than African American, Caucasian, or Mexican American
(excluded additional 1,130 from NHANES III), (3) age < 20 or
>39 (excluded additional 18,735 from NHANES III and 9,270
from CURES), (4) blood sugar < 50 mg/dL or >200 or known
diabetes (excluded additional 329 from NHANES III and 462
from CURES), and (5) BMI < 14 kg/m2 (excluded none from
NHANES III and additional 93 from CURES). There were a
total of 21,705 subjects (5,976 from NHANES III and 15,729
from CURES) who met the inclusion criteria. 2. Subjects and Methods 2.1. Study Populations. NHANES III [27] is a large cohort
representative of the US population, with minority groups
oversampled. It was conducted by the National Center For
Health Statistics and the Center for Disease Control and Pre-
vention on a nationwide probability sample of approximately
33,994 persons aged 2 months and over from mid-1988 to
mid-1994. The cross-sectional survey was designed to obtain
nationally representative information on the health and
nutritional status of the US population through interviews
and direct physical examinations. Written informed consent
was obtained from all participants and the National Center
for Health Statistics approved the protocol. Full details of the
study design, recruitment, and procedures are available from
the US Department of Health And Human Services [31].i 2.4. Statistical Methods. All analyses were sex-stratified and
performed using SAS version 9.1 (Cary, NC). We compared
the unadjusted prevalence of overweight and obesity, based
on BMI and waist categories, between AI and the three
US race groups using chi-square analysis. General linear
regression models were used to estimate unadjusted and
adjusted least squares means (±se) of the anthropometric
variables (dependent) by race and sex group. All 𝑃values
are 2-sided. Estimates were not weighted according to the
NHANES sampling scheme because our aim was to make
comparisons to the CURES population sample and not to
make estimations for the entire US population. p
CURES [31] is a large cross-sectional field survey of
representative samples of the area in and around Chennai, the
largest city in south India and the fourth largest city in India. This study recruited 26,001 subjects who were a random
sample of the population of Chennai (representing the urban
component) and villages around Chennai (representing the
rural component). The study commenced in August 2001
with the objective of comparing the prevalence of various
components of Insulin Resistance Syndrome and various
diabetes related complications. Ethical committee approval
was obtained prior to the start of the study and an informed
consent was obtained from all the study subjects. Details on
the study design, recruitment, and phases of the survey are
published elsewhere [31]. 3. Results Male
Female
African American
Mexican American
Caucasian
Asian Indian
African American
Mexican American
Caucasian
Asian Indian
𝑁
884
1043
799
7577
1131
1098
1021
8152
Height (cm)
176.9 (7.13)
170.0 (6.51)
177.5 (6.68)
164.7 (7.63)
163.6 (6.42)
157.4 (6.10)
164.0 (6.51)
156.3 (7.05)
Weight (kg)
82.5 (19.12)
75.4 (13.91)
81.3 (16.64)
59.2 (10.29)
74.5 (19.68)
67.3 (16.01)
67.3 (16.97)
54.7 (10.18)
BMI (kg/m2)
26.3 (5.56)
26.0 (4.30)
25.7 (4.70)
21.8 (3.80)
27.8 (7.04)
27.1 (6.15)
25.0 (6.08)
22.4 (4.45)
Waist (cm)
88.7 (14.54)
90.2 (11.17)
91.7 (12.71)
78.5 (10.10)
89.3 (16.20)
88.0 (14.29)
83.5 (14.86)
77.6 (10.59)
Hip (cm)
99.3 (11.13)
96.4 (7.84)
99.0 (8.86)
87.8 (9.74)
104.6 (13.82)
101.3 (12.38)
100.5 (12.61)
89.6 (11.33)
Waist/hip
0.89 (0.07)
0.93 (0.06)
0.92 (0.06)
0.90 (0.07)
0.85 (0.08)
0.87 (0.07)
0.83 (0.08)
0.87 (0.08)
BSA [32]
2.00 (0.24)
1.88 (0.19)
1.99 (0.22)
1.64 (0.16)
1.83 (0.25)
1.70 (0.21)
1.74 (0.22)
1.53 (0.15) 4 Journal of Obesity 100
90
80
70
60
50
40
30
20
10
0
(%)
African American
African American
Mexican American
Mexican American
Caucasian
Caucasian
Obese
Overweight
Normal
Men
Women
recommendations (Janssen et al, 2004) of
23 and 27.5 kg/m to define overweight and obesity, respectively. WHO Expert committee
∗Appropriate index for Asian populations based on
Asian Indian∗
Asian Indian∗
Asian Indian∗
Asian Indian∗
2
Figure 1: BMI categories stratified by ethnicity and sex. For example, the magnitude of the correlations between waist
and either body weight or BMI ranged from 0.56 to 0.63
in Chennai men and women, compared to 0.90 to 0.93 in
US men and women. The scattergrams in Figure 3, shown
for men, demonstrate the considerable population differences
in the association between waist and BMI between Chennai
and US Caucasian men. For any given BMI in AI there was
considerably more interindividual variation in the waist than
is observed in the US. Very similar results were observed for
women and for the other US ethnic groups (data not shown). g
Of note, age and height adjustment did not alter the corre-
lations in magnitude or direction in any considerable manner
(data not presented). In contrast, as presented in Tables 2
and 3, weight adjustment altered correlations between hip
and waist as well as between hip and WHR among the US
population. 3. Results Race- and sex-stratified sample sizes and unadjusted anthro-
pometric characteristics are shown in Table 1. Briefly, Asian
Indian men and women had substantially lower height,
weight, body mass index (BMI), WC, hip circumference
(HC), and body surface area relative to all US ethnic groups
(all sex-specific 𝑃values <0.0001). Asian Indian men had a
lower mean waist-to-hip ratio compared to the Caucasian and
Mexican American men (𝑃< 0.0001) but slightly larger mean
waist-to-hip ratio than the African American men (𝑃= 0.03). Asian Indian women had a higher mean waist-to-hip ratio
compared to the African American and Caucasian women
(𝑃< 0.0001) but similar to the Mexican American women
(𝑃= 0.43). 2.2. Anthropometric Assessments. In NHANES III, height was
measured to the nearest 0.1 cm with calibrated stadiometer,
without shoes. Weight was measured to the nearest 0.1 kg with
calibrated scale, allowing light clothing. WC was measured
to the nearest cm with tape measure at the highest point on
the iliac crest, while the subject was at minimum respiration,
allowing light clothing. HC was measured to the nearest cm
with a tape measure at maximum extension of the buttocks,
allowing light clothing. In CURES, height was measured
to the nearest cm with a tape measure, subjects standing
upright without shoes. Weight was measured to the nearest 3 3 Journal of Obesity Table 1: Mean (sd) anthropometric variables by population. 3. Results The strong correlations between hip and waist
were considerably attenuated by adjusting for weight in both
genders in the US population, whereas this adjustment had
little effect in the Asian Indian population (Tables 2 and 3). Of particular note the different direction of the correlation
between hips and WHR for AI (−0.12 for men, −0.23 for
women) was compared to US (0.40 for men, 0.26 for women). However, in the US the correlation between hips and WHR
was confounded by the high correlation between waist and
weight. As such, further adjustment for weight reversed the
direction of this correlation in the US (−0.37 for men, −0.53
for women). Weight adjustment marginally strengthened the
magnitude of the correlation in the AI, but because the
correlation between waist and weight is relatively low, the
direction of the association remained inversed (−0.29 in men,
−0.36 in women). 3.1. Ethnic Variations in BMI and WC Categories. In men and
women alike, as shown in Figure 1 based on BMI criteria,
the frequency of AI in the normal weight category (84.2%
for men, 77.4% for women) was significantly greater, and
the frequency of overweight and obesity was lower, than
any other ethnic group (𝑃< 0.0001 for all sex-stratified
comparisons). This remained true even after applying lower
cutoffs for the Asians Indians as suggested by the WHO
Expert Committee recommendations [25], except for the
frequency of overweight Asian Indian women which was
similar to the African American women (𝑃value = 0.69),
slightly less than Mexican American women (𝑃value =
0.03) and significantly less than Caucasian women (𝑃value
<0.0001). In Figure 2, it is seen that the Asian Indian men and
women have lower central obesity meeting NCEP’s metabolic
syndrome cutoff values for WC than all other groups (𝑃<
0.0001). When applying the ethnicity driven IDF cutoffs, the
differences between the AI and other groups were attenuated
but remained statistically significant (𝑃< 0.0001), even with
the lower cutoffs suggested for the Asian Indian men. 3.1. Ethnic Variations in BMI and WC Categories. In men and
women alike, as shown in Figure 1 based on BMI criteria,
the frequency of AI in the normal weight category (84.2%
for men, 77.4% for women) was significantly greater, and
the frequency of overweight and obesity was lower, than
any other ethnic group (𝑃< 0.0001 for all sex-stratified
comparisons). 3. Results This remained true even after applying lower
cutoffs for the Asians Indians as suggested by the WHO
Expert Committee recommendations [25], except for the
frequency of overweight Asian Indian women which was
similar to the African American women (𝑃value = 0.69),
slightly less than Mexican American women (𝑃value =
0.03) and significantly less than Caucasian women (𝑃value
<0.0001). In Figure 2, it is seen that the Asian Indian men and
women have lower central obesity meeting NCEP’s metabolic
syndrome cutoff values for WC than all other groups (𝑃<
0.0001). When applying the ethnicity driven IDF cutoffs, the
differences between the AI and other groups were attenuated
but remained statistically significant (𝑃< 0.0001), even with
the lower cutoffs suggested for the Asian Indian men. 3.3. The Waist-to-Weight Ratio (WWR). To further evaluate
possible differences between populations in central adiposity
while taking into account differences in total body mass, we
computed the WC to body weight ratio. The WWR followed
a normal distribution for all sex and population groups. The median WWR was highest among men and women
from India and was in fact higher than the 75th percentile
of all other groups. The means of WWR before and after
adjustment for height are shown in Table 4. As can be seen,
WWR was significantly higher (𝑃< 0.0001) in the AI than in
US for both sexes. The attenuation of these differences by age
and height adjustment was minimal. 4. Discussion The present study compares a number of traditional anthro-
pometric factors between young adults of the US repre-
sentative NHANES III survey, including three race/ethnic
groups, and the population-based CURES survey of AI. Our observations confirm the findings of others that the
AI tend to be much smaller than the US population in
all traditional anthropometric measurements—body weight,
body height, BMI, body surface area, hips, and WC. In
contrast, we observed the WHR, which some studies have
found to be higher in AI than in other groups [22, 24, 26], to
be somewhat higher in AI women compared to US women, 3.2. Correlations among Anthropometric Variables. Table 2
(men) and Table 3 (women) include the unadjusted sex- and
population-specific Pearson correlations among the various
anthropometric variables. We pooled the US race groups
within sex because the correlations did not vary in important
ways among US race groups. The magnitudes of these corre-
lations were all lower, without exception, in the AI compared
to the US, with most of the Asian-US differences being large. Journal of Obesity 5 Table 2: Pearson correlations—men. WAIST
HIP
WHR
WT
BMI
BSA
CURES
NHANES
CURES
NHANES
CURES
NHANES
CURES
NHANES
CURES
NHANES
CURES
NHANES
WAIST
0.72
0.18
0.42
0.84
0.60
0.90
−0.05
0.20
0.10
0.19
HIP
0.82
0.89
−0.29
−0.37
0.54
0.95
−0.01
0.20
0.05
0.20
WHR
0.45
0.77
−0.12
0.40
0.18
0.48
−0.06
0.09
0.09
0.15
WT
0.61
0.90
0.57
0.95
0.18
0.48
0.83
0.91
0.97
0.99
BMI
0.56
0.93
0.49
0.90
0.20
0.60
0.82
0.91
−0.03
0.36
BSA
0.57
0.84
0.54
0.92
0.16
0.41
0.97
0.98
0.66
0.82
HT
0.09
0.18
0.13
0.35
−0.03
−0.12
0.32
0.45
−0.26
0.05
0.53
0.61
Not bold: unadjusted. Bold: adjusted for age, height, and weight. WAIST: waist circumference; HIP: hip circumference; WHR: waist-to-hip ratio; WT: body weight; BMI: body mass index; BSA: body surface area; HT: height; CURES: Chennai Urban Rural Epidemiology Study;
NHANES: National Health and Nutrition Examination Survey. Table 2: Pearson correlations—men. 4. Discussion WAIST
HIP
WHR
WT
BMI
BSA
CURES
NHANES
CURES
NHANES
CURES
NHANES
CURES
NHANES
CURES
NHANES
CURES
NHANES
WAIST
0.72
0.18
0.42
0.84
0.60
0.90
−0.05
0.20
0.10
0.19
HIP
0.82
0.89
−0.29
−0.37
0.54
0.95
−0.01
0.20
0.05
0.20
WHR
0.45
0.77
−0.12
0.40
0.18
0.48
−0.06
0.09
0.09
0.15
WT
0.61
0.90
0.57
0.95
0.18
0.48
0.83
0.91
0.97
0.99
BMI
0.56
0.93
0.49
0.90
0.20
0.60
0.82
0.91
−0.03
0.36
BSA
0.57
0.84
0.54
0.92
0.16
0.41
0.97
0.98
0.66
0.82
HT
0.09
0.18
0.13
0.35
−0.03
−0.12
0.32
0.45
−0.26
0.05
0.53
0.61
Not bold: unadjusted. Bold: adjusted for age, height, and weight. WAIST: waist circumference; HIP: hip circumference; WHR: waist-to-hip ratio; WT: body weight; BMI: body mass index; BSA: body surface area; HT: height; CURES: Chennai Urban Rural Epidemiology Study
NHANES: National Health and Nutrition Examination Survey. 6 Journal of Obesity 6 Table 3: Pearson correlations—women. WAIST
HIP
WHR
WT
BMI
BSA
CURES
NHANES
CURES
NHANES
CURES
NHANES
CURES
NHANES
CURES
NHANES
CURES
NHANES
WAIST
0.65
−0.002
0.45
0.83
0.60
0.90
0.01
0.15
0.12
0.19
HIP
0.79
0.87
−0.36
−0.53
0.58
0.94
−0.02
0.23
0.07
0.10
WHR
0.40
0.69
−0.23
0.26
0.10
0.40
0.04
0.01
0.07
0.17
WT
0.63
0.90
0.62
0.94
0.09
0.40
0.86
0.94
0.97
0.99
BMI
0.57
0.92
0.60
0.93
0.04
0.46
0.86
0.94
−0.10
0.23
BSA
0.60
0.86
0.57
0.91
0.12
0.37
0.97
0.98
0.72
0.87
HT
0.06
0.07
−0.01
0.17
0.11
−0.11
0.20
0.29
−0.31
−0.05
0.42
0.44
Not bold: unadjusted. Bold: adjusted for age, height, and weight. WAIST: waist circumference; HIP: hip circumference; WHR: waist-to-hip ratio; WT: body weight; BMI: body mass index; BSA: body surface area; HT: height; CURES: Chennai Urban Rural Epidemiology Study;
NHANES: National Health and Nutrition Examination Survey. Journal of Obesity 7 7 Table 4: Mean (se) of waist-to-weight ratio by sex and population. Male
Female
African American
Mexican American
Caucasian
Asian Indian
African American
Mexican American
Caucasian
Asian Indian
Waist/weight
1.09 (0.006)
1.21 (0.006)
1.14 (0.006)
1.35∗(0.002)
1.23 (0.005)
1.33 (0.005)
1.27 (0.006)
1.45∗(0.002)
Waist/weight
1.21 (0.006)
1.27 (0.005)
1.26 (0.007)
1.37∗(0.002)
1.24 (0.005)
1.30 (0.005)
1.28 (0.005)
1.40∗(0.002)
adjusted for age and height
∗𝑃< 0.0001 compared to all other groups. 𝑃< 0.05 for all other sex-specific comparisons, except for Mexican American men and Caucasian men in the age and height-adjusted model. Table 4: Mean (se) of waist-to-weight ratio by sex and population. 4. Discussion Male
Female
African American
Mexican American
Caucasian
Asian Indian
African American
Mexican American
Caucasian
Asian Indian
Waist/weight
1.09 (0.006)
1.21 (0.006)
1.14 (0.006)
1.35∗(0.002)
1.23 (0.005)
1.33 (0.005)
1.27 (0.006)
1.45∗(0.002)
Waist/weight
1.21 (0.006)
1.27 (0.005)
1.26 (0.007)
1.37∗(0.002)
1.24 (0.005)
1.30 (0.005)
1.28 (0.005)
1.40∗(0.002)
adjusted for age and height
∗𝑃< 0.0001 compared to all other groups. 𝑃< 0.05 for all other sex-specific comparisons, except for Mexican American men and Caucasian men in the age and height-adjusted model. Table 4: Mean (se) of waist-to-weight ratio by sex and population. 8 Journal of Obesity 8 70
60
50
40
30
20
10
0
African American
African American
Mexican American
Mexican American
Caucasian
Caucasian
Asian Indian
Asian Indian
Meet IDF MS criteria
Meet IDF MS criteria
Male central obesity
≥90 Asian Indians
Female central obesity
Meet ATP III MS criteria
Meet ATP III MS criteria
(%)
ATP III: ≥102 cm
IDF: ≥94cm non-Asians
ATP III: ≥88cm
IDF: ≥80cm non-Asians
≥80 Asian Indians
Men
Women
Figure 2: Waist categories stratified by ethnicity and sex. Figure 2: Waist categories stratified by ethnicity and sex. 200
180
160
140
120
100
80
60
40
20
0
0
10
20
30
40
50
10
20
30
40
50
y = 2.57x + 25.6
R2 = 0.8671
y = 1.46x + 46.3
R2 = 0.3045
Waist (cm)
200
180
160
140
120
100
80
60
40
20
0
Waist (cm)
BMI (kg/m2)
BMI (kg/m2)
NHANES III
CURES
Figure 3: Correlations of waist circumference and BMI in US Caucasian and Asian Indian men. The corresponding 𝑅2 value for women of
NHANES III was 0.85, in comparison to 0.31 for CURES women. The 𝑅2 across the other two NHANES III race/ethnic groups for men and
women ranged from 0.81 to 0.88. 10
20
30
40
50
y = 1.46x + 46.3
R2 = 0.3045
200
180
160
140
120
100
80
60
40
20
0
Waist (cm)
BMI (kg/m2)
CURES 200
180
160
140
120
100
80
60
40
20
0
0
10
20
30
40
50
y = 2.57x + 25.6
R2 = 0.8671
Waist (cm)
(
)
BMI (kg/m2)
NHANES III Figure 3: Correlations of waist circumference and BMI in US Caucasian and Asian Indian men. The corresponding 𝑅2 value for women of
NHANES III was 0.85, in comparison to 0.31 for CURES women. 4. Discussion The 𝑅2 across the other two NHANES III race/ethnic groups for men and
women ranged from 0.81 to 0.88. of the present study, it appears that our novel measure-
ment, WWR, is a simple anthropometric index capturing
population differences in propensity for intra-abdominal fat
storage. The WWR would appear to sufficiently account for
the large confounding influence of overall body size and
general adiposity. For any given body weight, larger WC could
reflect larger intra-abdominal fat depots. Individuals with
relatively high body weights who tend to carry much of their but not different, or in fact lower, among AI men compared
to US men. In our analyses, the differences between means of
WHR, though significantly higher in AI women compared to
Caucasian and African American women, were small. It is possible, therefore, that if AI have a propensity
towards excess accumulation of visceral body fat, it is being
masked, perhaps especially in the men, by large differences in
overall body size between populations. Based on the findings 9 Journal of Obesity weight in muscle and subcutaneous fat depots peripherally
would be expected to have lower WWR and lower risk for
diabetes and CVD, hypothetically. Based on our observations,
the distribution of WWR in young adults from Chennai,
India, is considerably shifted to the right in comparison to
similarly aged adults from three race/ethnic groups of the US
population. measurement standards were described in the methods sec-
tion. Given that these differences were small, we believe that
the results are not materially biased by misclassification due
to such measurement differences. The fact that the CURES
survey was conducted in and around the southern Indian city
of Chennai begs the question of whether these results can be
generalized to the whole Asian Indian population.i An intriguing observation from comparisons performed
in present study was the vastly different correlation between
WC and BMI between young adults of Chennai, India,
compared to those from three US race/ethnic groups. In the
US over 80% of the variation (𝑟= ∼0.9) in WC is explained
by BMI, whereas in Chennai, India, these two parameters
shared less than 40% common variance (𝑟= ∼0.60). This
observation helps us to understand the basis for the higher
WWR in Chennai than in US. At any given BMI there is
clearly much more variation in the WC of Asian Indian
young adults than in US young adults. References [1] R. Sicree, J. Shaw, and P. Zimmet, “Diabetes and impaired
glucose tolerance,” in Diabetes Atlas, D. Gan, Ed., pp. 15–
103, International Diabetes Federation, Brussels, Belgium, 3rd
edition, 2006. [2] N. Abate and M. Chandalia, “Ethnicity and type 2 diabetes:
focus on Asian Indians,” Journal of Diabetes and Its Complica-
tions, vol. 15, no. 6, pp. 320–327, 2001. [3] S. R. Joshi, “Metabolic syndrome—emerging clusters of the
Indian phenotype,” Journal of Association of Physicians of India,
vol. 51, pp. 445–446, 2003. [4] R. Deepa, S. Sandeep, and V. Mohan, “Abdominal obesity,
visceral fat and type 2 diabetes—“Asian Indian phenotype”,” in
Type 2 Diabetes in South Asians: Epidemiology, Risk Factors and
Prevention, V. Mohan and G. H. R. Rao, Eds., pp. 138–152, Jaypee
Brothers Medical, New Delhi, India, 2006. Strengths of the present study are multiple. It is a
population-based and a large-scale study of AI who have
a high risk of diabetes; the study compares them with the
three main ethnic groups of the US NHANES population. The high quality standardization of anthropometric mea-
surements in both cohorts minimizes measurement errors
and biases. Despite several practical advantages of ecologic
studies, including low cost, there exist several methodological
limitations to any ecological study in its ability to make causal
inferences [32]. Ecological studies often suffer from biases
(ecological fallacy or aggregation bias), which represents the
failure of expected ecological effect estimates to reflect the
effect at the individual level. A potential strategy suggested
for minimizing ecological bias is to use smaller units in order
to make the groups more homogeneous with respect to the
exposure [32]. For this reason, we included only nondiabetic
young adults who were not underweight, which also helps
to minimize the effects of temporal ambiguity (presence
of disease effecting body habitus) and different cohort
effects between populations. Possible limitations of this study
originating from the somewhat different anthropometric [5] A. Ramachandran,
C. Snehalatha,
V. Viswanathan,
M. Viswanathan, and S. M. Haffner, “Risk of noninsulin dependent
diabetes mellitus conferred by obesity and central adiposity in
different ethnic groups: a comparative analysis between Asian
Indians, Mexican Americans and Whites,” Diabetes Research
and Clinical Practice, vol. 36, no. 2, pp. 121–125, 1997. [6] A. Raji, E. W. Seely, R. A. Arky, and D. C. Simonson, “Body fat
distribution and insulin resistance in healthy Asian Indians and
Caucasians,” Journal of Clinical Endocrinology and Metabolism,
vol. 86, no. 11, pp. Conflict of Interests The authors declare that there is no conflict of interests
regarding the publication of this paper. y
A strength of the WWR is that it retains simplicity in
measurement and calculation, making it desirable over WHR
and BMI in this regard, and allows comparisons between
people and populations varying in overall body size, making
it more useful than the WC alone. A possible limitation of the
WWR is that it does not consider height. To look further into
the usefulness of height, we did compute (data not presented)
a waist-to-BMI ratio (WBR). This ratio differentiated the two
populations on an ecological level in a similar manner as did
the WWR. Using such ratio did not seem to provide any
added benefit to the WWR, while it may be fraught with a
number of conceptual and statistical problems discussed by
others [35, 36]. Another conceivable limitation of the WWR
is that the denominator (weight) is highly correlated with the
numerator in many populations (see Tables 2 and 3). 4. Discussion Indeed, the variance
of WWR in the Chennai men and women was threefold
higher than that of the US men and women (0.03 versus 0.01). This observation suggests that AI may have a heightened
propensity for accumulating central adiposity, for any given
body size, than other populations. We believe that our findings in this study are provocative
and should stimulate further research into novel anthropo-
metric features that may differentiate populations at very
high risk for chronic disease, despite relatively low adiposity. The findings should motivate future coordinated research on
complex exposures, their social and behavioral determinants,
and possible interventions. Certainly, the WWR should be
validated using more precise clinical methods of measuring
fat depots, including DEXA and CT scan. The WWR should
be investigated in other studies in order to address possible
influences of age, ethnicity, gender and nutritional status, and
its relation to chronic diseases and mortality. References Viswanathan, “High prevalence of diabetes in an urban
population in South India,” British Medical Journal, vol. 297, no. 6648, pp. 587–590, 1988. [29] A. Misra, D. Athiko, R. Sharma, R. M. Pandey, and N. Khanna,
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population in India,” Diabetologia, vol. 40, no. 2, pp. 232–237,
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vascular Risk, vol. 3, no. 3, pp. 263–270, 1996. [15] K. Hughes, “Mortality from cardiovascular diseases in Chinese,
Malays and Indians in Singapore, in comparison with England
and Wales, USA and Japan,” Annals of the Academy of Medicine
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Potential effects of samsum ant, Brachyponera sennaarensis, venom on TNF-α/NF-κB mediated inflammation in CCL4-toxicity in vivo
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Lipids in health and disease
| 2,016
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© The Author(s). 2016 Open Access 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. Potential effects of samsum ant,
Brachyponera sennaarensis, venom on TNF-
α/NF-κB mediated inflammation in CCL4-
toxicity in vivo Jameel Al-Tamimi1, Ibrahim M. Alhazza1, Mohamed Al-Khalifa1, Ali Metwalli2, Ahmed Rady1 and Hossam Ebaid1* mi1, Ibrahim M. Alhazza1, Mohamed Al-Khalifa1, Ali Metwalli2, Ahmed Rady1 and Hossam Ebaid Jameel Al-Tamimi1, Ibrahim M. Alhazza1, Mohamed Al-Khalifa1, Ali Metwalli2, Ahmed Rady1 and Abstract Background: Ant venom shows antimicrobial, anti-parasitic and anti-inflammatory activities, both in vitro and in vivo. Our recent studies have confirmed the role of samsum ant venom (SAV) as a powerful antioxidant. This study aimed to
investigate whether SAV as a potential treatment for CCl4-induced acute liver toxicity in an animal (rat) model. Methods: Thirty-two rats were assigned into four groups; the first one served as the control. The second group received
a single dose of 1 ml/kg CCl4 in a 1:1 ratio with olive oil through an intraperitoneal injection. The third group received a
single dose of 1 ml/kg CCl4 and then treated with SAV at a dose of 100 μg SAV twice a week for three weeks. The fourth
group received a dose of 100 μg SAV only twice a week for three weeks. ELISA, RT-PCR and histopathological
examinations were applied. Results: Results showed that antioxidant enzymes were significantly reduced in the diseased animals. SAV was found
to significantly restore the oxidative stability in diseased animals. ELISA estimation and RT-PCR analysis also showed
significant upregulation of both nuclear factor (κB) NF-κB and inhibitor (κB) IκB, respectively, in the diseased animals
compared to the normal ones. The expression of tumour necrosis factor alpha (TNF-α) and pro-apoptotic
receptor (Fas) were also significantly up-regulated in the diseased rats. Interestingly, SAV was found to
significantly restore NF-κB, IκB and TNF-α in the diseased rats to the normal values. As a result, liver enzymes,
serum proteins and lipid concentrations were significantly improved by SAV in CCl4-animals in comparison with the
control ones. Moreover, SAV obviously improved the hepatic tissues of the same group was. Conclusion: SAV treatment restores the normal biochemical and oxidative stability by improving the TNF-α/NF-κB
mediated inflammation in CCL4-treated rats. Keywords: CCL4, Samsum ant venom, Antioxidant activity, NF-κB and IκB, TNF-α Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198
DOI 10.1186/s12944-016-0364-7 Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198
DOI 10.1186/s12944-016-0364-7 Open Access * Correspondence: hossamebaid@yahoo.com; hossamebaid1969@gmail.com
1Department of Zoology, College of Science, King Saud University, P.O. Box
2455, Riyadh 11451, Saudi Arabia
Full list of author information is available at the end of the article Sample collection In this context, this study provides insights into the
pharmacological applications of SAV by estimating the
inducible transcription nuclear factor-κB (NF-κB) which
is a central regulator of inflammatory and immune
responses. The inhibitor of κB (IκB) is a well-defined
regulator of NF-κB that resides in the cytoplasm and
prevents NF-κB from nuclear entry by sequestration. Animals were sacrificed by ether anesthesia after 24 h. of
the last SAV dose. Blood was collected by two separate
tubes, in tubes with EDTA, and centrifuged at 3000 rpm
for 10 min. The sera was separated and stored at −25 °C. The liver is gently excised and rinsed with BPS (7.4) to
eliminate blood contamination, dried by blotting with
filter paper was kept BPS. The tissue was then kept in
freezer directly at −80 °C until analysis of antioxidant
and oxidative stress. Blood biochemical parameters assay Blood biochemistry parameters of the study were esti-
mated and included cholesterol, triglyceride, total pro-
tein (TP) and albumin. In addition, some of blood
enzymes such as alanine aminotransferase (ALT), aspar-
tate aminotransferase (AST), alkaline phosphatase (ALP)
all
plasma
biochemical
parameters
analyzed
using
reagent kits (Quimica Clinica Aplicada S.A., Spain), by
Spectrophotometric
analyzer,
Pharmacia
Biotech,
(Ultrospec 2000, UV- Visible) Cambridge, England. Background pharmacologies. Ant venoms exhibit greater variability
in composition and function than do venoms from any
other arthropods [3]. Pharmaceutical scientists are constantly searching for
molecules of therapeutic benefit. Venoms are a promis-
ing source for the discovery of active molecules as they
offer potential biologically active properties [1], that may
be useful as new tools for the design of drugs [2]. Venoms exhibit a potential activities including anti-
microbial, haemolytic, cytolytic, paralytic and insecticidal Ant venom-toxic reactions are caused by substances,
including acids and alkaloids [4]. The venom of the ant
Odontomachus bauri has been demonstrated to show
antimicrobial activity against bacteria, as well as anti-
parasitic activity against Toxoplasma gondii infection [5]. The venom gland secretions of Samsum ant (SAV)
Pachycondyla species contain volatile components [6]. Carbon tetrachloride (CCl4) is a well-known chemical
compound causing hepatic injury [7]. It has been widely Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Page 2 of 11 Page 2 of 11 used to induce liver injury, fatty liver and liver fibrosis in
experimental animals, and a single dose of CCl4 can lead
to centrosomal necrosis and steatosis [8]. CCl4 is capable
of
reproducing
hepatic
cirrhosis
via
the
metabolic
changes resulting from free radicals [9]. Liver injury with
CCl4 includes the production of free radicals and the
activation of macrophages. intraperitoneal injection. The third group received a sin-
gle dose of 1 ml/kg CCl4 and then treated with SAV in
low dose of 100 μg/Kg body weight on five occasions
with two day intervals. The fourth group received a dose
of 100 μg SAV on five occasions with two day intervals. Methods Collection of the samsum ant dissection of the venom gland
Collected
ant’s
Samsum
colonies
from
Hotat
Bani
Tamim Governorate, East Riyadh, Kingdom of Saudi
Arabia. Extracted dirt block containing the nest ants and
put in a cloth bags were moved to the ant insectary in
the Zoology Department, King Saud University. In
addition, placed in plastic containers capacity of 20 × 70
cm, painted upper interior edges with grease to prevent
the exit of ants, and provided a glass tube with sugar so-
lution 10%, food added every day and consists of wheat
grains crushed or pills tile of the nest spray volumes of
water twice a day according. The insect is grabbing in
the area pregnant with forceps, grab the other machine
stinging other forceps, and then quietly removed the
separation of the venom gland. The venom glands were
homogenized and then centrifuged at 1000 rpm for 2
min. and the supernatant was collected in tube Eppendorf
stored at −25 °C in PBS buffer until use. Homogenate preparation The liver was perfused with (PBS) and homogenized,
and the obtained samples were centrifuged at 10000 × g
for 15 min at 4 °C. The supernatant was sucked and kept
on ice until used to assay GSH, CAT and MDA activity. Effect of SAV on TNF-α Because TNF-α is one of the inflammatory cytokines
which is controlled by NF-κB, it behaved in a similar
way to that of NF-κB. Results showed that the protein
level of TNF-α was significantly elevated in the CCl4
group compared to the control group. SAV, meanwhile,
was found to significantly restore the protein level of
TNF-α to the normal levels, as shown in Fig. 3. Experimental design p
g
Thirty-two Wister albino rats of male sex weighing
220–270 g (20 ± 1 weeks) were obtained from the
faculty of pharmacy, king Saud University, Saudi Arabia. The animals were acclimated to the laboratory condi-
tions two weeks before the start of experiment animals
were then housed in stainless steel wire cages (4 ani-
mals/cage), under pathogen-free conditions. Animals
were maintained were provided food and water. The
temperature was controlled at (22 ± 2 °C) with a relative
humidity of (45–65%) and lighting on a light/dark cycle
of 14/10 h. Animals were weighed before dosing and at
appropriate times and observed every second day to for
signs of ill health. The study protocol was approved by
the Animal Ethics Committee of the Zoology Depart-
ment in the College of Science at King Saud University. Rats were assigned into four groups; the first one served
as the control. The second group received a single dose
of 1 ml/kg CCl4 in a 1:1 ratio with olive oil through an Estimation of catalase (CAT) and GSH bioactivity
The activity of an antioxidant enzyme is assayed with
standard protocol for catalase (CAT) was done by
decomposition of hydrogen peroxide [11]. A glutathione
(GSH) assay was carried out as previously mentioned
[12]. Briefly, A 10% (w/v) homogenate of each frozen
tissue was prepared. The resulting supernatant was
boiled to deactivate and precipitate other proteins. GSH
concentrations were then measured by adding 100 μl of
boiled supernatant to 400 μl PBS. GSH concentrations
were then determined by measuring the absorbance
(OD) of the reaction after 1 min at 340 nm using a UV
Visible
Spectrometer
(Ultrospec
2000,
Pharmacia
Biotech). GSH standards were measured concurrently to
obtain a standard curve that was used to calculate GSH
concentrations in samples. Results were expressed as μg
GSH/g tissue. Page 3 of 11 Page 3 of 11 Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Histological sections Liver parts were collected from the sacrificed and
immersed in neutral buffered formalin 10% for at least
24 h. Tissues were fixed in Bouin’s fixative, processed
into paraffin, cut into 4 μm thick sections by a rotary
microtome were prepared (by Automatic tissue processor,
Auto technique). Sections were stained with Haematoxylin
and Eosin (H&E) for general histological architecture. The
light microscopic images of rat liver slices of control, CCl4,
CCl4 + 100 μg/kg SAV and 100 mg SAV were shown in
Figure. Sections were stained with Haematoxylin and Eosin
(H&E) for general histological architecture. Statistics Lipid peroxidation in homogenates was estimated spec-
trophotometrically following the method described by
Okhawa et al. [13, 14]. An aliquot of 0.5 ml of the result-
ing
supernatant
was
shaken
with
2.5
ml
of
20%
trichloroacetic acid. To the resulting mixture, 1 ml of
0.67%
thiobarbituric
acid
was
added,
shaken,
and
warmed for 30 min in a boiling water bath, and followed
immediately by rapid cooling in ice for 5 min. 4 ml of
n-butyl-alcohol was added and the sample was shaken
well. The resulting mixture was then centrifuged at
16,000 × g for 5 min. The resultant n-butyl-alcohol layer
was transferred to a separate tube and MDA content was
determined spectrophotometrically at 535 nm using a UV
Visible Spectrometer (Ultrospec 2000, Pharmacia Biotech). Data was analyzed using statistical package for social sci-
ences (SPSS) version 20.0. determined the statistical sig-
nificance of the differences between the averages of the
results of the various groups were the use of analysis of
variance
unidirectional
measurements
repeated
One
Way ANOVA -Post Hoc Tukey’s test method for pair-
wise comparisons. This was in the form of data repre-
sentation average ± standard error was adopted abstract
level (P <0.05). RT-PCR analysis of RNA expression of FAS and IκB RT PCR analysis of RNA expression of FAS and IκB
Quantification of mRNA expression by real-time poly-
merase chain reaction cDNA from the above preparation
was subjected to PCR amplification using 96-well optical
reaction plates in the ABI Prism 7500 System (Applied
Biosystems®). The 25-μl reaction mixture contained 0.1
μl of 10 μM forward primer and 0.1 μl of 10 μM reverse
primer (40 μM final concentration of each primer;
chosen from pubmed com), 12.5 μl of SYBR Green
Universal astermix, 11.05 μl of nuclease free water, and
1.25 μl of cDNA sample. The RT-PCR data was analyzed
using the relative gene expression method, as described
in Applied Biosystems ® User Bulletin No. 2. The data
are presented as the fold change in gene expression nor-
malized to the endogenous reference gene and relative
to a calibrator. Effect of SAV on NF-kB and IkB We estimated NF-κB, which is a central regulator of in-
flammatory and immune responses. IκB is a well-defined
regulator of NF-κB that resides in the cytoplasm and
prevents NF-κB from nuclear entry by sequestration. Results showed that the protein level of NF-κB and the
RNA expression of IκB are each significantly elevated
(p < 0.01) in the CCl4 group compared to the control
group. Interestingly, SAV was found to significantly
restore the protein level of NF-κB and the RNA
expression of IκB to normal levels, as shown in Fig. 2. Effect of samsum ant venom on oxidative stress Effect of samsum ant venom on oxidative stress The level of catalase (CAT), reduced glutathione (GSH),
and malondialdehyde (MDA) were taken as major
parameters to assess the antioxidant status in the major
organs of the liver of the treated animals. Samples in the
CCL4 group showed a significant decrease (p < 0.01) in
the level of CAT and GSH levels by 33% and 37.5%,
respectively, compared with the control group. The
CCL4 + SAV group, however, showed a significant in-
crease in these levels compared to the CCL4 group
(Fig. 1). The CCl4 treated group showed significantly in-
creased (p < 0.01) levels of MDA (by 86.83% of MDA in
liver samples compared to their respective controls. The
CCL4 + SAV group, however, showed a significant de-
crease by 7.6% compared to the CCL4 group (Fig. 1). ELISA estimation of FAS, NF-κB and TNF-α ELISA estimation of FAS, NF-κB and TNF-α The liver homogenate was tested for the protein level of
FAS, NF-κB and TNF-α by ELISA according to the man-
ufacturer’s instructions for the corresponding rat im-
munoassay kits (MyBioSource and Ray biotic, US). The
optical densities were measured at 450 nm. The detec-
tion limits were set according to the log-log correlative
coefficient of the standard curve. Effect of SAV on Fas Fas plays an important role in the regulation of immune
response and apoptosis via inflammatory cytokines such
as TNF-α. The Fas level in the CCl4 treated group
showed a
significant increase (p < 0.01) by 4.7% Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Page 4 of 11 Fig. 1 Estimation of catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) changes in the liver of rats challenged with CCL4 challenge
and treated with SAV. Values are expressed as the means ± standard errors. * shows statistically significant differences at P < 0.05 from the control. # shows
statistically significant differences at P < 0.05 between any group compared to the CCL4 group Fig. 1 Estimation of catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) changes in the liver of rats challenged with CCL4 challenge
and treated with SAV. Values are expressed as the means ± standard errors. * shows statistically significant differences at P < 0.05 from the control. # shows
statistically significant differences at P < 0.05 between any group compared to the CCL4 group compared to the control group, while CCL4 + SAV group
showed a decrease of 3% compared to CCL4 group and an
increase of 1.5% compared to the control (Fig. 4). rats showed several visible histological changes to the
liver, disruption of lobular architecture, including in-
flammatory cellular infiltrations, an increased number
of mitotic figures, hepatic cell necrosis and thick
vessel walls. This CCl4-induced damage to the liver
was significantly alleviated in the CCL4 + SAV treated
rats, with only minor side effects evident, such as
faintly stained nuclei. Among the SAV treated rats, a
general
hepatic
architecture
with
narrow
hepatic Histological sections The liver histology of the control group rats is shown in
Table 1 and Fig. 5, where normal hepatocyte, central
vein and histological architecture of hepatic tissues can
be observed. The histological features of the CCl4-treated Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Page 5 of 11 Fig. 2 Estimation of the protein level of inducible transcription nuclear factor-κB (NF-κB) by ELISA and the RNA expression of the inhibitor of κB
(IκB) by RT-PCR Fig. 2 Estimation of the protein level of inducible transcription nuclear factor-κB (NF-κB) by ELISA and the RNA expression of the inhibitor of κB
(IκB) by RT-PCR sinusoids and a large number of hepatocytes is shown
(Table 1, Fig. 5). Effect of samsum ant venom on liver function markers
In the present study, levels of AST, ALT and ALP in
plasma have been chosen as liver function markers. The
CCl4 treated group showed that ALT and AST levels
were significantly increased as compared to the control
group. This concentration was significantly decreased in
the CCL4 + SAV group, however (Table 2). On the other
hand, no significant changes were observed in the con-
centration of ALP in any of the investigated rats. Fig. 3 Changes in liver protein levels of TNF-a following CCL4 challenge
and treatment with SAV. Values are expressed as the means ± standard
errors. * shows statistically significant differences at P < 0.05 from the
control. # shows statistically significant differences at P < 0.05 between
any group compared to the CCL4 group Fig. 3 Changes in liver protein levels of TNF-a following CCL4 challenge
and treatment with SAV. Values are expressed as the means ± standard
errors. * shows statistically significant differences at P < 0.05 from the
control. # shows statistically significant differences at P < 0.05 between
any group compared to the CCL4 group Effect of samsum ant venom on blood lipid concentration
Blood lipid concentration is a significant indicator of the
ability of the liver to uptake different lipid derivatives. In
the CCL4 only treated group, a significant up regulation
of the cholesterol concentration in plasma was evident
(by 94% as compared to the control). In the CCL4 + SAV
group, however, this figure significantly decreased by 29% Fig. 3 Changes in liver protein levels of TNF-a following CCL4 challenge
and treatment with SAV. Values are expressed as the means ± standard
errors. Histological sections * shows statistically significant differences at P < 0.05 from the
control. # shows statistically significant differences at P < 0.05 between
any group compared to the CCL4 group Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Page 6 of 11 Fig. 4 Changes in liver protein levels (ELISA) and the RNA expression (RT-PCR) of Fas following CCL4 challenge and treatment with SAV. Values
are expressed as the means ± standard errors. * shows statistically significant differences at P < 0.05 from the control. # shows statistically significant
differences at P < 0.05 between any group compared to the CCL4 group Fig 4 Changes in liver protein levels (ELISA) and the RNA expression (RT-PCR) of Fas following CCL4 challenge and treatment with SAV Values Fig. 4 Changes in liver protein levels (ELISA) and the RNA expression (RT-PCR) of Fas following CCL4 challenge and treatment with SAV. Values
are expressed as the means ± standard errors. * shows statistically significant differences at P < 0.05 from the control. # shows statistically significant
differences at P < 0.05 between any group compared to the CCL4 group Effect of samsum ant venom on total protein and albumin
Total
protein
concentration
in
the
CCl4
group
showed a significant decrease by 24% compared to
the control group. While the CCL4 + SAV group
showed an increase by 16.6% compared to the CCL4
group. Similarly, albumin concentration in the CCl4
group
treated
showed
a
significant
decrease
by
23.33% compared to the control group, while SAV
was found to restore the concentration of the albu-
min to the control level (Table 2). Effect of samsum ant venom on total protein and albumin
Total
protein
concentration
in
the
CCl4
group
showed a significant decrease by 24% compared to
the control group. While the CCL4 + SAV group
showed an increase by 16.6% compared to the CCL4
group. Similarly, albumin concentration in the CCl4
group
treated
showed
a
significant
decrease
by
23.33% compared to the control group, while SAV
was found to restore the concentration of the albu-
min to the control level (Table 2). compared to the CCL4 treated group. Histological sections In a similar way, the
concentration of triglycerides in the CCL4 only treated
group showed significant elevation (p < 0.01) by 111.4%
compared to the control group, but this reading was sig-
nificantly decreased by 29.8% in the CCL4 + SAV group
compared to CCl4 treated group, although this was still
62.8% higher than the control concentration of triglycer-
ides (Table 2). Table 1 Histological features of the liver sections from the control,
CCL4-treated and CCL4 + SAV groups
Disruption
of lobular
architecture
Inflammatory
infiltrations
Mitotic
figures
Hepatcytic
necrosis
Dilated
CV
Control rats
0
0
0
0
0
CCl4-treated
rats
3
3
3
3
3
CCL4 + SAV
rats
1
1
0
1
1
CV Central vein Table 1 Histological features of the liver sections from the control,
CCL4-treated and CCL4 + SAV groups Effect of SAV and CCL4 on blood parameters The CCL4 group showed a significant increase in the
total number of white blood cells compared with the
control group. SAV was found to improve the total
blood cell count in the CCL4 rats. Furthermore, there
was an obvious improvement in the differential white
blood counts in the rats treated with SAV together with
CCL4 (Table 3). Page 7 of 11 Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Fig. 5 Representative histological features of the liver sections from control animal group (a, H&E, X200; b H&E, X400). A representative section
from CCL4-treated group showing the increased vacuolation (c, d; H&E X400). SAV showed a remarkable improvement in the histological architecture
of CCL4 + 100 SAV group (e, f; H&E X400). 100 SAV group (g, h; H&E X400) Fig. 5 Representative histological features of the liver sections from control animal group (a, H&E, X200; b H&E, X400). A representative section
from CCL4-treated group showing the increased vacuolation (c, d; H&E X400). SAV showed a remarkable improvement in the histological architecture
of CCL4 + 100 SAV group (e, f; H&E X400). 100 SAV group (g, h; H&E X400) Effect of SAV and CCL4 on the weight of the rats Effect of SAV and CCL4 on the weight of the rats 6 The average body weights of the rats in the different rat groups
as measured every third day from the beginning until the end of the
experiment until the end. * shows statistically significant differences at
P < 0.05 from the control. # shows statistically significant differences at
P < 0.05 between any group compared to the CCL4 group that resides in the cytoplasm and prevents NF-κB from
nuclear entry by sequestration. NF-κB is required for the
induction of pro-inflammatory cytokines, such as IL-1b,
TNF-α and IL-6 [27]. We addressed the role of NF-κB
in the present study. The oxidative stability induced by
SAV appeared to mediate the activation of NF-κB, lead-
ing to a normalization in the regulation of the inflamma-
tory cascade and the stimulation of tissue repair in SAV
rats. Additionally, gene expression of pro-inflammatory
cytokines, including TNF-α, and the protein levels of
NF-κB, were significantly increased in tissues of the
CCl4 group. Here, the imbalance between the production of ROS
and the level of protection afforded by cellular antioxi-
dants cause an oxidative stress [24]. The release of oxy-
gen radicals causes tissue damage during inflammation
[25]. We found that the level of MDA was elevated in
diseased rats while, SAV restored the oxidative markers
in these rats. Previous studies have shown that SAV
plays an active role in scavenging free radicals [26]. Indeed, a significant enhancement of glutathione and
catalase was observed in the SAV supplemented rats
in this study. In the same way, SAV has a potential impact in stimu-
lating the characteristic anti-inflammatory cytokines. SAV restored normal levels of TNF-α in diseased rats. The expression of the chemokines and adhesion mole-
cules necessary for the recruitment of inflammatory cells
to the site of injury is modulated by TNF-α [28, 29]. Effect of SAV and CCL4 on the weight of the rats The results showed (Fig. 6) the average body weight of
the rats based on measurements taken every third day
from the beginning of the experiment until the end. The
results showed a significant decrease (p < 0.05) in the
weight of rats in the CCL4 group, as well as, initially, in
the CCL4 + SAV group compared to the control group. It was also observed that animals in the CCL4−group ex-
hibited decreased appetite, diarrhoea, and a lack of activ-
ity. A significant improvement in the body weight after
treatment with SAV was observed in the CCL4-treated
rats (CCL4 + SAV), in particular during the last week of
the experiment. Recent studies have demonstrated that natural products
and/or functional food-supplementation enhances the
immune system, prevents DNA damage, and decreases
the risk of a wide range of diseases [15–17]. Ant venoms
are characterized by their high specificity and potency in
acting on molecular targets and the immune system
[18–21]. SAV was found to have the potential capability
to restore oxidative stability after CCL4 acute nephrotox-
icity [22, 23] in rats in a dose dependent manner [10]. The low dose of 100 μg/Kg body weight on three occa-
sions with two day intervals resulted in the best out-
comes from the point of view of the structure of the Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Page 8 of 11 Table 2 Liver functions, cholesterol, triglycerides, total protein
and albumin levels in different rat groups
Control
CCL4
CCL4 + SAV
SAV
ALT [IU/L]
42 ± 5.5
139 ± 9.3*
102 ± 8.7*#
60 ± 4.2#
ALP [IU/L]
180 ± 10.8
190 ± 9.7
185 ± 12.4
170 ± 10.3
AST [IU/L]
50 ± 3.2
165 ± 11.6*
110 ± 12.3*#
52 ± 6.3#
Cholesterol mg/dl
130 ± 12.5
255 ± 14.9
180 ± 11.1
135 ± 9.2
Triglycerids mg/dl
35 ± 5.2
75 ± 7.6
60 ± 5.6
30 ± 2.3
Total protein g/dl
6.5 ± 0.5
4.9 ± 0.2
5.5 ± 0.3
6.8 ± 0.4
Albumin
g/dl
3.5 ± 0.1
3 ± 0.06
3.2 ± 0.12
3.3 ± 0.15
Values are means ± standard errors. * shows statistically significant differences
at P < 0.05 from the control. Effect of SAV and CCL4 on the weight of the rats # shows statistically significant differences at
P < 0.05 between any group compared to the CCL4 group Table 2 Liver functions, cholesterol, triglycerides, total protein
and albumin levels in different rat groups Fig. 6 The average body weights of the rats in the different rat groups
as measured every third day from the beginning until the end of the
experiment until the end. * shows statistically significant differences at
P < 0.05 from the control. # shows statistically significant differences at
P < 0.05 between any group compared to the CCL4 group Values are means ± standard errors. * shows statistically significant differences
at P < 0.05 from the control. # shows statistically significant differences at
P < 0.05 between any group compared to the CCL4 group renal tissue, with a partial nut not complete recovery
[10]. Thus, the low dose may be applied for a longer
time in order to achieve a complete structural and func-
tional recovery. It is important to report that low doses
of SAV might help the rodents to adapt and optimize
their physiological systems, as is evident from the histo-
pathological evaluation in the present study. Thus, we
used here SAV at a dose of 100 μg SAV five occasions
with two day intervals. Many physiological indicators
were measured, such as blood lipid profile, oxidative
stress, antioxidant enzymes, glutathione, TNF-α, Fas and
NF-κB. renal tissue, with a partial nut not complete recovery
[10]. Thus, the low dose may be applied for a longer
time in order to achieve a complete structural and func-
tional recovery. It is important to report that low doses
of SAV might help the rodents to adapt and optimize
their physiological systems, as is evident from the histo-
pathological evaluation in the present study. Thus, we
used here SAV at a dose of 100 μg SAV five occasions
with two day intervals. Many physiological indicators
were measured, such as blood lipid profile, oxidative
stress, antioxidant enzymes, glutathione, TNF-α, Fas and
NF-κB. Fig. 6 The average body weights of the rats in the different rat groups
as measured every third day from the beginning until the end of the
experiment until the end. * shows statistically significant differences at
P < 0.05 from the control. # shows statistically significant differences at
P < 0.05 between any group compared to the CCL4 group Fig. haemoglobin, RBC red blood cells, WBC white blood cell, Neut neutrophil, Lymph lymphocytes, Mono monocytes
ues are means ± standard errors. *shows statistically significant differences at P < 0.05 from the control. # shows statistically significant
ween any group compared to the CCL4 group p
y
p
y
p
y
y
ard errors. *shows statistically significant differences at P < 0.05 from the control. # shows statistically significant differences at P < 0.0
ared to the CCL4 group Effect of SAV and CCL4 on the weight of the rats The
extrinsic signalling cascade is activated by extracellular
signals via cytokines, which include members of the NF-κB, which is stimulated by oxidative stress, is a
central regulator of inflammatory and immune re-
sponses, while IκB is a well-defined regulator of NF-κB Table 3 Changes in blood parameters caused by CCL4 challenge and SAV treatment
Blood parameters
Control
CCL4
CCL4 + 100 μg SAV
100 μg SAV
RBC X106/mm3
8.45 ± 0.11
7.75 ± 0.27
7.22 ± 0.27
7.20 ± 0.13
Hb mg/100 mg
16.10 ± 0.18
15.32 ± 0.26
14.8 ± 0.63
14.83 ± 0.23
WBC x103/mm3
9.60 ± 1.71
16.36 ± 1.17*
11.88 ± 0.97*#
10.55 ± 1.37#
Neut. x103/mm3
29.88 ± 1.25
32.72 ± 2.71
10.8 ± 0.17*#
9.55 ± 1.03*#
Lymph. x103/mm3
65.63 ± 1.30
63.10 ± 2.97
80.6 ± 0.40*#
83.60 ± 1.23*#
Mono. x103/mm3
4.50 ± 0.22
4.18 ± 0.33
8.6 ± 0.35*#
6.85 ± 0.87*#
Hb haemoglobin, RBC red blood cells, WBC white blood cell, Neut neutrophil, Lymph lymphocytes, Mono monocytes
Values are means ± standard errors. *shows statistically significant differences at P < 0.05 from the control. # shows statistically significant differences at P < 0.05
between any group compared to the CCL4 group Table 3 Changes in blood parameters caused by CCL4 challenge and SAV treatment
Blood parameters
Control
CCL4
CCL4 + 10 Hb haemoglobin, RBC red blood cells, WBC white blood cell, Neut neutrophil, Lymph lymphocytes, Mono monocytes
Values are means ± standard errors. *shows statistically significant differences at P < 0.05 from the control. # shows statistically significant differences at P < 0.05
between any group compared to the CCL4 group Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Page 9 of 11 Page 9 of 11 TNF super family, e.g., TNF and Fas ligand, and their cell
surface receptors. Fas is one of several important genes
involved in the initiation or activation of apoptotic sig-
nalling pathways. Fas also plays an important role in the
regulation of immune response. Here, the Fas protein
was elevated in the diseased rats, which may indicate its
role in the pathology of liver. Interestingly, gene expres-
sion of the pro-apoptotic receptor Fas was significantly
up-regulated following CCL4 induction in rats. The
elevated Fas level either indicates the increased suscepti-
bility of CCl4 to apoptosis [30] or points to the pro-
survival action of Fas itself. Effect of SAV and CCL4 on the weight of the rats Thus, oxidative stress may
induce cell apoptosis via Fas up-regulation and the Fas-
mediated apoptotic pathway. Park et al. [31] found that
deletion of Fas protects cells from the cytotoxic effects. In this context, it is interesting that the expression of
Fas in hepatic tissue was significantly increased in CCL4
rats, while SAV treatment was found to restore the Fas
level by a significant degree. is in accordance with a recent study that found that SAV
had a hypolipidemic effect in rats after induced disrup-
tion of these parameters by LPS injection [39]. This is
also consistent with the results reported in [40], which
showed an improvement in the antioxidant levels in the
serum of mice after injecting insect venom, as well as
with other previous results [41, 42], which confirmed
the ability of hornet venom to activate antioxidant
enzymes, and the ability of bee venom to stimulate these
same enzymes. There is an inverse correlation between
blood lipid profile and T-cell proliferative capacity in
dogs, where the reduction in total blood cholesterol,
LDL and non-HDL-cholesterol levels was correlated
with an increase in T-cell proliferation [43]. As it has a
hypolipidemic effect, SAV therefore, can encourage the
immune response. Conclusion Lopes KS, Rios RV, de Carvalho Lima CN, Linhares MI, Torres AFC, Havt A,
Quinet YP, de Franca Fonteles MM, Martins MC. The effects of the Brazilian
antDinoponera quadriceps venom on chemically induced seizure models. Neurochem Int. 2013;63:141–5. 3. Blum MS. Ant venoms: chemical and pharmacological properties. Toxicol
Toxin Rev. 1992;11:115–64. 4. Potiwat R, Sitcharungsi R. Ant allergens and hypersensitivity reactions in
response to ant stings. Asian Pacific Allergy Immunol. 2015;33:267–75. 5. Silva MF, Mota CM, Miranda VD, Oliveira Cunha AD, Silva MC, Naves KS,
Oliveira FD, Silva DA, Mineo TW, Santiago FM. Biological and Enzymatic
Characterization of Proteases from Crude Venom of the Ant Odontomachus
bauri. Toxins (Basel). 2015;7:5114–28. 6. Nikbakhtzadeh MR, Tirgari S, Fakoorziba MR, Alipour H. Two volatiles from
the venom gland of the Samsum ant. Pachycondyla sennaarensis Toxicon. 2009;54:80–2. 7. Brattin WJ, Glende EA, Recknagel RO. Pathological mechanisms in carbon
tetrachloride hepatotoxicity. Free Radic Biol. 1985;1:27–38. 8. Fujii T, Fuchs BC, Yamada S, Lauwers GY, Kulu Y, Goodwin JM, Lanuti M,
Tanabe K. Mouse model of carbon tetrachloride induced liver fibrosis:
Histopathological changes and expression of CD133 and epidermal growth
factor. BMC Gastroenterol. 2010;10:79. 9. Boveris A, Koch OR, Stoppani OM. Decreased rate of hydrogen-peroxide
production by liver-mitochondria in chronic experimental alcoholism. Med
Aires. 1978;38:647–51. 10. Ebaid H, Al-Tamimi J, Hassan I, Alhazza I, Al-Khalifa M. Antioxidant Bioactivity
of Samsum Ant (Pachycondyla sennaarensis) Venom Protects against CCL 4-
Induced Nephrotoxicity in Mice. Oxid Med Cell Longev. 2014;2014:763061. 11. Aebi H. [13] Catalase in vitro. Methods Enzymol. 1984;105:121–6. 12. Jollow DJ, Mitchell JR, al Zampaglione N, Gillette JR. Bromobenzene-induced liver
necrosis. Protective role of glutathione and evi dence for 3, 4-bromobenzene
oxide as the hepatotoxic metabolite. Pharmacology. 1974;11:151–69. 13. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by
thiobarbituric acid reaction. Anal Biochem. 1979;95:351–8. 14. Shimada K, Fujikawa K, Yahara K, Nakamura T. Antioxidative properties of
xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Agric
Food Chem. 1992;40:945–8. 15. Oyaizu M. Studies on products of browning reaction–antioxidative activities
of products of browning reaction prepared from glucosamine. Eiyogaku
zasshi= Japanese Nutr. 1986;6:307–15. 16. Ebaid H, Badr G, Metwalli A. Immunoenhancing property of dietary un-
denatured whey protein derived from three camel breeds Biologia normal inflammatory events of the healing process and
then induced normal tissue formation. Availability of data and materials 16. Ebaid H, Badr G, Metwalli A. Immunoenhancing property of dietary un-
denatured whey protein derived from three camel breeds. Biologia. 2012;67/2:425–33. All data generated or analysed during this study are included in this
published article. All data generated or analysed during this study are included in this
published article. 17. Badr G, Bashandy S, Ebaid H, Mohany M, Sayed D. Vitamin C
supplementation reconstitutes polyfunctional T cells in streptozotocin-
induced diabetic rats. Eur J Nutr. 2012;51:623–33. Author details
1D
f 1Department of Zoology, College of Science, King Saud University, P.O. Box
2455, Riyadh 11451, Saudi Arabia. 2Department of Food Science, College of
Agriculture and Food Science, King Saud University, Riyadh, Saudi Arabia. 24. Shen W, Shi D, Wand D, Guo Y, Hai S, Yue Z. Quinestrol Treatment Induced
Testicular Damage via Oxidative Stress in Male Mongolian Gerbils (Meriones
unguiculatus). Exp Anim. 2011;60:445–53. Received: 20 May 2016 Accepted: 8 November 2016 Received: 20 May 2016 Accepted: 8 November 2016
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necrosis. Protective role of glutathione and evi dence for 3, 4-bromobenzene
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care, analysed the biochemical data, prepared the results, figures, and the
oxidative stress tests. HE designed this study, supervised the practical work
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peel extract against carbon tetrachloride-induced liver injury in rats. Environ
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interpretation of data and in writing the manuscript. 15. Oyaizu M. Studies on products of browning reaction–antioxidative activities
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of Scientific Research at king Saud University for funding this Research group
NO: RGP - 1435-093. 13. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by
thiobarbituric acid reaction. Anal Biochem. 1979;95:351–8. 14. Shimada K, Fujikawa K, Yahara K, Nakamura T. Antioxidative properties of
xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Agric
Food Chem. 1992;40:945–8. Conclusion This result was
confirmed by the improvement in the histological archi-
tecture of hepatic tissues as well as the normalization of
liver functions. Taken together, the results of this work
support the hypothesis that the oxidative stability and
reduction in inflammation induced in CCl4 rats by SAV
contributes to the accelerated restoration of tissue struc-
ture and functions (Fig. 7). In conclusion, SAV can
effectively protect against CCl4-induced damage, and the
mechanisms underlying this protective effect are poten-
tially associated with improving the TNF-α/NF-κB medi-
ated inflammation in CCL4-treated rats. The potential
role of this natural product, SAV, needs to be intensively
investigated in future research focusing on patient-
oriented outcomes. 2. Lopes KS, Rios RV, de Carvalho Lima CN, Linhares MI, Torres AFC, Havt A,
Quinet YP, de Franca Fonteles MM, Martins MC. The effects of the Brazilian
antDinoponera quadriceps venom on chemically induced seizure models. Neurochem Int. 2013;63:141–5. 3. Blum MS. Ant venoms: chemical and pharmacological properties. Toxicol
Toxin Rev. 1992;11:115–64. 3. Blum MS. Ant venoms: chemical and pharmacological properties. Toxicol
Toxin Rev. 1992;11:115–64. 4. Potiwat R, Sitcharungsi R. Ant allergens and hypersensitivity reactions in
response to ant stings. Asian Pacific Allergy Immunol. 2015;33:267–75. 5. Silva MF, Mota CM, Miranda VD, Oliveira Cunha AD, Silva MC, Naves KS,
Oliveira FD, Silva DA, Mineo TW, Santiago FM. Biological and Enzymatic
Characterization of Proteases from Crude Venom of the Ant Odontomachu
bauri. Toxins (Basel). 2015;7:5114–28. 4. Potiwat R, Sitcharungsi R. Ant allergens and hypersensitivity reactions in
response to ant stings. Asian Pacific Allergy Immunol. 2015;33:267–75. 5. Silva MF, Mota CM, Miranda VD, Oliveira Cunha AD, Silva MC, Naves KS,
Oliveira FD, Silva DA, Mineo TW, Santiago FM. Biological and Enzymatic
Characterization of Proteases from Crude Venom of the Ant Odontomachus
bauri. Toxins (Basel). 2015;7:5114–28. 6. Nikbakhtzadeh MR, Tirgari S, Fakoorziba MR, Alipour H. Two volatiles from
the venom gland of the Samsum ant. Pachycondyla sennaarensis Toxicon. 2009;54:80–2. 7. Brattin WJ, Glende EA, Recknagel RO. Pathological mechanisms in carbon
tetrachloride hepatotoxicity. Free Radic Biol. 1985;1:27–38. 8. Fujii T, Fuchs BC, Yamada S, Lauwers GY, Kulu Y, Goodwin JM, Lanuti M,
Tanabe K. Mouse model of carbon tetrachloride induced liver fibrosis:
Histopathological changes and expression of CD133 and epidermal growth
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production by liver-mitochondria in chronic experimental alcoholism. Med
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Brattin WJ, Glende EA, Recknagel RO. Pathological mechanisms in carbon
tetrachloride hepatotoxicity. Free Radic Biol. 1985;1:27–38. 6.
Nikbakhtzadeh MR, Tirgari S, Fakoorziba MR, Alipour H. Two volatiles from
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2009;54:80–2. 3.
Blum MS. Ant venoms: chemical and pharmacological properties. Toxicol
Toxin Rev. 1992;11:115–64. Conclusion In CCl4 treated rats, oxidative stress causes tissue dam-
age [23]. On the other hand, SAV greatly stimulated the The results for the CCL4-injected rats revealed a clear
decline in the number of neutrophils and organ func-
tions, as has previously been found in [32, 33]. The
CCL4-treated rats also showed an increase in the con-
centration of liver enzymes and a decrease in total pro-
tein and albumin. This may be due to releasing of many
liver enzymes into the blood in the event of damage to the
membranes of liver cells [34]. In contrast, in the CCL4+
SAV group, there was an evident improvement in both the
blood profile and the liver function, indicating a positive
role for SAV in tissue repair. The evidence of the restor-
ation of liver function is particularly strikingly revealed in
the improvement in albumin levels, which is one of the
main goals of liver treatments. This improvement is due
to the ability of SAV to reform the liver tissue through its
ability to activate antioxidant enzymes and reduce oxida-
tive stress in these tissues. These results are similar to
those of Kim et al. [35] who proved the ability of bee
venom to lower the concentration of liver enzymes after a
significant increase in the serum of CCL4–injected mice. Fig. 7 Diagrammatic summary of the relationship between CCl4
effects and SAV treatment of the rats Here, a single intraperitoneal dose of CCl4 caused
severe hepatotoxicity in rats, as shown by the significant
elevation of the serum lipid profile and the increased in-
cidence of histopathological hepatic injury. This is con-
sistent with previous studies that have showed that
increased serum lipids is the most important mechanism
by which CCL4 increases oxidation in cellular mem-
branes [36, 37]. The damage in cell membranes may be
due to increased ROS that affects cell metabolism and
increases the oxidation of lipids, DNA and, protein, and
this has been confirmed by numerous studies [32, 38]. We found that SAV was effective in rejuvenating the
activity of antioxidant enzymes that had been lowered in
the hepatic tissues of CCL4−animals. This has been at-
tributed to the role of SAV inhibited free radicals. This Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 Page 10 of 11 2. References 1. Casewell NR, Wüster W, Vonk FJ, Harrison RA, Fry BG. Complex cocktails: the
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Pol. 2012;59:323–31. 28. Bickel M, Nothen SM, Freiburghaus K, Shire D. Chemokine expression in
human oral keratinocyte cell lines and keratinized mucosa. Dent Res. 1996;75:1827–34. 29. Zhang X, Kohli M, Zhou Q, Graves DT, Amar S. Short- and longterm effects
of IL-1 and TNF antagonists on periodontal wound healing. Immuno. 2004;173:3514–23. 30. Komatsu K, Manabe N, Kiso M, Shimabe M, Miyamoto H. Soluble Fas (FasB)
regulates luteal cell apoptosis during luteolysis in murine ovaries. Mol
Reprod Dev. 2003;65:345–52. 31. Park YJ, Lee S, Kieffer TJ, Warnock GL, Safikhan N, Speck M, Hao Z, Woo M,
Marzban L. Deletion of Fas protects islet beta cells from cytotoxic effects of
human islet amyloid polypeptide. Diabetologia. 2012;55:1035–47. 32. Taniguchi M, Takeuchi T, Nakatsuka R, Watanabe T, Sato K. Molecular
process in acute liver injury and regeneration induced by carbon
tetrachloride. Life Sci. 2004;75:1539–49. 33. Baligar NS, Aladakatti RH, Ahmed M, Hiremath MB. Hepatoprotective activity
of the neem-based constituent azadirachtin-A in carbon tetrachloride
intoxicated Wistar rats. Can Physiol Pharmacol. 2014;92:267–77. 34. Stacey NH, Haschek WM, Winder C. Systemic toxicology. Occup Toxicol
London Taylor Fr. 1993;42:37–76. 35. Kim SJ, Park JH, Kim KH, Lee WR, Chang YC, Park KK, Lee KG, Han SM, Yeo
JH, Pak SC. Bee venom inhibits hepatic fibrosis through suppression of pro-
fibrogenic cytokine expression. Am J Chin Med. 2010;38:921–35. 36. Demirdag K, Bahcecioglu IH, Ozercan IH, OZDEN M, Yilmaz S, Kalkan A. Role
of L-carnitine in the prevention of acute liver damage induced by carbon
tetrachloride in rats. Gastroenterol Hepatol. 2004;19:333–8. 37. Manibusan MK, Odin M, Eastmond DA. Postulated carbon tetrachloride
mode of action: a review. Environ Sci Heal Part. 2007;25:185–209. 38. Yuan C, Li Z, Yi M, Wang X, et al. Effects of Polysaccharides from Selenium-
enriched Pyracantha fortuneana on Mice Liver Injury. Med Chem (Los Angeles). 2015;11:780–8. 39. Jiang H, Wu FR, Gao JR, Chen JF. Dynamic study on curative effect of
Shuganjianpifang against hepatic fibrosis induced by CCl4 in rats. Zhong
yao cai= Zhongyaocai= Chinese Med Mater. 2014;37:1815–9. 40. Kuriakose GC, Kurup GM. Antioxidant activity of Aulosira fertilisima on CCl4
induced hepatotoxicity in rats. Indian Exp Biol. 2008;46:52. 41. Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 References Patella V, Incorvaia C, Minciullo PL, Oricchio C, Saitta S, Florio G, Saija A,
Gangemi S. Oxidative stress markers in patients with hymenoptera venom
allergy. Allergy Asthma Proc. 2015;36:e9–e13. 42. Colinet D, Cazes D, Belghazi M, Gatti JL, Poirie M. Extracellular superoxide
dismutase in insects characterization, function, and interspecific variation in
parasitoid wasp venom. Biol Chem. 2011;286:40110–21. 43. Nunes EA, Bonatto SJ, de Oliveira HH, Rivera NL, Maiorka A, Krabbe EL,
Tanhoffer RA, Fernandes LC. The effect of dietary supplementation with 9-
cis:12-trans and 10-trans:12-cis conjugated linoleic acid (CLA) for nine
months on serum cholesterol, lymphocyte proliferation and
polymorphonuclear cells function in Beagle dogs. Res Vet Sci. 2008;84:62–7. • We accept pre-submission inquiries
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https://openalex.org/W2713001338
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https://europepmc.org/articles/pmc5787849?pdf=render
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English
| null |
Production of human vitronectin in <i>Nicotiana benthamiana</i> using the <scp>INPACT</scp> hyperexpression platform
|
Plant biotechnology journal
| 2,017
|
cc-by
| 9,776
|
Summary y
Human vitronectin (hVN) is a glycoprotein that functions as a cell adhesion molecule and a
regulator of coagulation in blood plasma and the extracellular matrix. In vitro, hVN is added to
serum-free media in order to promote the adhesion of animal cells to tissue culture surfaces
and the proliferation of undifferentiated stem cells. Here, we report the production of hVN in
Nicotiana benthamiana using the inducible In Plant ACTivation (INPACT) hyperexpression
platform. N. benthamiana plants were transformed with an INPACT expression cassette
encoding hVN, and both the Tobacco yellow dwarf virus Rep/RepA activator and Tomato bushy
stunt virus p19 gene under the transcriptional control of the ethanol-inducible AlcR:alcA gene
switch. hVN expression was maximal 4–5 days postactivation of the INPACT platform with a
dilute ethanol solution, and crude yields of the recombinant protein reached a maximum of
643 78 mg/kg fresh weight. A three-stage purification protocol was developed using
heparin and polyhistidine tag affinity binding and size exclusion filtration, resulting in a plant-
made hVN product of >90% purity. Storage conditions for plant-made hVN were identified
that maximized the capacity of the recombinant protein to promote cell adhesion. Critically,
plant-made hVN was shown to be functionally equivalent to commercial, plasma-derived hVN
at promoting one-half maximal attachment of murine fibroblast cells (BALB-C/3T3) in serum-
free medium at <0.1 lg/cm2 to tissue culture plasticware. The INPACT platform represents an
attractive means of producing large quantities of functional, animal-free hVN for in vitro
applications. Keywords: biopharming, INPACT,
transgene expression, Nicotiana
benthamiana, vitronectin, inducible. promote cell adhesion and induce cell spreading (Underwood and
Bennett, 1989). Recombinant VN and its variants have also been
used in combination with defined culture media to maintain
pluripotency and growth of pluripotent stem cells (Li et al., 2010). Fusion of hVN to insulin-like growth factor (IGF), IGF-binding
proteins and epidermal growth factor significantly enhances
proliferation and migration of primary skin keratinocytes and the
re-epithelialization of wounds (Hollier et al., 2005; Upton et al.,
2008; Xie et al., 2010). As a result, clinical hVN-based treatments
for improved wound management and healing have been
developed, particularly where cell proliferation is required and/
or wound repair has been delayed, such as in burns or ulcers
(Upton et al., 2011). Further, hVN and its derivatives have been
used to promote osseointegration of implantable devices by
improving surface–cell interactions and increasing implant con-
nectivity with surrounding bone (Cacchioli et al., 2009). Summary hVN in its
native form is traditionally purified from human blood plasma. As
such, the protein must undergo strict regulatory testing for
infectious agents and has become prohibitively expensive for
applications in translational animal research. ª 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited. Production of human vitronectin in Nicotiana
benthamiana using the INPACT hyperexpression platform Benjamin Dugdale1,*, Maiko Kato1, Pradeep Deo1, Manuel Plan1,†, Mark Harrison1, Robyn Lloyd1, Terry Walsh1,
Robert Harding1 and James Dale1 1Centre for Tropical Crops and Biocommodities, Queensland University of Technology (QUT), Brisbane, QLD, Australia Received 4 April 2017;
revised 13 June 2017;
accepted 16 June 2017. *Correspondence (Tel +61 7 3138 1661;
fax + 61 7 3138 4132; email
b.dugdale@qut.edu.au)
†Present address: Metabolomics Australia
(UQ Node), Australian Institute for
Bioengineering and Nanotechnology, The
University of Queensland, St Lucia, QLD,
4072, Australia. Received 4 April 2017;
revised 13 June 2017;
accepted 16 June 2017. *Correspondence (Tel +61 7 3138 1661;
fax + 61 7 3138 4132; email
b.dugdale@qut.edu.au)
†Present address: Metabolomics Australia
(UQ Node), Australian Institute for
Bioengineering and Nanotechnology, The
University of Queensland, St Lucia, QLD,
4072, Australia. Keywords: biopharming, INPACT,
transgene expression, Nicotiana
benthamiana, vitronectin, inducible. Plant Biotechnology Journal (2018) 16, pp. 394–403 Received 4 April 2017;
revised 13 June 2017;
accepted 16 June 2017.
*Correspondence (Tel +61 7 3138 1661;
fax + 61 7 3138 4132; email
b.dugdale@qut.edu.au)
†Present address: Metabolomics Australia
(UQ Node), Australian Institute for
Bioengineering and Nanotechnology, The
University of Queensland, St Lucia, QLD,
4072, Australia. doi: 10.1111/pbi.12779 doi: 10.1111/pbi.12779 Production of transgenic N. benthamiana plants
containing an enhanced INPACT platform encoding hVN Production of transgenic N. benthamiana plants
containing an enhanced INPACT platform encoding hVN Production of transgenic N. benthamiana plants
containing an enhanced INPACT platform encoding hVN Synchronized activation of transgene amplification and expres-
sion from the INPACT platform is strongly dependent on the
regulated expression of Rep/RepA activator genes. As such, the
identification of elite N. benthamiana parent lines transformed
with the ethanol-inducible TYDV Rep/RepA cassette (pAlc-Rep/
RepA; Figure 1) was critical. Elite lines must satisfy two major
criteria: (i) minimal Rep/RepA expression in the absence of the
ethanol inducer molecule but rapid activation postethanol appli-
cation, and (ii) minimal negative physiological impact of Rep/RepA
accumulation on the plant, as overexpression of these gene
products can be phytotoxic and cause rapid yellowing and
necrosis (Dugdale et al., 2013). Transgenic N. benthamiana
plants (NbAlc-1, -2, -3, -4 and -5) containing the ethanol-
inducible
TYDV
Rep/RepA
cassette
were
generated
using
Agrobacterium tumefaciens-mediated leaf disc transformation,
acclimatized in soil and activated with a 5% (v/v) ethanol foliar
spray. Three days postethanol application, RNA was extracted
from leaves and used as the template in a reverse transcription-
polymerase chain reaction (RT-PCR) with primers specific for Rep/
RepA gene sequences. Rep/RepA transcripts, indicated by an
~750-bp RT-PCR product, were detected in four of the five plants
(NbAlc-1,
-2,
-4
and
-5)
following
ethanol
application
(Appendix S1). No PCR product was observed in RT-PCRs without
reverse transcriptase, indicating the absence of contaminating
gDNA. RNA extracted from N. tabacum line NtSRN-2 (a tobacco
line containing the same pAlc-Rep/RepA cassette) provided the
positive control for the RT-PCR. This tobacco line has been
previously shown to express Rep/RepA by quantitative real-time
PCR (qRT-PCR) following ethanol induction (Dugdale et al.,
2013). Based upon the abundance of Rep/RepA transcripts in
the RT-PCR and the absence of an abnormal phenotype associ-
ated with Rep/RepA accumulation, NbAlc-1 was selected as the
elite line for supertransformation with the modified INPACT
platform encoding hVN. We recently described the In Plant ACTivation (INPACT)
platform, an inducible, high-level expression system for trans-
genic plants based upon the disaggregated DNA genome of a
geminivirus, Tobacco yellow dwarf virus (TYDV) (Dugdale et al.,
2013, 2014). The INPACT platform is unique in that the gene of
interest is split and arranged such that its expression only occurs
from extrachromosomal episomes that are released from the host
chromosome in the presence of the TYDV-encoded replication-
associated proteins, Rep and RepA. Production of human vitronectin in plants 395 Production of human vitronectin in plants 395 processes are typically required for animal-derived protein prod-
ucts destined for use in animal or human therapeutics. However,
the economic feasibility of plant-based protein production relative
to conventional bacteria, yeast or insect cell bioreactor systems is
fundamentally dependent upon recombinant protein yield. The
use of plant viral vectors to amplify transgene copy number and
virus-derived gene products to suppress post-transcriptional gene
silencing (PTGS), an innate plant defence pathway that can
specifically target transgene mRNA for degradation, has improved
recombinant protein yields such that plants are becoming a
competitive platform for the production of biologically equivalent
proteins. Recent ‘deconstruction’ of the genomes of both DNA
and RNA plant viruses has allowed the development of plant viral
transgene expression vectors adapted for either short-term,
transient or long-term, stable protein production in plants
(reviewed in Lico et al., 2008 and Mortimer et al., 2015). Introduction Human vitronectin (hVN), formerly known as serum spreading
factor, is one of the major cell adhesion proteins found in the
blood and the extracellular matrix (Conlan et al., 1988; Shaffer
et al., 1984). Nascent hVN is converted to its mature form via
cleavage of a 19-amino acid secretion signal peptide at the
N-terminus. Circulating hVN occurs predominantly as a mono-
meric polypeptide (75 kDa) or as two polypeptides (65 and
10 kDa) linked by a disulphide bond (Tollefsen et al., 1990) and
provides a regulatory link between a wide range of biological
activities, including tissue repair, angiogenesis, haemostasis and
metastasis (Schvartz et al., 1999). hVN interacts with diverse
proteins via conserved regions located throughout the polypeptide
chain (Jin and Varner, 2004; Preissner, 1991; Zhou et al., 2003)
and the carboxyl-terminal region contains a cryptic, arginine-rich
domain that binds heparin, an anticoagulant. This domain is only
exposed after hVN binding to the thrombin–antithrombin III
complex in vivo or denaturation with urea, heat, or acid in vitro,
and it is this conformational change that activates self-association
of hVN into multimers with significantly increased affinity for
heparin compared to the monomeric form (Preissner, 1991). Plant-based protein production is a convenient means of
manufacturing therapeutics and nontherapeutics normally iso-
lated from animal sources, including blood plasma or tissues. Production in plants minimizes the risk of contamination with
animal pathogens and obviates the need for expensive serological
screening and more extensive purification. Such quality control Vitronectin (VN) has been isolated from a range of mammalian
sources and utilized for both research and clinical applications
because of its adhesive properties. VN is most commonly used in
animal cell culture to coat tissue culture surfaces in order to 394 Production of transgenic N. benthamiana plants
containing an enhanced INPACT platform encoding hVN Temporal control of Rep/
RepA expression is achieved using the ethanol-responsive alc
gene switch. Essentially, the INPACT platform provides the
benefits of transient transgene expression in a stably transformed
plant, thereby disconnecting plant growth from recombinant
protein production. Nicotiana benthamiana transformed with an
INPACT platform encoding hVN and activated with a dilute
ethanol solution reached maximum crude yields of ~100 mg hVN/
kg fresh weight (FW) (Dugdale et al., 2013). In the present study,
we have integrated the gene encoding Tomato bushy stunt virus
(TBSV) p19, a suppressor of PTGS, into the INPACT platform and
describe
its
effects
on
recombinant
hVN
accumulation
in
N. benthamiana. The inclusion of TBSV p19 into the INPACT
expression platform significantly increased maximum production
of recombinant hVN by ~sixfold. A simple, three-stage hVN
purification process was developed, and the ability of plant-made
hVN to promote the attachment of murine fibroblast cells to
tissue culture surfaces was compared to commercial hVN isolated
from human plasma. The results demonstrate the potential of the
INPACT expression platform for biopharming animal proteins in
planta. Agrobacterium-mediated transformation was used to super-
transform leaf discs from NbAlc-1 with the modified INPACT
platform encoding hVN and containing the TBSV p19 gene
under
the
transcriptional
control
of
the
alcA
promoter
(pINPACT-hVN2, Figure 1). To facilitate accumulation of hVN,
the native N-terminal secretion signal was preserved and an ER alcAP
nosT
nosT
35SP
alcR
TYDV Rep/RepA
TYDV LIR
alcAP
TBSV p19
nosT
nosT
Syntron 3’ half
TYDV SIR
hVN exon 2
Syntron 5’ half
TYDV LIR
hVN exon 1
35SP
pAlc-Rep/RepA
pINPACT-hVN2
Native secretion signal
6XHIS-KDEL
Figure 1 Schematic representation of the ethanol-inducible Rep/RepA activator cassette (pAlc-Rep/RepA) and the modified INPACT cassette encoding
hVN and p19 (pINPACT-hVN2). 35SP = CaMV 35S promoter, nosT = nopaline synthase gene terminator, alcAP = alcA:minimal CaMV 35S promoter fusion,
alcR = gene encoding the alcohol receptor transcription factor, TYDV Rep/RepA = gene encoding the Tobacco yellow dwarf virus Rep/RepA activator
proteins, TBSV p19 = gene encoding the Tomato bushy stunt virus 19 K protein, TYDV LIR = Tobacco yellow dwarf virus large intergenic region,
syntron = synthetic intron, hVN exon = part of gene encoding the human vitronectin protein, TYDV SIR = Tobacco yellow dwarf virus small intergenic
region, 6XHIS = polyhistidine affinity tag, KDEL = ER retention signal. Identification of elite INPACT supertransformed lines Detached leaves from the eleven transgenic N. benthamiana
lines were excised and tested for ethanol-induced accumulation
of recombinant hVN. Leaves were incubated on MS0 solid
media with small wells containing 5% (v/v) ethanol. Total
soluble protein (TSP) was extracted 5 days postactivation and
recombinant hVN levels determined by immunoblotting with an
hVN-specific monoclonal antibody. One line, T0-2, was identi-
fied as a high hVN-expressing INPACT plant (results not shown)
and grown to maturity. Southern hybridization analysis using
probes specific for either the nptII selection gene (within the
pAlc-Rep/RepA T-DNA) or hVN gene (within the pINPACT-hVN2
T-DNA) showed this elite line contained a single integrated copy
of
both
the
pAlc-Rep/RepA
and
pINPACT-hVN2
cassettes
(Appendix S2). Line T0-2 was selfed, and the resulting 16 T1
generation events were screened for hVN accumulation using
the same method as was used to analyse the T0 events. Three
events expressing the highest levels of hVN (T1-8, T1-13 and T1-
15) were selected by immunoblotting (Appendix S3). These lines
were selfed and three T2 generation events (T2-1, T2-2 and T2-3)
identified as high-expressing hVN plants. All generations of
transgenic plants developed normally and appeared phenotyp-
ically similar to wild-type N. benthamiana plants in tissue culture
and soil. Normal growth and development of transgenic plants
was also observed in subsequent T3 generation plants grown in
soil (Appendix S4). Figure 2 RT-PCR to detect p19 and Rep/RepA expression in high-
expressing INPACT lines following ethanol activation. Detached leaves of T2
generation INPACT lines (T2-1, T2-2, and T2-3) were activated with a 0.5%
(v/v) ethanol solution and sampled at 0 and 5 days postactivation. RNA was
isolated from leaf samples and p19 (top panel), Rep/RepA (middle panel)
and actin housekeeping (bottom panel) gene expression detected by RT-
PCR. Amplified products were electrophoresed through a 1.5% agarose
and stained with SYBR Safe DNA gel stain (Life Technologies). For PCR
controls, water was used as the negative control (ve) and 50 ng of
plasmid DNA containing the p19 gene (with syntron), TYDV RepRepA or
actin genes were used as the positive controls (+ve). RT-PCR without
reverse transcriptase (); RT-PCR with reverse transcriptase (+);
M = GeneRuler 1-kb DNA ladder (Life Technologies); WT, wild type. Molecular weights marked in kb. Ethanol-activated expression of p19 and Rep/RepA in
elite INPACT plants Detached leaves from T2 progeny plants, designated T2-1, T2-2
and T2-3, were activated in vitro by incubation in liquid MS0
media containing 0.5% (v/v) ethanol for 5 days. RNA was
extracted from leaves pre- (Day 0) and postethanol (Day 5)
activation and analysed by RT-PCR (Figure 2). p19 transcripts
(as indicated by a ~ 550-bp RT-PCR product) were detected in
all transgenic progeny at Day 0, suggesting ‘leaky’ expression
in the absence of the ethanol inducer molecule (Figure 2a). The
relative
abundance
of
these
RT-PCR
products,
however,
increased by Day 5 suggesting that the addition of ethanol
does increase alcA-directed p19 expression. In contrast, no Rep/
RepA expression was observed prior to ethanol activation, and
Rep/RepA transcripts were relatively abundant after activation
(as indicated by a ~450-bp RT-PCR product; Figure 2b). The
sizes of the RT-PCR products for both p19 and Rep/RepA were
smaller than the PCR products amplified using plasmid DNA as
templates,
indicating
correct
processing
of
both
the
syn-
thetic and TYDV Rep introns, respectively. No RT-PCR products
were observed in reactions using wild-type N. benthamiana
RNA
as
the
template. RT-PCR
products
(~400 bp)
were
obtained
from
all
plant
RNAs
using
primers
designed
to
amplify the actin housekeeping gene (Figure 2c). No RT-PCR
products
were
observed
in
the
absence
of
the
reverse
transcriptase enzyme. Production of transgenic N. benthamiana plants
containing an enhanced INPACT platform encoding hVN TYDV LIR
alcAP
TBSV p19
nosT
nosT
Syntron 3’ half
TYDV SIR
hVN exon 2
Syntron 5’ half
TYDV LIR
hVN exon 1
35SP
pINPACT-hVN2
Native secretion signal
6XHIS-KDEL Figure 1 Schematic representation of the ethanol-inducible Rep/RepA activator cassette (pAlc-Rep/RepA) and the modified INPACT cassette encoding
hVN and p19 (pINPACT-hVN2). 35SP = CaMV 35S promoter, nosT = nopaline synthase gene terminator, alcAP = alcA:minimal CaMV 35S promoter fusion,
alcR = gene encoding the alcohol receptor transcription factor, TYDV Rep/RepA = gene encoding the Tobacco yellow dwarf virus Rep/RepA activator
proteins, TBSV p19 = gene encoding the Tomato bushy stunt virus 19 K protein, TYDV LIR = Tobacco yellow dwarf virus large intergenic region,
syntron = synthetic intron, hVN exon = part of gene encoding the human vitronectin protein, TYDV SIR = Tobacco yellow dwarf virus small intergenic
region, 6XHIS = polyhistidine affinity tag, KDEL = ER retention signal. nt Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 16, 394–403 ª 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 16, 394–403 ª 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley Benjamin Dugdale et al. 396 0.5
0.5
0.5
– + –
+ – + – + – + – + – + – + –ve +ve
M
WT T2-1 T2-2 T2-3 WT T2-1 T2-2 T2-3
DAY 0
DAY 5
(a)
(b)
(c) retention signal (KDEL) added to the C-terminus. For purifica-
tion purposes, a C-terminal polyhistidine affinity tag (6XHIS) was
also included. Following cleavage of the 19-amino acid secre-
tion signal, plant-made hVN has an approximate size of 469
amino acids and an estimated glycan-free molecular weight of
53.63 kDa. Eleven
independent
N. benthamiana
lines
were regenerated on media containing both kanamycin and
hygromycin. (a) Identification of elite INPACT supertransformed lines ª 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 16, 394–403 Estimation of hVN crude yield Yield estimates of plant-made hVN were obtained using T3
generation plantlets. Seed from three independent T2 generation
plants (T2-1, T2-2 and T2-3) were sown onto MSO media
containing both kanamycin and hygromycin antibiotics and
100% of the seed germinated within 1 week. This suggested
all T3 generations plants contained both gene cassettes. Eight
seedlings representing each T2 parent plant were randomly
selected. Detached leaves from in vitro plants were activated for
5 days in liquid MS0 media containing 0.5% (v/v) ethanol. Leaf
material from the eight seedlings was pooled (designated Batches
T3-1, T3-2 and T3-3) and hVN levels in the TSP compared to
known quantities of commercial, pure hVN (Promega) by SDS-
PAGE and immunoblotting (Figure 3c). The use of denaturing
buffer to extract plant-made hVN and the immediate analysis of
these extracts by SDS-PAGE resulted in a single 75-kDa band on
the immunoblot. The commercial hVN standard appeared as a
doublet because human-derived hVN is clipped into two major
products of 75 kDa and 65 kDa. A ChemiDoc imaging system
(Bio-Rad) was used to estimate the amount of hVN in TSP extracts
by comparing signal intensity to the hVN standard curve ranging
from 50 to 500 ng. This entire process was performed three times
on separate occasions. The estimated hVN yields (expressed as
mean standard error) from Batches T3-1, T3-2 and T3-3 over
three independent experiments were 577 162, 709 155,
642 40 mg/kg (FW) of leaf, respectively (Table 1). Statistical
analysis indicated there was no significant difference in yields
between batches of T3 generation seedlings (P > 0.05), suggest-
ing consistent activation and expression in this generation of
plants. The overall estimated average hVN yield from T3 gener-
ation plants was 643 78 mg/kg FW of leaf. 3 Figure 3 Time course and dose–response of ethanol activation for
maximal INPACT-based hVN expression and crude yield estimation. Detached leaves of elite T0 generation INPACT line, T0-2, were activated in
liquid MS0 media containing ethanol. For time-course kinetics, leaves were
incubated in 0.5% (v/v) ethanol solution and sampled 3, 4, 5, 6, and
7 days postactivation (Panel a). For dose–response, leaves of line T0-2 were
incubated in different concentrations (0.1, 0.25, 0.5, 1.0 and 2.0% v/v) of
ethanol and sampled 5 days postactivation (Panel b). Samples in Panels a
and b were extracted under nondenaturing conditions and TSP was
normalized by Bradford protein assay. Estimation of hVN crude yield TSP (5 lg) was separated by SDS-
PAGE. For estimation of crude hVN yields in elite INPACT plants, detached
leaves from three batches (marked T3-1, T3-2 and T3-3) of T3 generation
plantlets (eight plants from each T2 parental line) were incubated in 0.5%
(v/v) ethanol solution and sampled 5 days postactivation (Panel c). TSP was
extracted under denaturing conditions. TSP (5 lg) was separated by SDS-
PAGE. A standard curve of hVN (Promega) ranging from 50 to 500 ng was
included for comparative yield estimates. Levels of hVN expression were
determined by immunoblotting. Immunoblot signals were detected and
measured using a ChemiDoc imaging system (Bio-Rad). Molecular weights
marked in kDa. Production of human vitronectin in plants Production of human vitronectin in plants 39 397 T3-1 T3-2 T3-3 50 100 200 400 500
hVN standard (ng)
50
3 4 5 6 7
Days postethanol
application
0.1 0.25 0.5 1.0 2.0
% ethanol
103
77
50
103
77
(a)
(c)
(b) Kinetics of recombinant hVN accumulation and ethanol
dose–response The kinetics of recombinant hVN accumulation were assessed by
incubating transgenic leaf material from elite T0 generation line T0-
2 in liquid MS0 solution containing 0.5% (v/v) ethanol for 3, 4, 5, 6
and 7 days. hVN accumulation in leaf total soluble protein (TSP)
extracts was measured by immunoblotting (Figure 3a). Maximum
accumulation of the 75-kDa form of recombinant hVN was
observed 4–5 days after INPACT activation based on immunoblot
signal intensities. Ethanol dose–response was assessed by incu-
bating leaf material from the same plant in liquid MS0 solution
containing increasing concentrations of ethanol (0.1%, 0.25%,
0.5%, 1.0% and 2.0% (v/v)). Five days after activation, hVN
accumulation was measured in leaf TSP extracts by immunoblot-
ting (Figure 3b). Maximum accumulation of the 75-kDa form of
recombinant hVN was observed using 0.5% (v/v) ethanol based on
immunoblot signal intensity. Interestingly, high molecular weight
hVN forms and an ~60 kDa hVN degradation product were also
observed after immunoblotting. These extraneous hVN forms are
likely multimers of hVN formed by self-association under nonde-
naturing extraction conditions and a proteolytic cleavage product
as a result of the freeze-and-thaw process prior to SDS-PAGE and
immunoblotting, respectively. Purification of recombinant hVN from N. benthamiana Human vitronectin was purified from N. benthamiana using a
three-stage process based upon both affinity chromatography
and size exclusion filtration. Samples from each key step
(Figure 4) and all steps (Appendix S5) of the purification process
were separated by SDS-PAGE and visualized using Coomassie
Blue dye. N. benthamiana leaf proteins were solubilized in 9 M
urea to induce the conformational change in hVN that increases
heparin affinity (Figure 4, lane 1). Denatured hVN was resolved
from the majority of contaminating N. benthamiana leaf proteins
by heparin affinity chromatography (Figure 4, lane 2). The purity
of hVN after elution from the heparin affinity matrix was ~70%. hVN was further purified using metal affinity chromatography
(Figure 4, lane 3). The purity of hVN after elution from the metal
affinity matrix with 150 mM imidazole was ~80%–90%. hVN was
separated from low molecular weight (<30 kDa) contaminants
and concentrated to ~0.5 mg/mL using centrifugal ultrafiltration Table 1 Estimation of crude hVN yields in T3 generation INPACT plants
Yield (mg hVN/kg FW leaf)
T3 Generation plants
Experiment 1
Experiment 2
Experiment 3
Mean Standard Error
Batch T3-1
370.9
973.5
385.6
577 162
Batch T3-2
874.0
923.2
330.8
709 155
Batch T3-3
581.6
740.1
605.2
642 40
Average overall yield
643 78 Table 1 Estimation of crude hVN yields in T3 generation INPACT plants stimation of crude hVN yields in T3 generation INPACT plant ª 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 16, 394–403 Benjamin Dugdale et al. 398 (Figure 4, lane 4). The purity of concentrated hVN was estimated
to be >90% at a final yield of between 30 and 128 mg/kg FW of
transgenic N. benthamiana leaf. increasing concentrations of both plant-made hVN and commer-
cial hVN. Maximum fibroblast adhesion (4 9 104 cells/well) was
observed at 0.4 lg hVN/cm2, and half-maximum fibroblast
adhesion occurred at ~0.05 lg hVN/cm2. The capacity of plant-
made hVN to promote fibroblast adhesion was statistically
equivalent to that of the commercial product over all concentra-
tions tested (P > 0.05). The identity of the purified protein from transgenic N. ben-
thamiana leaf was confirmed by N-terminal amino acid sequenc-
ing (Figure 5). No amino acid was detected on the fifth of seven
cycles of Edman degradation. Discussion Plant-
made hVN was further subjected to size exclusion filtration using an
Amicon Ultra Centrifugal Filter (Ultracel-30K) in order to remove low
molecular weight protein contaminants and concentrate the product (lane
4). Protein samples representing each major step of the 3-stage extraction
protocol were electrophoresed through a 12% SDS-polyacrylamide gel
and stained with Coomassie Brilliant Blue R-250. M = prestained SDS-
PAGE standards, low range (Bio-Rad). Molecular weights marked in kDa. The INPACT platform provides the benefits of high-level,
transient transgene expression in a stably transformed plant,
whereby extrachromosomal copies of the INPACT transgene
expression cassette are released, amplified, and transcribed only
in the presence of ethanol and the Rep/RepA activator proteins. However, transient overexpression of heterologous genes can
trigger PTGS, an innate plant pathogen defence mechanism that
can significantly reduce steady state levels of recombinant protein
accumulation (Voinnet, 2001; Voinnet et al., 1998; Waterhouse Discussion Vitronectin is primarily used to promote cell attachment and
proliferation in vitro but also has other therapeutic and nonther-
apeutic applications. These applications, however, are limited by
the high cost of the native protein due to the extensive
purification and serological testing required to ensure the
absence of bloodborne pathogens. As such, in planta production
of recombinant hVN that is functionally equivalent and free from
animal pathogens by virtue of its source offers a cost-effective
alternative. We have previously demonstrated inducible produc-
tion of recombinant hVN in transgenic N. benthamiana plants
using the In Plant ACTivation (INPACT) platform (Dugdale et al.,
2013). Soil-acclimatized plants or detached leaves were activated
with ethanol, and maximal hVN expression was detected by
immunoblotting 4–5 days postapplication. Recombinant hVN
was partially purified from leaves using heparin affinity chro-
matography, and crude yields were estimated at ~100 mg hVN/
kg FW of leaf. In the present study, we have dramatically
improved hVN yield by modification of the INPACT platform,
developed an efficient recombinant hVN purification protocol,
and demonstrated that recombinant hVN produced in transgenic
N. benthamiana is functionally equivalent to commercial, human
plasma-derived VN. Figure 4 Purification of recombinant hVN from N. benthamiana using a
three-stage extraction protocol. Total leaf protein solubilized in 9 M urea
(lane 1) was passed through a column containing Heparin Sepharose 6
Fast Flow resin and bound hVN eluted under high salt (500 mM NaCl)
conditions (lane 2). HIS-tagged hVN was then immobilized in TALON
Superflow Metal Affinity Resin and eluted using imidazole (lane 3). Plant-
made hVN was further subjected to size exclusion filtration using an
Amicon Ultra Centrifugal Filter (Ultracel-30K) in order to remove low
molecular weight protein contaminants and concentrate the product (lane
4). Protein samples representing each major step of the 3-stage extraction
protocol were electrophoresed through a 12% SDS-polyacrylamide gel
and stained with Coomassie Brilliant Blue R-250. M = prestained SDS-
PAGE standards, low range (Bio-Rad). Molecular weights marked in kDa. Figure 4 Purification of recombinant hVN from N. benthamiana using a
three-stage extraction protocol. Total leaf protein solubilized in 9 M urea
(lane 1) was passed through a column containing Heparin Sepharose 6
Fast Flow resin and bound hVN eluted under high salt (500 mM NaCl)
conditions (lane 2). HIS-tagged hVN was then immobilized in TALON
Superflow Metal Affinity Resin and eluted using imidazole (lane 3). Purification of recombinant hVN from N. benthamiana However, the presence of Cys-S-b-
propionamide (Cys-S-Pam), the product of cysteine alkylation by
acrylamide under alkaline conditions, after the fifth Edman
degradation cycle, suggested the presence of a cysteine residue. The resulting amino terminal sequence is identical to that
predicted following cleavage of the native 19-amino acid hVN
secretion signal (Figure 5). Plant-made, purified hVN was stored for 7 weeks as a liquid at
4 °C, a frozen liquid at 80 °C, and freeze dried powder at
80 °C to determine the effects of long-term storage on protein
activity. The capacity of plant-made hVN to promote cell adhesion
in tissue culture after storage was compared with commercial,
plasma-derived hVN (Promega) (Figure 6b). Maximum fibroblast
adhesion for all hVN forms was observed at ~1.0 lg hVN/cm2. Half-maximum fibroblast adhesion for stored plant-derived hVN
was ~0.2 lg hVN/cm2, compared to ~0.1 lg hVN/cm2 for the
commercial control. The concentrations of both commercial and
plant-made hVN that promoted maximum and half-maximum cell
adhesion
were
significantly
higher
than
was
observed
in
Figure 6a, most likely because of variations in tetrazolium salt
uptake
by
cells
using
MTT
[3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide] versus MTS [3-(4,5-dimethylthia-
zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium]. Of the three conditions tested, storage of plant-made
hVN as a frozen liquid at 80 °C was the most effective at
retaining adhesion promoting activity. Activity of hVN stored in
this manner was statistically equivalent to the commercial product
in the range of 0.2–1.6 lg/cm2. Adhesive properties of plant-made hVN The capacity of plant-made hVN to promote cell adhesion in
tissue culture was assessed using murine fibroblast cells (BALB-C/
3T3) (Figure 6a). Commercial, plasma-derived hVN (Promega)
was used as a control. Fibroblast binding increased with M 1 2 3 4
103
81
47
34
27
17 M 1
103
81
47
34
27
17 3 ª 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 16, 394–403 ª 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists a MAPLRPLLILALLAWVALA /DQESCKG…
DQES─KG… Figure 5 Comparison of the N-terminal sequence of plant-made hVN
with native hVN. The native hVN 19 amino acid secretion signal is
underlined and cleavage site marked (/). The blank amino acid read at
position 5 of plant-derived hVN is marked (─). Production of human vitronectin in plants 399 (a)
(b) (a) bushy stunt virus (TBSV), is known to sequester double-stranded,
small interfering RNA duplexes with high affinity, and co-
expression of this protein has been shown to significantly increase
transient heterologous gene expression (Sainsbury and Lomonos-
soff, 2008; Sainsbury et al., 2009). However, p19 itself is
phytotoxic and the generation of stably transformed plants
expressing p19 is challenging (Scholthof, 2007). While a recom-
binant p19 mutant (R43W) that does not induce phytotoxicity has
been developed (Saxena et al., 2011), this mutant form confers
only a modest (1.7-fold) increase in transgene expression levels. Therefore, in order to fully exploit the benefits of the TBSV p19
protein in transgenic plants, we integrated ethanol-inducible
expression of the TBSV p19 gene into the INPACT platform for
the production of hVN. We observed low level, ‘leaky’ p19
transcription in the absence of ethanol induction but there was
no evidence of a negative impact upon phenotypic parameters
such as plant growth or development in the T0 to T3 generation
events. Further, we demonstrated that co-expression of p19 and
hVN increased recombinant hVN accumulation up to sixfold,
relative to the unmodified INPACT platform. This suggests that
PTGS is likely a limiting factor for INPACT-based recombinant
protein expression, but it can be overcome by inducible expres-
sion of a PTGS suppressor gene. (b) Intracellular targeting of recombinant proteins can significantly
influence the steady state levels to which they accumulate in
planta (Harrison et al., 2011; Streatfield et al., 2003). We
previously tested whether localized accumulation of recombinant
hVN in intracellular compartments greatly influenced its yield, by
targeting the protein to the cytoplasm, endoplasmic reticulum
(ER), apoplast, mitochondria or chloroplast. We found that hVN
retention in the ER resulted in maximum accumulation in
N. tabacum leaves (results not shown). Native hVN is glycosylated
at three sites which accounts for as much as 30% of the mass of
the mature protein (Schvartz et al., 1999), and changes in hVN
glycosylation alter its ability to form multimers and bind collagen
(Sano et al., 2007). MAPLRPLLILALLAWVALA /DQESCKG…
DQES─KG… However, glycosylation does not appear
essential for some of its biological activities as glycan removal can
increase collagen binding (Sano et al., 2007) and bacteria-made
VN has been used in chemically defined animal cell culture
systems (Chen et al., 2011). In the present study, the native hVN
amino-terminal secretion signal was preserved so that the
recombinant protein would enter the ER and an ER retention
signal (KDEL) added to the carboxyl-terminus of hVN to enhance
accumulation. Figure 6 Adhesive properties of plant-made hVN and effects of long-
term storage. Panel (a): The ability of plant-made hVN (open circles) to
promote the attachment of BALB-C/3T3 cells in serum-free medium to
tissue culture plasticware was compared to commercial, plasma-derived
hVN (closed circles). MTS absorbance readings at 490 nm are directly
proportional to the number of viable cells bound to the plasticware
surface. Panel b: Plant-made hVN was stored long term for 7 weeks as (i) a
liquid at 4 °C (open triangles), (ii) frozen liquid at 80 °C (open circles) or
(iii) lyophilized at 80 °C (closed triangles) and its ability to promote the
attachment of BALB-C/3T3 cells to tissue culture plasticware compared to
commercial, plasma-derived hVN (closed circles). MTT absorbance
readings at 570 nm are directly proportional to the number of viable cells
bound to the plasticware surface. Samples were analysed in triplicate, and
error bars indicate mean standard deviation. We have developed a small-scale production system using
leaves harvested from transgenic plants maintained in vitro and
activated with ethanol in solution. Elite T0 generation N. ben-
thamiana INPACT events expressing high levels of recombinant
hVN were micro-propagated in tissue culture using a rapid axillary
shoot induction method (Deo et al., 2015) that allowed gener-
ation of ~200 individual plants. We routinely produced ~150 g of
N. benthamiana leaf mass per week using a rotating subculture
system and accumulated >3 kg of leaves in a 5-month period. Detached leaves were activated in tissue culture containing a
liquid growth media supplemented with 0.5% (v/v) ethanol and
harvested for purification after 5 days when hVN accumulation
was at its peak. Activated leaves could be stored at 80 °C or
used immediately for hVN extraction. Laboratory-scale purification of recombinant hVN was routinely
undertaken using 10–30 g of N. benthamiana leaves, but we
have successfully scaled up purification to 100 g quantities of
leaves in the present study. ª 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 16, 394–403 rnal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 16, 394–403 Vector construction Leaf samples were immediately snap-frozen in liquid nitrogen
following harvesting. Tri reagent (Sigma-Aldrich, Castle Hill, NSW,
Australia) was used to extract total RNA from tissue according to the
manufacturer’s instructions and the method of Azevedo et al. (2003). Oligo (dT) 18 primer was used to synthesize first-strand
complementary DNA from total RNA using M-MLV Reverse Tran-
scriptase (Promega, Alexandria, NSW, Australia) according to the
manufacturer’s instructions. Reactions were also prepared without
reversetranscriptasetoconfirmtheabsenceofcontaminatinggDNA. PCRs were performed using GoTaq Green master mix (Promega) and
the following cycling conditions 94 °C for 5 min followed by 29
cyclesof94 °Cfor30 s,55 °Cfor30 s,72 °Cfor30 s,and72 °Cfor
10 min. Primer sets were as follows; TYDV Rep/RepA–Rep/RepA-F
(50-TCAGACTGGCAACCTATT-30) and Rep/RepA-R (50-GCGAACT
ATTATCCAGAC-30), wild-type TBSV p19–P19-F (50-CCATGGAA
AGGGCTATTCAGGGAAATGATG-30) and P19-R (50-GAGCTCTC
ACTCGGATTCTTTCTCAAAGTC-30), and actin–Actin-F (50-CTATTCT
CCGCTTTGGACTTGGCA-30) and Actin-R (50-AGGACCTCAGGAC
AACGGAAACG-30). The CaMV 35S promoter (35SP) controlling expression of the
hygromycin
B
phosphotransferase
(hph)
selection
gene
in
pCAMBIA1300 was replaced with the nopaline synthase gene
promoter (nosP). The nosP and hph genes were amplified by PCR
and fused using overlapping PCR with the following primer pairs;
hph-F
(50-TCTCCGCTCATGATCATGAAAAAGCCTGAACTCACC
GCGA-30) and hph-R (50-CTCGAGCTTGTCGATCGACAGATCCG
GTCGGCATC-30), and nosP-F (50-GAATTCTCTAGACACGTGAGA
TCCGGTGCAGATTATTTGGATTGAGAGTG-30) and nosP-R (50-TT
CAGGCTTTTTCATGATCATGAGCGGAGAATTAAGGGAG-30). The
resulting nosP-hph fusion was ligated into pCAMBIA1300 using
XhoI and EcoRI restriction sites. An INPACT expression cassette
encoding the GUS reporter gene was excised from pINPACT-GUS
(Dugdale et al., 2013) and ligated into the above vector using
EcoRI/HindIII restriction sites. The nearly complete INPACT cassette
encoding hVN (with native secretion signal, KDEL retention signal
and polyhistidine affinity tag) was then excised from pINPACT-
hVN (Dugdale et al., 2013) and ligated into the vector using SwaI/
PacI restriction sites to create pINPACT-hVN-nos. Wild-type TBSV
p19 (GenBank Accession M21958.1) was codon modified to
include human and N. tabacum first preferred codons and an 84-
bp synthetic intron (syntron; Dugdale et al., 2013) between the
AG/GT at nucleotide position 201. The modified p19 gene was
chemically synthesized by GeneArt (Life Technologies, Mount
Waverley, VIC, Australia) and ligated upstream of the nopaline
synthase gene terminator (nosT) in the plasmid pACN2 using
unique PstI restriction sites. The final INPACT hVN expression MAPLRPLLILALLAWVALA /DQESCKG…
DQES─KG… Interestingly, higher plants encode et al., 1998). In order to overcome this, virus-encoded suppres-
sors of PTGS can be co-expressed with the gene of interest
(Voinnet et al., 1999). One such suppressor, p19 from Tomato Benjamin Dugdale et al. 400 hVN analogues that function in plasma membrane–cell wall
adhesion (Zhu et al., 1993), bacterium–plant interaction (Wagner
and Matthysse, 1992) and pollen tube extension (Sanders et al.,
1991). One such protein, tobacco PVN1 (plant vitronectin-like 1),
is predominantly localized to the cell wall and has been shown to
bind both glass and heparin (Zhu et al., 1994). N. benthamiana
encodes a protein with 97% similarity to tobacco PVN1 (Nakasugi
et al., 2013) and contains an RYD motif with functional similarity
to the hVN RGD binding domain. Therefore, a second affinity
chromatography step (i.e. immobilized metal) was included in our
hVN purification process to minimize the likelihood of contam-
ination with N. benthamiana PVN1. We observed that recombi-
nant hVN yield was significantly affected by the age and
physiological status of plants in vitro. Leaves obtained from
freshly propagated N. benthamiana in vitro plants were generally
larger, appeared healthier and yielded the maximum level of
>90% pure recombinant hVN (128 mg/kg FW). In contrast, leaves
from the third harvest and beyond were generally smaller, more
chlorotic and had reduced hVN content (30 mg/kg FW). vector was constructed by three-way ligation of the following
fragments: Pmll/XbaI digested pINPACT-hVN-nos backbone, PmlI/
BamHI
digested
alcA
promoter
sequence
and
BamHI/XbaI
digested p19-nosT sequence from pACN2. The resulting vector
was designated pINPACT-hVN2 (Figure 1). Construction of the vector pAlc-Rep/RepA, a pBIN-based vector
backbone containing (i) the TYDV Rep/RepA activator genes
downstream of the alcA promoter, (ii) the alcR transcription
factor gene under the transcriptional control of 35SP and (iii) the
neomycin phosphotransferase (nptII) resistance gene for kana-
mycin selection of transformed plant cells, has been previously
described (Figure 1; Dugdale et al., 2013). Stable transformation of N. benthamiana All
vectors
for
stable
transformation
were
mobilized
into
Agrobacterium tumefaciens (strain LBA4404) by electroporation. A. tumefaciens-mediated transformation of N. benthamiana leaf
discs and their regeneration were as described by Horsch et al. (1985). Transgenic plants containing the pAlc-Rep/RepA cassette
were selected and regenerated in media containing kanamycin
(200 lg/mL). Ethanol-inducible expression of the Rep/RepA genes
in these lines was assessed by RT-PCR. Leaves from event NbAlc-1
were subsequently used for supertransformation with recombi-
nant A. tumefaciens harbouring pINPACT-hVN2. To ensure
supertransformed plants contained both pAlc-Rep/RepA and
pINPACT-hVN2 expression cassettes and were independent
events, plantlets were excised from different leaf pieces and
regenerated in media containing both kanamycin (200 lg/mL)
and hygromycin (25 lg/mL). In vitro or soil-acclimatized plants
were maintained in a controlled environment chamber with a 16-
h photoperiod at 25 °C and grown to the 8- to 10-leaf stage prior
to harvest and ethanol activation. Optimization of the molecular features of the INPACT platform
resulted in a sixfold increase in recombinant hVN accumulation
compared to the unmodified INPACT platform, and the develop-
ment of an efficient extraction protocol produced a plant-made
hVN of >90% estimated purity that was functionally equivalent to
the native human protein. While transient agroinfiltration-based
expression remains the preferred method for rapid recombinant
protein production in plants, there is still an obvious need for
extended, large-scale production capacities. The elite transgenic
INPACT plants and the seed bank generated in this study
represent a permanent genetic resource for the consistent and
high-level production of this valuable protein into the future. ª 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 16, 394–403 Cell adhesion assay Plant-made and plasma-derived hVN (Promega) were diluted in
Dulbecco’s PBS (DPBS, Life Technologies) to concentrations
ranging between 1.6, 0.800, 0.400, 0.200, 0.100, 0.050,
0.02500, 0.0125 and 0 lg/cm2 in 100 lL. Wells of a Nunc
MaxiSorp flat-bottom 96-well plate (Sigma) were coated with
100 lL of each hVN dilution in triplicate. Plates were incubated
for 2 h at room temperature then rinsed 3 times with 250 lL of
DPBS per well. After rinsing, 200 lL of DPBS blocking solution
(DPBS with 2 mg/mL bovine serum albumin (BSA)) was added to
each well and the plate was incubated for 1 h at room
temperature. The BSA blocking solution was removed prior to
adding animal cells. Three-day-old BALB-C/3T3 cells were har-
vested by trypsinization and pelleted by centrifugation at 200 g
for 5 min at room temperature. Cells were resuspended in 1 mL
Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v)
foetal bovine serum (Life Technologies). Viable cell counts were
estimated using trypan blue exclusion dye (Life Technologies) and
cell concentrations adjusted to 4 9 105 cells/mL. An aliquot
(100 lL) of cell suspension (4 9 105 cells/mL) was added to each
hVN-coated well, and the plate was incubated at 37 °C with 5%
(v/v) CO2 for 1 h. Unattached cells were gently aspirated from
the wells using a multichannel pipette and the attached cells
gently washed 3 times with 250 lL of serum-free DMEM (Sigma)
per well. Cell densities were measured using either MTS or MTT
substrate methodologies. For MTS substrate, 100 lL of serum-
free DMEM and 20 lL of CellTiter 96 AQueous One Solution
Cell Proliferation Assay reagent (Promega) was added to each
well and incubated at 37 °C with 5% (v/v) CO2 for 1 h. Absorbance at 490 nm (compared to absorbance at a reference
wavelength of 690 nm) was measured using a Beckman Coul-
ter
TM plate reader and data averaged using three replicates. Mean
absorbance at 490 nm versus the hVN concentration was plotted
in order to determine ED50. For the MTT substrate, 10 lL of MTT
(5 mg/mL) in DPBS was added to each well and incubated at A column of TALON Superflow Metal Affinity Resin (Sigma)
was prepared with a packed bed volume equivalent to 1 mL/5 g
leaf material and equilibrated with three column volumes of HS
Elution buffer. Purification of hVN from N. benthamiana leaves Purification of hVN from N. benthamiana leaves Leaves were ground to a powder in liquid nitrogen using a mortar
and pestle. For rapid hVN detection, TSP was extracted in five
volumes of either 200 mM phosphate buffer (pH 7) or extraction
buffer (9 M urea, 50 mM phosphate, 10 mM b-mercaptoethanol,
pH 7) and separated by SDS-PAGE for immunoblotting. For
laboratory-scale purification, extraction buffer containing EDTA-
free Complete Protease Cocktail Inhibitor (Roche, Castle Hill, NSW,
Australia) was added to the leaf powder at a ratio of 7.5 mL per
gram FW. The concentration of urea was increased from 8 M (that
used to isolate hVN from blood plasma (Yatohgo et al., 1988)), to
9 M in order to compensate for high leaf water content. The
resulting slurry was agitated at 15 rpm on an orbital wheel shaker
for 15–30 min at room temperature. Larger plant debris was
removed by filtration through Miracloth (VWR, Murarrie, QLD,
Australia) prior to clarification by centrifugation at 20 000 g for
20 min at 20 °C. A Heparin Sepharose 6 Fast Flow (GE Healthcare,
Mansfield, QLD, Australia) column was prepared with a packed
bed volume equivalent to 1 mL/5 g starting leaf material and
equilibrated with three column volumes of HS buffer (8 M urea,
50 mM phosphate, pH 7). The supernatant was loaded directly
onto the equilibrated Heparin Sepharose. The Heparin Sepharose
was washed with five column volumes of HS buffer and bound
hVN eluted using five column volumes of HS Elution buffer (HS
Column buffer supplemented with 500 mM NaCl). Production of human vitronectin in plants 5% (v/v) ethanol. For time-course kinetics, dose–response, yield
estimates and laboratory-scale hVN purification, leaves from
tissue culture N. benthamiana plants were harvested, placed in a
sterile 500-mL tissue culture vessel, and immersed in 150 mL of
MS0 media (Murashige and Skoog, 1962) containing 0.5% (v/v)
ethanol. The vessel was agitated on a flatbed rotary shaker
(60 rpm) for 5 days with a 16-h photoperiod at 25 °C. For time-
course kinetics, sampling time was varied (3, 4, 5, 6, and 7 days
postactivation), and for dose–response, ethanol concentration
was varied (0.1%, 0.25%, 0.5%, 1.0%, and 2.0% (v/v)). Excess
liquid was removed prior to extraction or freezing in liquid
nitrogen and storage at 80 °C. Samples (5 lL) from each step of the purification protocol were
collected and stored on ice prior to PAGE analysis. Purified plant-
made hVN was quantified using the Bradford Protein Assay
microtitre plate procedure (Bio-Rad, Regents Park, NSW, Aus-
tralia) according to the manufacturer’s instructions and known
amounts of commercial purified hVN (Promega). Absorbance at
595 nm was determined using a Beckman Coulter
TM AD200 plate
reader, and samples were analysed in triplicate. PAGE, immunoblotting, N-terminus sequencing and
yield estimation PAGE, immunoblotting, N-terminus sequencing and
yield estimation PAGE and immunoblotting for detection of hVN was carried out
as described by Dugdale et al. (2013). For amino-terminal
sequencing, 3 lg of plant-made hVN was subjected to SDS-
PAGE, transferred to PVDF membrane and stained with Ponceau
dye. The major 75-kDa band was excised from the PVDF
membrane, and the first seven amino-terminal residues were
sequenced using an Applied Biosystems 494 Precise Protein
Sequencing System (Australian Proteome Analysis Facility, Mac-
quarie University, NSW). For yield estimation, Clarity Western ECL
substrate (Bio-Rad) was formulated according to the manufac-
turer and applied as a 1 : 10 dilution to the membrane. Signal
strength was detected using a ChemiDoc imaging system (Bio-
Rad) and yield calculated from a hVN standard curve ranging
from 50 to 500 ng. Average plant-made hVN yield is presented as
mean standard error. Ethanol activation The INPACT platform was activated in whole or detached leaves
using three different methods. For soil-acclimatized plants, 5% (v/
v) ethanol in water was applied as a foliar spray and root drench. For rapid screening of transgenic lines, leaves of in vitro N. ben-
thamiana plants were ethanol activated in sealed Petri dishes. Leaves were placed adaxial side down onto solid MS0 media
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column was washed with five column volumes of HS Elution
buffer and bound HIS-tagged hVN eluted using five column
volumes of TALON Elution buffer (HS Elution buffer supple-
mented with 150 mM imidazole). An Amicon Ultra Centrifugal Filter unit (Ultracel-10K, Millipore)
was equilibrated using 3 mL of HS Elution buffer and centrifu-
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TALON column was diluted 1 : 2 with HS Elution buffer (to
decrease the imidazole concentration to 75 mM) then loaded
onto the Ultracel-10K filter and centrifuged at 5000 g for 10 min
at 18 °C. The filter was washed five to 6 times with 3–4 mL of HS
Elution buffer and centrifuged at 5000 g for 10 min at 18 °C
until the final retained volume was between 100 and 500 lL. A Puradisc FP 30 PTFE, 0.2-lm sterile syringe filter (Thermo
Fisher, Scoresby, VIC, Australia) was equilibrated with 1 mL of HS
elution buffer. hVN retained after size exclusion filtration was
sterilized using the equilibrated filter. Purified plant-made hVN
was either stored short-term at 4 °C or long term at 80 °C with
or without freeze drying. An Amicon Ultra Centrifugal Filter unit (Ultracel-10K, Millipore)
was equilibrated using 3 mL of HS Elution buffer and centrifu-
gation at 5000 g for 5 min at 18 °C. The eluent from the
TALON column was diluted 1 : 2 with HS Elution buffer (to
decrease the imidazole concentration to 75 mM) then loaded
onto the Ultracel-10K filter and centrifuged at 5000 g for 10 min
at 18 °C. The filter was washed five to 6 times with 3–4 mL of HS
Elution buffer and centrifuged at 5000 g for 10 min at 18 °C
until the final retained volume was between 100 and 500 lL. A Puradisc FP 30 PTFE, 0.2-lm sterile syringe filter (Thermo
Fisher, Scoresby, VIC, Australia) was equilibrated with 1 mL of HS
elution buffer. hVN retained after size exclusion filtration was
sterilized using the equilibrated filter. Purified plant-made hVN
was either stored short-term at 4 °C or long term at 80 °C with
or without freeze drying. Benjamin Dugdale et al. 402 402 37 °C with 5% (v/v) CO2 for 3–4 h until a dark precipitate
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(2010) Development of a three-dimensional human skin equivalent wound
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expression in transgenic N. benthamiana parent lines following
ethanol activation. Yatohgo, T, Izumi, M, Kashiwagi, H and Hayashi, M (1988) Novel purification of
vitronectin from human plasma by heparin affinity chromatography. Cell
Struct. Funct. 13, 281–292. Appendix S2 Southern hybridization to determine copy number. Appendix S3 Identification of high hVN-expressing T1 generation
INPACT lines. Appendix S3 Identification of high hVN-expressing T1 generation
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NC (1993) Enrichment of vitronectin- and fibronectin-like proteins in NaCI-
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A Combined Ultrafiltration/Diafiltration Step Facilitates the Purification of Cyanovirin-N From Transgenic Tobacco Extracts
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Frontiers in bioengineering and biotechnology
| 2,019
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cc-by
| 8,316
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ORIGINAL RESEARCH
published: 09 January 2019
doi: 10.3389/fbioe.2018.00206 Keywords: cyanovirin-N, host cell protein, particle size distribution, plant-derived biopharmaceuticals, protein
purification, regenerated cellulose, RuBisCO, zeta potential Reviewed by: Reviewed by:
Audrey Yi-Hui Teh,
St George’s, University of London,
United Kingdom
Eirini Theodosiou,
Aston University, United Kingdom
*Correspondence:
Johannes F. Buyel
johannes.buyel@rwth-aachen.de Reviewed by:
Audrey Yi-Hui Teh,
St George’s, University of London,
United Kingdom
Eirini Theodosiou,
Aston University, United Kingdom
*Correspondence:
Johannes F. Buyel
johannes.buyel@rwth-aachen.de Reviewed by:
Audrey Yi-Hui Teh,
St George’s, University of London,
United Kingdom
Eirini Theodosiou,
Aston University, United Kingdom The production of biopharmaceutical proteins in plants offers many advantages over
traditional expression platforms, including improved safety, greater scalability and
lower upstream production costs. However, most products are retained within plant
cells or the apoplastic space instead of being secreted into a liquid medium, so
downstream processing necessarily involves tissue and cell disruption followed by
the removal of abundant particles and host cell proteins (HCPs). We investigated
whether ultrafiltration/diafiltration (UF/DF) can simplify the purification of the model
recombinant protein cyanovirin-N (CVN), an ∼11 kDa HIV-neutralizing lectin, from
tobacco extracts prior to chromatography. We compared different membrane types and
process conditions, and found that at pH 8.0 and 50 mS cm−1 an UF step using a
100 kDa regenerated cellulose membrane removed more than 80% of the ∼0.75 mg
mL−1 total soluble protein present in the clarified plant extract. We recovered ∼70%
of the CVN and the product purity increased ∼3-fold in the permeate. The underlying
effects of tobacco HCP retention during the UF/DF step were investigated by measuring
the zeta potential and particle size distribution in the 2–10,000 nm range. Combined
with a subsequent 10 kDa DF step, this approach simultaneously reduced the process
volume, conditioned the process intermediate, and facilitated early, chromatography-free
purification. Due to the generic, size-based nature of the method, it is likely to be
compatible with most products smaller than ∼50 kDa. *Correspondence:
Johannes F. Buyel
johannes.buyel@rwth-aachen.de *Correspondence:
Johannes F. Buyel
johannes.buyel@rwth-aachen.de Specialty section:
This article was submitted to
Process and Industrial Biotechnology,
a section of the journal
Frontiers in Bioengineering and
Biotechnology Received: 12 October 2018
Accepted: 12 December 2018
Published: 09 January 2019 ORIGINAL RESEARCH
published: 09 January 2019
doi: 10.3389/fbioe.2018.00206 Patrick Opdensteinen 1,2, Juliana I. Clodt 3, Catherine R. Müschen 1, Volkan Filiz 3 and
Johannes F. Buyel 1,2* Edited by:
Thomas Bartholomäus Brück,
Technische Universität München,
Germany Edited by:
Thomas Bartholomäus Brück,
Technische Universität München,
Germany Edited by:
Thomas Bartholomäus Brück,
Technische Universität München,
Germany Edited by:
Thomas Bartholomäus Brück,
Technische Universität München,
Germany 1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany, 2 Institute for Molecular
Biotechnology, RWTH Aachen University, Aachen, Germany, 3 Institute of Polymer Research, Helmholtz-Zentrum Geesthacht,
Geesthacht, Germany Abbreviations: CIP, cleaning in place; CMC, critical micelle concentration;
CP, concentrated permeate; CVN, cyanovirin-N; DoE, design of experiments;
DSP, downstream processing; HCPs, host cell proteins; MWCO, molecular
weight cut-off; NWP, normalized water permeability; pI, isoelectric point;
PS-b-P4VP, polystyrene-block-poly(4-vinylpyridine); PSBC, PS-b-P4VP-diblock
copolymers; RC, regenerated cellulose; RuBisCO, ribulose-1,5-bisphosphate
carboxylase/oxygenase; SD, standard deviation; SDS, sodium dodecylsulfate;
UF/DF, ultrafiltration/diafiltration; TSP, total soluble protein; USP, upstream
production. Citation: Opdensteinen P, Clodt JI,
Müschen CR, Filiz V and Buyel JF
(2019) A Combined
Ultrafiltration/Diafiltration Step
Facilitates the Purification of
Cyanovirin-N From Transgenic
Tobacco Extracts. Front. Bioeng. Biotechnol. 6:206. doi: 10.3389/fbioe.2018.00206 January 2019 | Volume 6 | Article 206 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org HCP Removal by UF/DF Opdensteinen et al. INTRODUCTION Here we report the purification of recombinant cyanovirin-
N (CVN), an ∼11 kDa HIV-neutralizing lectin, from Nicotiana
tabacum (tobacco) extracts using different UF/DF membranes in
a design-of-experiments (DoE) approach (Figure 1). The effect of
pore sizes, separation conditions and additives on product purity
and recovery were evaluated using CVN as a model recombinant
protein. The effect of integrating UF/DF is discussed in terms of
overall process economics. Plants are advantageous over cell-based approaches for the
production of biopharmaceutical proteins due to the inability
of plants to support the replication of mammalian viruses, the
greater scalability of whole plants compared to fermenters, and
the lower capital and operating costs of upstream production
(Tuse, 2011; Buyel et al., 2017). However, recombinant proteins
produced in plants typically accumulate inside the plant cells
and must be released by disruption, which also generates
large amounts of particulates and abundant soluble host cell
proteins (HCPs). Despite recent advances in the removal of such
impurities (Buyel et al., 2015), downstream processing (DSP) in
plant-based systems remains challenging due to the abundance of
soluble HCPs in the clarified plant extract (Wilken and Nikolov,
2012; Buyel, 2015), especially if affinity purification steps such as
Protein A chromatography are not available for product capture. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is
the major HCP in tobacco, accounting for up to 29% of the total
soluble protein (TSP). This is equivalent to ∼0.9 g L−1 or 3.5 g
kg−1 biomass based on a combined analysis using Bradford assay
for total protein quantitation and densitometric analysis of LDS-
PAA gels to obrain relative protein abundance (Buyel et al., 2013). RuBisCO and other HCPs can thus reduce the product-specific
binding capacity during early capture steps (Buyel and Fischer,
2014d), increasing the costs for the corresponding equipment
and media. Several methods have been developed to remove
HCPs before, during or after extraction, including centrifugal
extraction (Turpen, 1999), rhizosecretion (Drake et al., 2009),
precipitation (Holler et al., 2007), pH shift (Hassan et al., 2008;
Buyel and Fischer, 2014b), and heat treatment (Buyel et al., 2014a;
Menzel et al., 2016), but these methods are not applicable to
all products, for example due to thermal or pH sensitivity as
observed for a malaria vaccine candidate expressed in Nicotiana
benthamiana (Menzel et al., 2018). HIGHLIGHTS and conditioning of process intermediates (Cromwell et al.,
2006). Size-based purification is often prudent because many
HCPs form multimers, e.g., a ∼560 kDa hetero-hexadecamer
in the case of RuBisCO (Buyel et al., 2015), which can thus
be separated from smaller recombinant proteins. However, the
separation performance is reduced by membrane fouling (Hadidi
and Zydney, 2014), which involves the deposition of suspended
or dissolved substances on the membrane surface or in its pores
(Koros et al., 1996). Fouling can be prevented or minimized by
the careful selection and adjustment of membrane properties
and filtration conditions, such as pore size, hydrophilicity,
transmembrane pressure, and pH (Koros et al., 1996; Dosmar,
2005; Cromwell et al., 2006). - Recombinant cyanovirin-N can be purified from clarified
plant extracts by UF/DF. - Recombinant cyanovirin-N can be purified from clarified
plant extracts by UF/DF. y
- A regenerated cellulose membrane with a 100-kDa cut-off
achieved efficient purification. - Buffer pH and detergents influenced the zeta potential and
particle size distribution of tobacco HCPs. - UF/DF-based purification of recombinant proteins can
simplify downstream processing. Extract Clarification Extracts were clarified using a BP-410 bag filter (Fuhr, Klein-
Winternheim, Germany) and a double-layer PDH4 depth filter
(Pall, Dreieich, Germany) as previously described (Buyel and
Fischer, 2014a), and in selected cases were also filtered using
an Emphaze AEX Hybrid purifier (3 M, Neuss, Germany). The
extracts and filtrates were monitored for turbidity, pH and
conductivity, and were passed through a Satopore Capsule
0.20-µm filter (Sartorius-Stedim Systems GmbH, Göttingen,
Germany) before UF/DF. In contrast, ultra-/diafiltration (UF/DF) is a gentle, size-based
separation method that can also be used for the concentration Plant Cultivation and Extraction Transgenic tobacco (Nicotiana tabacum cv. Petit Havana SR1)
variety expressing CVN was cultivated as previously described
(Buyel and Fischer, 2012) and harvested after 52–60 days. The
plant material was stored at −20◦C and extraction was carried
out as previously reported (Buyel et al., 2014b) using three
volumes of extraction buffer (3 mL g−1 biomass) and the same
pH and conductivity as in the subsequent UF/DF step. We used
citric acid buffer for pH 4.0–5.5, phosphate for pH 7.0–8.0, and
glycine for pH 9.0, as well as conductivities in the 15–100 mS
cm−1 range (equivalent to ∼125–1,325 mM sodium chloride). Additives were included before or after extraction in different
concentrations (Table S1). Regeneration of UF/DF Membranes g
UF/DF membranes were washed with (i) extraction buffer, (ii)
1 M sodium hydroxide, and (iii) ultra-pure water before storage
in 20% [v/v] aqueous ethanol. The ultra-pure water wash was
carried out using 20 L wash volume per m2 membrane area
whereas 10 L m−2 was used in all other steps. The normalized
water permeability (NWP) values of the membrane before and
after use, and after regeneration, were calculated for runs with
ultra-pure water using Equation 2 (Nestola et al., 2014). We used
a temperature correction factor of 1.072 at 22◦C as provided by
the manufacturer, which differed slightly from published values
(Kestin et al., 1978). Ultrafiltration/diafiltration area (Rangou et al., 2014) were made from polystyrene-block-
poly(4-vinylpyridine) (PS-b-P4VP) isoporous diblock copolymer
(PSBC), polyacrylonitrile (PAN) or polyvinylidene difluoride
(PVDF) with 50% (m/m) titan dioxide and fed with 50 mL of the
extract. By default, four cycles of 4-fold feed concentration were
conducted per run. After each concentration cycle, buffer with
the same pH and conductivity as the feed was added to restore
the feed starting volume. If not mentioned otherwise (e.g., in a
DoE context) a pH of 7.5 and a conductivity of 50 mS cm−1 was
used. The membrane molecular weight cut-off(MWCO) values
were transformed to pore sizes using Equation 1 (Erickson, 2009;
Zhang et al., 2018). The CVN band intensity was used as a surrogate for
product concentrations and was determined by densitometry
using ImageJ software (National Institutes of Health, Maryland,
USA). Scanned western blot images were transformed to 16-
bit grayscale, black-white inverted and the band intensity
was quantified. The TSP was determined using the Bradford
assay as described (Buyel and Fischer, 2014b). Native PAGE
was conducted using 4–16% gradient gels according to the
manufacturer’s protocol (Thermo Fisher Scientific, Waltham,
United States). Pore size [nm] = 0.09 × (MWCO [Da])0.44
(1) (1) Ultrafiltration/diafiltration A Sartocon Slice 200 bench-top system (Sartorius) was used for
all UF/DF experiments with a transmembrane pressure of 1.1 bar
and a flow rate of 250 mL min−1 if not required otherwise by the
DoE setup. Standard regenerated cellulose (RC) and polyether
sulfone (PESU) membranes (Sartorius) with a 200 cm² filter
area were fed with 200 mL of clarified plant extract. Our custom
membranes with a pore size of 8.5–100 nm and a 17 cm² surface January 2019 | Volume 6 | Article 206 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 2 Opdensteinen et al. HCP Removal by UF/DF FIGURE 1 | Extraction and filtration process flow for the purification of cyanovirin-N starting with transgenic plants (A), extraction (B), bag filtration (C), depth filtration
(D), a first (E) and second (F), ultra-/diafiltration step, and process intermediate (G). The effect of detergent addition (H) at two process stages was investigated. FIGURE 1 | Extraction and filtration process flow for the purification of cyanovirin-N starting with transgenic plants (A), extraction (B), bag filtration (C), depth filtration
(D), a first (E) and second (F), ultra-/diafiltration step, and process intermediate (G). The effect of detergent addition (H) at two process stages was investigated. Gel Electrophoresis and Protein Detection
Process samples were characterized by lithium dodecylsulfate
polyacrylamide gel electrophoresis and subsequent protein
staining with Simply Safe-Stain or western blotting as previously
described (Buyel and Fischer, 2014c). CVN was detected
using
the
primary
rabbit-anti-CVN
polyclonal
antibody
300i072BαCVN at a concentration of 0.08 µg mL−1 and a
secondary
goat-anti-rabbit
antibody
labeled
with
alkaline
phosphatase (Jackson Immuno Research Laboratories, West
Grove, USA) diluted 1:5,000 in 5% [m/v] milk powder in PBS-T. area (Rangou et al., 2014) were made from polystyrene-block-
poly(4-vinylpyridine) (PS-b-P4VP) isoporous diblock copolymer
(PSBC), polyacrylonitrile (PAN) or polyvinylidene difluoride
(PVDF) with 50% (m/m) titan dioxide and fed with 50 mL of the
extract. By default, four cycles of 4-fold feed concentration were
conducted per run. After each concentration cycle, buffer with
the same pH and conductivity as the feed was added to restore
the feed starting volume. If not mentioned otherwise (e.g., in a
DoE context) a pH of 7.5 and a conductivity of 50 mS cm−1 was
used. The membrane molecular weight cut-off(MWCO) values
were transformed to pore sizes using Equation 1 (Erickson, 2009;
Zhang et al., 2018). Block Copolymer Membranes Rapidly Clog
During the Processing of Clarified Tobacco
Extracts Our analysis of tobacco extracts revealed that the major plant
HCP RuBisCO formed oligomers larger than 480 kDa even after
extraction (Figure 2A). We therefore concluded that size-based
separation from recombinant proteins such as the ∼11 kDa
lectin CVN should be feasible by UF/DF, as previously speculated
(Buyel et al., 2015). Therefore, we investigated different
membrane materials, molecular weight cut-off(MWCO) ratings
and process conditions, first in terms of the NWP to assess
the compatibility of the membrane materials with plant-derived
feed streams. We found that hydrophilic RC membranes with a
MWCO of 100 kDa (300 kDa RC not available) had a significantly
higher NWP before (∼25%) and after regeneration (∼16%)
than the widely-used PESU membranes (100 and 300 kDa
MWCO) despite the larger pore size of the latter (Figure S1). We
concluded that RC was less susceptible to membrane fouling than
PESU under our process conditions, which is favorable because
it ensures a stable permeate flux and reproducible conditions
between experiments, and reduces operational costs arising from
the need for extensive membrane cleaning (Sommerfeld and
Strube, 2005). Our results support previous studies in which
RC also outperformed PESU and other synthetic membranes
(Amanda and Mallapragada, 2001; Susanto et al., 2007). We also
found that TSP recovery in the permeate was higher for the 100
kDa RC membrane than the PESU counterpart with the same
MWCO (Table 1). This may reflect subtle pore size variations
between the membranes, or may indicate a concentration
polarization effect that can prevent effective passage of molecules
through a PESU membrane (van den Berg and Smolders, 1989). Also, membrane-protein interactions that cause fouling may be
responsible for the differences between the membranes. These
interactions can be driven by either hydrophobic forces resulting
from conformational changes of the proteins (Truskey et al.,
1987) or by electrostatic attraction (Palecek and Zydney, 1994). Both mechanisms can potentially contribute to the differences
in fouling we observed between RC, PESU PSBC membranes
because the latter two are formed from polymers that contain
aromatic groups that can interact with hydrophobic amino acids
in proteins. Block Copolymer Membranes Rapidly Clog
During the Processing of Clarified Tobacco
Extracts Additionally, the apparent zeta-potential of RC
membranes was about half of that of the PESU counterparts
(Figure S3G) reducing the strength of potential electrostatic
interactions with HCPs, half of which (mass wise) should be We also tested five novel polymer membrane materials (Table 1),
but none of the combinations of pore size and membrane
thickness achieved the selective retention of tobacco HCPs. Interestingly, even though the calculated MWCO rating of some
of the membranes was close to or larger than that of the 300
kDa PESU membrane, only a few HCPs were observed in the
corresponding permeates. As a result, the increase in CVN purity
was a marginal ∼1.3-fold (Table 1). Furthermore, membranes B,
C, and D rapidly clogged during loading with the filtered tobacco
extract. The brownish discoloration of these membranes (data
not shown) indicated substantial fouling, which may explain
the clogging and the unexpected retention of HCPs and CVN. Furthermore, the use of Equation 1 for the transformation of pore
sizes into MWCO ratings may have been an oversimplification. Comparison with the RC and PESU membranes indicated that
increasing the PSBC membrane thickness could potentially
improve CVN purification, i.e., the PSBC membrane thickness
was only about 25% of that of the other membranes (Table 1)
and thickness can affect the selectivity of membranes (Kanani
et al., 2010). In any case, the tested block copolymer membranes
did not appear suitable for the purification of recombinant
CVN from plant extracts. We therefore proceeded with a more
detailed investigation of RC as the most promising membrane
material. Design of Experiments positively charged at our experimental pH of 7.5 according to
their isoelectric point (Figure 2D). Design Expert 8.0 was used to set up and analyze IV-optimal
designs consisting of 16–18 runs as described before (Buyel and
Fischer, 2014e). The factors were pH (4.0, 4.5, 6.25. 7.0, 8.0,
and 9.0), conductivity (15, 50, and 100 mS cm−1), detergent
concentration (0, 10, 50, and 90% critical micellar concentration,
CMC), and detergent type (coded as the positive and negative
charge (−1, 0, +1) of the corresponding detergent molecules
(Table S1). Apart from the HCPs, plant extracts contain a vast number
of other molecules, including DNA and cell wall fragments,
carbohydrates and pigments. Colloids larger than 0.2 µm along
with most pigments were removed during extract filtration
using according adsorptive depth and membrane filters leaving
only two populations of colloids of ∼10 nm and ∼150 nm
(Figure S2C) in the clarified extract. Whereas, the former
matched with the expected size of protein oligomers (2–10 nm),
the identity of the latter colloid population remained unknown
but based on its marginal volumetric fraction (Figure S2D) we
deemed it unlikely to affect the UF/DF. UF/DF Performance Evaluation TSP recovery in the UF/DF permeate was calculated using
Equation 3. TSP
% initial
=
Pn
1 (Pi × VPi)
(F × VF)
× 100%
(3) (3) NWP
L
s · m2 · Pa
=
PF · TCF
TMP · membrane area
(2) NWP
L
s · m2 · Pa
=
PF · TCF
TMP · membrane area
(2) (2) where Pi, protein concentration in the ith permeate [g L−1]; F,
protein concentration in feed [g L−1]; VPi, volume of the ith
permeate [L]; and VF, feed volume [L]. Here, i = 1, 2, 3, 4. where PF, permeate flow [g s−1]; TCF, temperature correction
factor [–]; and TMP, transmembrane pressure [Pa]. The recovery of CVN after UF/DF was calculated as the
fraction of CVN mass in permeate and feed represented by the
densiometric CVN signal derived from the western blots of the
corresponding samples. CVN purity was calculated as the ratio of
the CVN signal derived from the western blots to the TSP value
derived from the Bradford assay. The purity increase after UF/DF
was calculated as the ratio of the CVN purity in the permeate and
feed. Washing and data recording for the calculation of NWP values
was conducted at a flow rate of 250 mL min−1 (inlet pressure =
0.7 bar) and a transmembrane pressure of 0.3 bar at 22◦C. Data were recorded with Satorius WinWedge software
(SartoWedge PC interface software) and the corresponding
Microsoft Excel worksheet. January 2019 | Volume 6 | Article 206 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 3 HCP Removal by UF/DF Opdensteinen et al. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org A 100 kDa MWCO Membrane Facilitates
the Selective Retention of HCPs Because the major tobacco HCP RuBisCO (apparent size, ∼560
kDa) was retained by a 100 kDa membrane (Figure 2A), we
next investigated RC membranes with MWCOs in the 30–
100 kDa range as well as a 300 kDa PESU membrane (this
MWCO rating was not available for RC membranes) to identify
conditions suitable for the efficient separation of HCPs from
CVN. Regardless of the conductivity (15–100 mS cm−1) and pH
(pH 4.0–9.0), <1% (n = 3) of the TSP (including the product)
passed through the 30 kDa membrane whereas close to all (98
± 1%; ±SD, n = 2) passed through the 300 kDa membrane
(Figure 2B). Only the 100 kDa membrane exhibited some degree
of selectivity in terms of HCP retention. We therefore used a DoE January 2019 | Volume 6 | Article 206 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 4 HCP Removal by UF/DF Opdensteinen et al. URE 2 | Separation of tobacco HCPs by UF/DF. (A) Native PAGE of clarified tobacco extract and UF/DF samples using a 100 kDa RC membrane (pH 7.6, 50 mS
1). The black arrow indicates RuBisCO (∼560 kDa). (B) LDS-PAGE analysis of the protein composition of UF/DF samples (pH 7.0 and 50 mS cm−1) after
ration using different membranes. (C) Response surface of TSP recovery in the UF/DF permeate of a 100 kDa RC membrane relative to the load, showing
ndence on pH and conductivity. (D) The pI-dependent distribution of tobacco HCPs and the corresponding mass fractions in plant extracts (Buyel et al., 2013). bin width was 0.5. The permeate TSP recoveries were predicted by the model displayed in (C) for 50 mS cm−1. MWCO, molecular weight cut-off; PESU,
ether sulfone; RC, regenerated cellulose; PSBC, polystyrene block copolymer; L, load; R, retentate; P, permeate. FIGURE 2 | Separation of tobacco HCPs by UF/DF. (A) Native PAGE of clarified tobacco extract and UF/DF samples using a 100 kDa RC membrane (pH 7.6, 50 mS
cm−1). The black arrow indicates RuBisCO (∼560 kDa). (B) LDS-PAGE analysis of the protein composition of UF/DF samples (pH 7.0 and 50 mS cm−1) after
separation using different membranes. (C) Response surface of TSP recovery in the UF/DF permeate of a 100 kDa RC membrane relative to the load, showing
dependence on pH and conductivity. (D) The pI-dependent distribution of tobacco HCPs and the corresponding mass fractions in plant extracts (Buyel et al., 2013). A 100 kDa MWCO Membrane Facilitates
the Selective Retention of HCPs The bin width was 0.5. The permeate TSP recoveries were predicted by the model displayed in (C) for 50 mS cm−1. MWCO, molecular weight cut-off; PESU,
polyether sulfone; RC, regenerated cellulose; PSBC, polystyrene block copolymer; L, load; R, retentate; P, permeate. concentration in the feed at pH 8.0 (0.75 ± 0.17 g L−1; ±SD,
n = 5) compared to pH 4.5 (0.14 ± 0.02 g L−1; ±SD, n = 2)
may have caused stronger concentration polarization at high
pH, further inhibiting recovery by preventing protein transport
through the membrane (Jang et al., 2009). At pH 4.0, the
absolute protein concentrations of 0.07 ± 0.05 g L−1 (±SD, n
= 6) was close to the quantitation limit of the Bradford assay,
probably limiting the predictive power of the DoE model. We
assume that the low TSP concentrations at pH <5.0 reflected the
protein aggregation we observed by dynamic light scattering, and
the subsequent removal of these aggregates during clarification
(Figure 3A). Also, by analyzing the UF/DF permeates with
LDS-PAGE we found that for a pH of ∼4 the fraction of
proteins smaller than 20 kDa was substantially higher than at
higher pH (Figure S3A–E). Therefore, the share of proteins
able to pass the membranes was higher at low pH which likely approach to characterize HCP separation using this membrane
in more detail, which yielded a predictive model of good quality
(Figure 2C and Table S2). We found that the TSP recovery
(excluding the product) in the permeate decreased from ∼60 to
20% as the pH increased, which coincided with the distribution
of the pI values of tobacco HCPs, i.e., high recovery at a pH close
to the pI value representing many tobacco HCPs (Figure 2D). We speculated that this was the joined result of two effects. On the one hand, electrostatic protein-membrane interactions
should be low close to the pI, thus increasing recovery, as
previously reported (Fane et al., 1983; Burns and Zydney, 1999). This hypothesis was supported by our observation that the
absolute zeta-potential of plant extract and purified RuBisCO
decreased at lower pH values (Figure S2A and Table S3). Also,
the zeta potential of the membranes decreased with decreasing
pH (Figure S3G). On the other hand, the higher absolute TSP January 2019 | Volume 6 | Article 206 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 5 HCP Removal by UF/DF Opdensteinen et al. A 100 kDa MWCO Membrane Facilitates
the Selective Retention of HCPs TABLE 1 | Properties and performances of different membranes in terms of CVN purification. ID
Material [–]
Membrane
area [m2]
MWCO [kDa]a
Pore radius
[nm]
Membrane
thickness
[µm]
Permeate
CVN recovery
[% initial]
Permeate TSP
recovery [%
initial]
CVN Purity
increase [–]
Completed
UFDF cycles
[–]
A
PSBC
1.7·10−3
∼150
8.5
50
73.90
93.92
0.79
4
B
PSBC
1.7·10−3
∼325
12.0
11
92.89
78.73
1.18
2b
C
PSBC
1.7·10−3
∼1,200
21.0
41
66.20
68.43
0.97
2b
D
PSBC
1.7·10−3
∼3,500
34.0
31
81.03
62.54
1.30
3b
E
PAN
1.7·10−3
∼215
10.0
40
101.22
92.29
1.10
4
F
PVDF + 50% TiO2
1.7·10−3
∼40,000
100.0
40
52.49
79.67
0.66
4
n.a. RC
0.02
30
∼4.2
180
n.a. 0.42
n.a. 3
n.a. RC
0.02
100
∼7.1
180
69.75
17.06
3.14
4
n.a. PESU
0.02
100
∼7.1
120
45.22
10.82
3.49
4
n.a. PESU
0.02
300
∼11.6
120
n.a. 97.90
n.a. 3
aMWCO was estimated based on Equation 1 for block copolymer materials. bThe run was terminated due to the blocking of the membrane. TABLE 1 | Properties and performances of different membranes in terms of CVN purification. aMWCO was estimated based on Equation 1 for block copolymer materials. bThe run was terminated due to the blocking of the membrane. FIGURE 3 | Effect of pH on protein aggregation and CVN purification using a 100 kDa RC membrane. (A) A shift in particle size distribution in clarified tobacco
extracts occurred when reducing the pH from 8.0 to 5.0, as determined by dynamic light scattering. (B) Western blot (purple) and staining with Coomassie Brilliant
Blue (blue) overlay of process samples from extraction to concentrated UF/DF-purified CVN (indicated by a black arrow). (C) The pH-sensitive formation of aggregates
and the dispersion of said aggregates by adding the detergent SDS to the clarified tobacco extract. CMC, critical micellar concentration; CP, concentrated permeate;
E1–E4, homogenate, conditioned extract, bag filtrate, depth filtrate; MWCO, molecular weight cut-off; r, calculated hydrodynamic particle radius; RC, regenerated
cellulose; PSBC, polystyrene block copolymer; L, load; R1–R4, retentates after diafiltration steps 1–4; P, permeate. FIGURE 3 | Effect of pH on protein aggregation and CVN purification using a 100 kDa RC membrane. (A) A shift in particle size distribution in clarified tobacco
extracts occurred when reducing the pH from 8.0 to 5.0, as determined by dynamic light scattering. Conditions Supporting High CVN Recovery
and High CVN Purity Do Not Overlap Conditions Supporting High CVN Recovery
and High CVN Purity Do Not Overlap g
The highest CVN recovery of 89 ± 5% (±SD, n = 2, Table 2)
was observed at pH 4.5 and 50 mS cm−1, and was thus close to
the theoretical pI of CVN (∼5.0), a phenomenon observed for
other proteins before, such as monomers and dimers of bovine
serum albumin (BSA), and the human DNase dornase alfa (Fane
et al., 1983; Burns and Zydney, 1999). In contrast, the greatest
increase in CVN purity (3.14 ± 0.42, SD, n = 3) was achieved at
pH 8.0 and 50 mS cm−1, but the CVN recovery was only ∼70%
(Table 2, Figure 3B). Interestingly, conditions supporting the
greatest increase in CVN purity were associated with the highest
absolute zeta potentials (Figure S2A, Table S3). We speculated
that increasing the zeta potential artificially could therefore
improve the purity of CVN even further. The zeta potential
describes the electric potential of a moving, dispersed colloid
(Hunter, 2013), and is thus affected by molecules binding to the
colloid surface. We therefore tested several substances (Table S1)
including various detergents known to interact with proteins
or to improve UF/DF performance, in order to determine their
effect on CVN purification (Jang et al., 2009). Because detergents
such as SDS can interfere with protein quantitation methods
such as the Bradford assay, we used appropriate controls to
compensate for any offsets. Adding the zwitterionic detergent Empigen-BB (90% CMC)
or the non-ionic detergent Triton X-100 (90% CMC) increased
the purity of CVN about 5- and 4-fold, respectively, whereas
the cationic detergent CTAB did not improve CVN recovery or
purity (Figure 4). y
The addition of detergents reduced the average protein size
in clarified extracts as determined by dynamic light scattering
compared to the pH 8.0 standard (native size) from 7.84 ±
3.66 (mode ± standard deviation; n = 3 with >12 analytical
replicates each; applies to all following sizes) to 5.85 ± 2.66 nm
(Figures S2C,D). Our interpretation was that these results
indicated the partial disassembly of oligomeric HCPs, which we
confirmed for purified RuBisCO (native = 6.77 ± 2.37 nm, 90%
CMC SDS = 4.36 ± 1.29 nm; pH 8.0, Figure S2B) and which
was in agreement with previous reports (Jang et al., 2009). A 100 kDa MWCO Membrane Facilitates
the Selective Retention of HCPs (B) Western blot (purple) and staining with Coomassie Brilliant
Blue (blue) overlay of process samples from extraction to concentrated UF/DF-purified CVN (indicated by a black arrow). (C) The pH-sensitive formation of aggregates
and the dispersion of said aggregates by adding the detergent SDS to the clarified tobacco extract. CMC, critical micellar concentration; CP, concentrated permeate;
E1–E4, homogenate, conditioned extract, bag filtrate, depth filtrate; MWCO, molecular weight cut-off; r, calculated hydrodynamic particle radius; RC, regenerated
cellulose; PSBC, polystyrene block copolymer; L, load; R1–R4, retentates after diafiltration steps 1–4; P, permeate. FIGURE 3 | Effect of pH on protein aggregation and CVN purification using a 100 kDa RC membrane. (A) A shift in particle size distribution in clarified tobacco
extracts occurred when reducing the pH from 8.0 to 5.0, as determined by dynamic light scattering. (B) Western blot (purple) and staining with Coomassie Brilliant
Blue (blue) overlay of process samples from extraction to concentrated UF/DF-purified CVN (indicated by a black arrow). (C) The pH-sensitive formation of aggregates
and the dispersion of said aggregates by adding the detergent SDS to the clarified tobacco extract. CMC, critical micellar concentration; CP, concentrated permeate;
E1–E4, homogenate, conditioned extract, bag filtrate, depth filtrate; MWCO, molecular weight cut-off; r, calculated hydrodynamic particle radius; RC, regenerated
cellulose; PSBC, polystyrene block copolymer; L, load; R1–R4, retentates after diafiltration steps 1–4; P, permeate. caused the increased permeate recovery we observed under these
conditions. TSP recovery in the permeate (Figure 2C). Even though salt can
reduce the electrostatic exclusion of proteins from membrane
pores and thus increase recovery (van Eijndhoven et al., 1995;
Zeman et al., 1996), we assume that conductivities of ∼100 mS Conductivity had a relevant effect only at pH values above
7.0, where conductivities higher than 50 mS cm−1 reduced the January 2019 | Volume 6 | Article 206 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 HCP Removal by UF/DF Opdensteinen et al. TABLE 2 | Recovery and increase in purity of CVN in UF/DF permeates using a 100 kDa RC membrane. Standard deviations result from two or three runs. Conditions Supporting High CVN Recovery
and High CVN Purity Do Not Overlap In
the feed, this can increase the effective quantity of colloids that
block membrane pores, which in turn can increase concentration
polarization during UF/DF, allowing only small proteins such
as CVN to pass through the membrane. Electrostatic repulsion
between the membrane and charged HCPs (due to discord
between the pH and pI, or interaction with the detergents) is
also possible, because the charged detergent SDS increased the
absolute zeta potential (Table S3) and was associated with higher
purity as we speculated. In contrast, a pore-narrowing effect
caused by the adsorption of more protein on the pore walls of
the ultrafiltration membrane as the quantity of colloids increased A 100 kDa MWCO Membrane Facilitates
the Selective Retention of HCPs pH [–]
Conductivity
[mS cm−1]
TSP recovery
[% load]
CVN recovery
[% load]a
CVN purity
increase [–]a
Number
of runs [–]
Zeta potential tobacco extract,
monomodal analysis mode [mV ]
4.50
50
63.02 ± 8.22
89.10 ± 4.70
1.43 ± 0.26
2
−4.27 ± 0.67 (pH 5.0, 7.57 mS cm−1)
6.25
50
46.80 ± 4.43
77.88 ± 5.03
1.68 ± 0.27
2
−9.94 ± 0.57 (pH 6.5, 6.53 mS cm−1)
8.00
50
22.30 ± 2.02
69.75 ± 6.68
3.14 ± 0.42
3
−13.07 ± 0.80 (pH 8.0, 6.32 mS cm−1)
6.25
15
47.22
67.49
1.42
1
n.a. 8.00
15
21.74
42.95
1.98
1
n.a. aValues ± standard deviation ncrease in purity of CVN in UF/DF permeates using a 100 kDa RC membrane. Standard deviations result from two or three runs. cm−1 (∼1.5 M sodium chloride) triggered protein adsorption to
the membranes via hydrophobic interactions as described before
(Fane et al., 1983). aggregation. However, when we subsequently added increasing
amounts of SDS, the opacity gradually reduced, especially at pH
6.5 where the liquid became transparent again. Dynamic light
scattering confirmed that the particle size distribution after the
addition of SDS had a profile similar to that before the pH
shift, indicating the presence of proteins (2–10 nm) and small
particles (∼80 nm) (Figures S2C,D). We used a DoE approach
to characterize the effect of the detergent concentration and
charge, which resulted in a model with good predictive quality
(Figure 4 and Tables S4, S5). The highest CVN purity increase
was ∼20-fold using SDS at 90% CMC, which was seven times
higher than without detergents. However, the CVN recovery was
only ∼22% instead of 70% in a detergent-free setup. The highest
CVN recovery in the presence of detergents was 62% when
the negatively charged detergent SDS was added at 10% CMC
(Figure 4C), which was slightly less than the ∼70% observed
for the detergent-free setup. The increase in purity under these
conditions was only ∼3.4-fold, which was comparable to that of
the detergent-free setup (∼3.1-fold). January 2019 | Volume 6 | Article 206 Detergents Can Increase the Purity but Not
the Recovery of CVN During UF/DF The simplest way to add detergents to our process was to include
them in the extraction buffer. This increased the purity by 3.0 ±
0.6 (±SD, n = 2) and was thus comparable to the detergent-free
approach. However, the CVN recovery in the UF/DF permeate
was reduced to 42 ± 10% (±SD, n = 3) which was 30% lower
than without detergents. In addition, the intense green color
of the UF/DF feed indicated the presence of plant pigments
(Figure S3F) which can interfere with protein purification, e.g.,
through covalent binding to the product (Barros et al., 2011;
Wilken and Nikolov, 2012). We therefore added the detergent after clarification. When
we adjusted the pH of the clarified tobacco extract to 6.5 and
5.0 (starting from 8.0), the opacity of the liquid increased
within seconds (Figure 3C) which we attributed to protein January 2019 | Volume 6 | Article 206 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 7 HCP Removal by UF/DF Opdensteinen et al. FIGURE 4 | Response surface for CVN recovery and purity based on detergent charge and concentration. The detergent charge was coded as zero (0, charge type
not present) or 1.0 (charge type present) for negative and positive charges, with the zwitterionic detergent having a charge coding of [1.0, 1.0]. The model for CVN
recovery is depicted in the bottom row of panels (C and D), and the purity model is shown in the top row (A and B). Red dots represent actual experiments. FIGURE 4 | Response surface for CVN recovery and purity based on detergent charge and concentration. The detergent charge was coded as zero (0, charge type
not present) or 1.0 (charge type present) for negative and positive charges, with the zwitterionic detergent having a charge coding of [1.0, 1.0]. The model for CVN
recovery is depicted in the bottom row of panels (C and D), and the purity model is shown in the top row (A and B). Red dots represent actual experiments. seemed less likely, because this phenomenon is prevented by
detergents (Brink et al., 1993). 3) or the CVN recovery (∼55%) or the increase in purity (2-fold). Removing phenolic compounds and DNA using a specialized
Emphaze filter also had no effect on TSP (103%) or CVN recovery
(55%) or the increase in purity (3.15-fold). Detergents Can Increase the Purity but Not
the Recovery of CVN During UF/DF We concluded that,
in our process, phenolic compounds and DNA did not have
a relevant effect on UF/DF performance using a 100 kDa RC
membrane. Anti-foaming Agents and Phenolic
Impurities Do Not Affect CVN Recovery or
the Increase in CVN Purity We also tested whether the addition of polyvinylpolypyrrolidone
(PVPP) could improve CVN recovery or purity because it is
known to remove phenolic substances and high-molecular-mass
compounds from plant extracts that may interfere with UF/DF
(Loomis, 1974). In this context, we also tested the anti-foaming
agent Polaxamer 188 (Pluronic F68), which can affect protein
permeation during UF/DF (Kloosterman et al., 1988). When
Polaxamer 188 was added prior to UF/DF, CVN recovery was
33.94 ± 0.74% (SD, n = 2) and the increase in purity was
3.4 and 2.4 at 10 and 90% CMC, respectively, thus offering no
improvement compared to an additive-free setup. Adding PVPP
before UF/DF did not affect the TSP (97 ± 6% initial, ±SD, n = Economic Relevance of UF/DF Purification
Steps Evaluating the economics of UF/DF is dependent on the process
and product. In the case of plant-derived biopharmaceutical
proteins for which no affinity purification step is available,
UF/DF showed three clear advantages: (i) the large and expensive
columns required for HCP binding (Buyel and Fischer, 2014b)
can be avoided, (ii) processing times can be reduced, e.g.,
for 600 L plant extract from ∼12 h in the case of a packed-
bed column (5-L column, 30 cm bed height, 300 cm h−1
linear flow rate) to ∼1 h (UF/DF with 2 m2 filter area, 1.25 January 2019 | Volume 6 | Article 206 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 8 HCP Removal by UF/DF Opdensteinen et al. bar transmembrane pressure), and (iii) the conditioning of
process intermediates can be combined with concentration
(Lightfoot et al., 2008), allowing the use of smaller and thus
less expensive equipment due to the smaller volume streams. Based on the current CVN recoveries and purities (Figure 4), a
detailed analysis of a hypothetical three-stage purification process
(Table S6) revealed that incorporating UF/DF before the first
chromatographic purification step can reduce the duration of
DSP from 14 to 9 h with cost savings of about 15%. For this
calculation, we assumed that UF/DF will increase the CVN
recovery of the first chromatographic step from 0.5 to 0.7 due
to the lower HCP burden. In the calculation, this allowed the
omission of a third chromatography step, which was otherwise
necessary to achieve a product purity >95%, but will require
experimental verification once a full process is set up for
CVN. Additional benefits of the UF/DF setup include the cost-
saving potential of the smaller-scale equipment and therefore
the smaller process footprint, e.g., due to an increased product-
specific binding capacity of chromatography columns. However,
this was not taken into account for the cost calculation because
the effect will strongly depend on the pH at which a plant-based
process is operated, i.e., at high pH the benefits of UF/DF will
be substantial because large quantities of HCPs can be removed
from the process intermediate whereas under acidic conditions a
pH shift alone can be sufficient to remove HCPs. the purity of CVN, but at the expense of product recovery,
which fell from ∼70 to 20%. Economic Relevance of UF/DF Purification
Steps Therefore, selection of the MWCO
should be accompanied by a careful adjustment of the separation
conditions, which will help to control and improve recombinant
protein purification from plant extracts using UF/DF in the
future. Additionally, UF/DF may help to save 10–15% in DSP
costs with additional savings being possible due to the smaller
volumes and hence the smaller footprint of DSP equipment. CONCLUSIONS Among the membranes we tested, regenerated cellulose (RC)
was the most suitable material for the purification of CVN
from clarified tobacco extracts due to low membrane fouling. Unfortunately, RC membranes are currently not available with
MWCOs between 100 and 300 kDa, which could improve the
separation of HCPs from plant-derived recombinant proteins
smaller than 50 kDa. SUPPLEMENTARY MATERIAL In addition to the MWCO, more tobacco HCPs were retained
at high pH, increasing the purity of CVN by about 3-fold
(3.14 ± 0.42; ±SD, n = 3) using a 100 kDa RC membrane
at pH 8.0 and 50 mS cm−1. Adding the negatively charged
detergent SDS resulted in an additional >20-fold increase in The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fbioe. 2018.00206/full#supplementary-material FUNDING This work was funded by the Fraunhofer-Gesellschaft Internal
Programs under Grant No. Attract 125-600164 and DFG grant
RTG 2375. ACKNOWLEDGMENTS The authors acknowledge Dr. Audrey Teh for providing the
transgenic seeds of tobacco plants expressing CVN as well as
Dr. Tobias Schleuss and Dr. Bjoern Hansmann from Sartorius
for sharing the membrane zeta potentials. We would also like
to thank Dr. James B. McMahon for providing the anti-CVN
antibody 300i072BαCVN. We are grateful to Ibrahim Al Amedi
for cultivating the tobacco plants used in this investigation. We wish to thank Dr. Richard M. Twyman for editorial
assistance. AUTHOR CONTRIBUTIONS PO conducted the UF/DF experiments and analyzed the process
samples. JC cast the custom membranes. CM prepared the
protein solutions and purified protein samples. VF designed
the custom membranes. JB designed and analyzed the UF/DF
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462–466. doi: 10.5483/BMBRep.2009.42.7.462 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Kanani, D. M., Fissell, W. H., Roy, S., Dubnisheva, A., Fleischman, A.,
Zydney, A. L. (2010). Permeability—Selectivity analysis for ultrafiltration:
effect of pore geometry. J. Membr. Sci. 349:405. doi: 10.1016/j.memsci.2009. 12.003 Copyright © 2019 Opdensteinen, Clodt, Müschen, Filiz and Buyel. 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) and the copyright owner(s) 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. Kestin, J., Sokolov, M., and Wakeham, W. A. (1978). Viscosity of liquid
water in the range −8◦C to 150◦C. J. Phys. Chem. Ref. Data 7, 941–948. doi: 10.1063/1.555581 Kloosterman, J., Wassenaar, P. D., Slater, N. K. H., and Baksteen, H. (1988). The
effect of anti-foam agents on the ultrafiltration of a protease solution. Bioprocess
Eng. 3, 181–185. doi: 10.1007/BF00379655 January 2019 | Volume 6 | Article 206 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 10
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Culturalisme et réflexion existentialiste— Une lecture de Retour de manivelle de Julien Kilanga Musinde
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Culturalisme et réflexion existen-
tialiste—Une lecture de Retour de
manivelle de Julien Kilanga Musinde Culturalism and existentialist thought—
A reading of Julien Kilanga Musinde’s Retour
de manivelle Culturalism and existentialist thought—
A reading of Julien Kilanga Musinde’s Retour
de manivelle
Published in 2008, the novel Retour de manivelle by Julien Kilanga Musinde revives the unfinished debate related to the search
of identity in African writing. The universe depicted represents the modern society and Musinde’s main character is changing as
fast as he relocates to a different society. The author depicts this flexibility as a strength that commands the adaptability of the
character without suppressing the initial culture of the protagonist. Musinde chooses to freely express his fantasy and, at the same
time, integrate his subjective world vision and multidimensional scholarship in the interpretation of the identity. The question of
culture being central to the novel, the paper is aimed at demonstrating, however, that the culture that is depicted as both
exclusive and dynamic in Musinde’s work should be understood mainly in cyclic perception in which both the starting and the
arrival points are joining in a unique individual subjectivity, such a subjectivity having the potential of engendering a new
discourse by attempting to juxtapose conflicting ways of life. The paper also demonstrates how Musinde distances himself from the
materialistic world vision commonly expressed by existentialists and Epicurean philosophers. This attitude allows the author to
reflect on the interconnectivity between the immediate empirical reality and the world beyond from the perspective of a
transnational African intellectual in a globalizing world. Keywords: Congolese literature, cultural identity, existentialism,
globalisation, Julien Kilanga Musinde, meaning of death. De manière générale la littérature africaine moderne a la réputation d’être ancrée dans
l’immédiateté des faits de la vie sociopolitique si bien qu’un lecteur avisé y découvrirait
des redites nombreuses et prédictibles. Il n’est pas surprenant de constater que les
écrivains africains ont tendance, dans la majorité des cas, à puiser aux mêmes sources
d’inspiration qui peuvent se résumer en la mégestion des affaires de l’État et la
dénonciation des maux sociaux comme la corruption, la dictature, le néocolonialisme,
etc. Aussi le rôle de l’écrivain et celui de l’historien se confondent-ils parfois lorsque
les questions socio-politiques préoccupent tous les deux. Patrick Kabeya Mwepu
Patrick Kabeya Mwepu né en RDC,
Docteur en langue et literature françaises
(Université du Cap), professeur associé et
directeur de l’École des langues (Univer-
sité Rhodes), auteur d’un livre et de
plusieurs articles en literature africaine
francophone, rédacteur-adjoint de la
revue French Studies in Southern Africa.
E-mail: p.mwepu@ru.ac.za TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013
DOI: http://dx.doi.org/10.4314/tvl.v50i1.8 Culturalisme et réflexion existen-
tialiste—Une lecture de Retour de
manivelle de Julien Kilanga Musinde Bien que les tendances de la
littérature africaine moderne soient de nos jours variées, on remarque cependant que
généralement, comme le constate Madebe dans son étude sémiotique sur la littérature
francophone, “la littérature africaine ne s’affirme que par sa dépendance à l’Histoire
et par conséquent, au politique, les productions littéraires se définissant elles-mêmes
comme de prises de position face à l’Histoire”(Madebe 93). Cette dépendance à l’Histoire TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013
DOI: http://dx.doi.org/10.4314/tvl.v50i1.8 9 7 qui tient l’écrivain africain comme “un nœud gordien”(Nganang 88), est l’une des
caractéristiques majeures de l’écriture africaine postcoloniale. Pourtant, la lecture de Retour de manivelle de Julien Kilanga Musinde laisse voir au
lecteur que l’auteur des “Vagissements”possède un avis contraire dans l’orientation
de sa plume et dans la vision qu’il a pour la littérature de son pays, la République
Démocratique du Congo, et de l’Afrique en général.1 Sur les traces de ses concitoyens
et prédécesseurs, à savoir Vumbi Yoka Mudimbe et George Ngal, Musinde tente de
remettre les pendules à l’heure en rappelant une conception philosophique de la
littérature dans un monde où elle semble tomber en désuétude. Dans ce roman publié
en 2008, l’auteur, par le truchement de son héros, sent l’envie de se démarquer d’une
écriture axée généralement sur le quotidien qu’il considère comme étant “déjà
dénaturé”(119). Pour Musinde, la prospérité d’une culture (littérature) est fonction de
la prospérité d’une nation (119). Il en déduit qu’une écriture engagée dans le quotidien
serait décadente étant donné qu’elle serait encline à décrire “les malheurs de l’époque,
les guerres, la faim, la pauvreté, la violence et la dictature”(119). Par contre, Musinde
se penche à sa manière dans ce roman sur des questions culturelles et philosophiques
livrant ainsi au lecteur sa vision ontologique. On peut voir que l’auteur présente au
public un roman à thèse destinée à promouvoir sa vision de l’essence de l’être. Retour
de manivelle se présente aussi comme une définition du soi et de l’autre qui ne manque
pas de ressusciter la question existentialiste sur le sens de la mort. Toutefois, l’auteur
présente sa vision du monde, donnant implicitement au lecteur une appréhension
imagée des questions de haute portée philosophique. Concernant l’histoire, le roman Retour de manivelle est centré sur la problématique
de l’identité culturelle dans une société mondialisée. Culturalisme et réflexion existen-
tialiste—Une lecture de Retour de
manivelle de Julien Kilanga Musinde Le héros, Josué, raconte sa
vie selon la perspective d’un adulte : une naissance modeste au village avant de
rejoindre la ville, une évolution scolaire exceptionnelle, la chance d’occuper des
postes de responsabilité, etc. Le retour au village natal du héros, déjà adulte et ayant
acquis un statut de fonctionnaire international, semble être le moment fort du roman
qui voit le protagoniste ressusciter, au propre comme au figuré, parmi les siens au
terme d’un bref séjour dans le “royaume”des morts. La question de l’identité culturelle La quête de l’identité en littérature africaine est aussi vieille que la littérature elle-
même. Dans le roman Un homme pareil aux autres, par exemple, René Maran mettait
déjà sur scène un personnage problématique. Le héros Jean Veneuse déclarait ce qui
suit : “Venu à Bordeaux tout enfant, à une époque où il aurait été difficile d’y trouver
huit ou dix nègres, mes meilleurs amis sont des Blancs. Je pense et vis à la française. La
France est ma religion. Je ramène tout à elle. Enfin, hormis ma couleur, je me sais
Européen” (Maran 184). TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 9 8 Ce passage traduit non seulement la perte d’une identité traditionnelle mais aussi
l’acquisition d’une nouvelle identité par un même sujet. Il s’agit d’une représentation
romancée de la déchéance qui menace certaines cultures dans un contexte de
cohabitation et d’affrontement culturel. Or, il n’est pas dans l’intérêt d’un peuple de
voir sa culture disparaître. Comment donc prévenir une menace culturelle imminente
dans une société africaine postcoloniale tournée vers l’ailleurs ? Préconiserait-on de
vivre en ermite pour mieux conserver sa culture ? Dans le roman Retour de manivelle,
Musinde s’interroge et tente de répondre à sa manière à cette préoccupation. Dès le
début du roman, on peut lire ce qui suit : “Mon fils, je sais que tu veux aller vers ces
pays lointains au contact d’autres cultures. Je ne veux pas te l’interdire. Mais il faut
d’abord assimiler ta propre culture avant d’aller vers celles des autres.” (10) Tels sont les propos que le père de Josué, le héros du roman, adresse à son fils à la
veille d’un périple qui le conduirait loin de sa terre natale. Ces instructions font
partie des préparatifs du voyage et méritent d’être interprétés, à l’instar de ceux du
roman de Kourouma, comme étant un effort pour “adoucir, voire détourner le mauvais
sort”(Kourouma 151) au voyageur. Situés au début du roman (à la deuxième page du
livre), ces propos posent indirectement la problématique d’une ontologie sous-tendue
par une essence culturelle qui fait partie intégrante de l’identité du héros, qui ici
pourrait symboliser l’intellectuel africain moderne. Il est évident que le personnage
central, Josué, est un jeune qui n’a pas encore pénétré profondément sa culture. Ainsi
les propos “il faut d’abord assimiler ta culture”sonnent comme une obligation morale
faite par un maître (le père) à un novice (le fils). TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 La question de l’identité culturelle Loin d’être une simple exhortation,
ces propos sont à comprendre comme une coercition morale que viennent renforcer,
sur le plan stylistique, les impératifs du genre “regarde cet arbre” (10), “regarde ces
feuilles”, “regarde-les bien”(11), etc. Ces impératifs mettent l’emphase sur l’initiation
au secret de la nature au terme de laquelle, en guise de couronnement rituel, l’initié
est convié à “boire du vin de palme dans une corne de buffle”(11). La réflexion autour de ce symbolisme naturel pourrait révéler le fait que la tendance
actuellement pour les jeunes et les intellectuels africains est d’aller vers les autres à la
découverte du monde. Ce rapetissement de l’espace, comme le disait Senghor, “tend
à provoquer une réaction d’autodéfense contre l’uniformisation”(Biondi 122). Le rite
consistant à “boire dans la corne du buffle”constitue un rappel à l’ordre lancé à
l’intellectuel africain, l’invitant à composer avec la nature (le temple) (13) et la terre
natale ; cette dernière à son tour renvoie non seulement aux sentiments patriotiques
qui devraient caractériser l’intellectuel africain dans sa rencontre avec l’”autre
monde”mais aussi à l’attachement indéfectible à l’histoire et au passé que l’écrivain
considère comme un creuset de forces sacrées. C’est dans ce sens que l’on pourrait
comprendre l’évocation du mot “ancêtres”qui vient à son tour renforcer l’idée du
sacré reprise dans la séquence “temple où toute prière adressée aux ancêtres trouve
une place de choix”(13). La sympathie entre l’homme et la nature (qui est sacrée) se TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 9 9 rapproche de la vision baudelairienne selon laquelle le spirituel et le matériel
communient harmonieusement comme on pourrait le lire dans le poème
“Correspondances”du recueil Les Fleurs du Mal : rapproche de la vision baudelairienne selon laquelle le spirituel et le matériel
communient harmonieusement comme on pourrait le lire dans le poème
“Correspondances”du recueil Les Fleurs du Mal : La Nature est un temple où de vivants piliers Laissent parfois sortir de confuses paroles ; L’homme y passe à travers des forêts de symboles Qui l’observent avec des regards familiers. (Baudelaire 85) Dans l’univers de Musinde, la “nature sacrée”de Baudelaire acquiert une seconde
dimension : elle devient spécifique. La spécificité de ce “temple”est exprimée par les
démonstratifs du genre : “cet arbre”, “cette corne de buffle”, “cette forêt”, etc. La question de l’identité culturelle La
spécification de la nature, ou mieux son individualisation, est une expression imagée
de “l’instinct de la terre”qui voit le personnage de Josué “se perdre”à travers le monde
et “se retrouver”une fois dans son terroir. Musinde garde ainsi sa vision du monde
enracinée dans une culture spécifiée dont les instruments de transport vers l’au-delà
sont prédéterminés. Et l’auteur de conclure : “la vie et le développement de ce pays
dépend (sic) largement de l’observance équilibrée des directives de la main de la
tradition.”(125). Dans cette perspective, les impératifs absolus ramenant le héros à la tradition de sa
terre natale préviendraient-ils les “citoyens”de la disparition de leur culture nationale
lorsqu’elle sera confrontée à une nouvelle culture ? Même sans se déplacer, la menace
d’une “mort culturelle”se fait parfois sentir localement étant donnés les interactions
multiformes et l’influence des médias. Le souci du père est de ne pas voir sa culture
disparaître. Ce même souci qui a été éprouvé par Senghor, par exemple, constitue un
alibi pour ceux qui veulent voir rayonner leur propre culture, en dépit de la multiplicité
des menaces. C’est avec regret que Senghor exprimait sa tristesse de voir disparaître à
petit feu la culture de son pays dans le milieu même à partir duquel elle aurait dû
irradier : “Ma tristesse le jour où Joal, mon village natal, me recevait pour fêter mes
soixante-dix ans ! Les jeunes filles ne savaient plus chanter, créer des chants gymniques
à la gloire de leurs ‘champions noirs et élancés’. Ni les jeunes hommes danser la danse
de la victoire en secouant leurs clochettes de bronze” (Biondi 122–23). Dans cette perspective, l’univers de Musinde constitue une subjectivité visant à
revaloriser ou à promouvoir une vision du monde menacée ou mal représentée suite
au désintéressement de ceux qui auraient dû la promouvoir ou à la mauvaise foi et
l’ignorance de ceux qui l’appréhendent in vitro. La question de l’identité culturelle Dans ce sens, l’effort de Musinde est
de placer son imaginaire dans un contexte postcoloniale que Moura présente de la
sorte : Aux yeux de la critique postcoloniale, l’œuvre vise à se situer dans le monde en se
branchant sur un ensemble socio-culturel enraciné en un territoire, ce branchement
étant fréquemment rendu difficile en raison d’une (tenace) hiérarchisation Aux yeux de la critique postcoloniale, l’œuvre vise à se situer dans le monde en se
branchant sur un ensemble socio-culturel enraciné en un territoire, ce branchement étant fréquemment rendu difficile en raison d’une (tenace) hiérarchisation TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 100 européenne—que ce soit la dévalorisation pure et simple du “primitif”—des
traditions concernées. La scénographie postcoloniale a d’abord cette particularité
que l’œuvre vise à légitimer la culture dont elle émane en se donnant pour le
prolongement actuel de ses traditions. (Moura 111) Partant de cet appel à l’approfondissement préalable de sa propre culture, Musinde
relance le débat on ne peut plus culturaliste, débat qui depuis un certain temps a été
évincé, à quelques exceptions près, par le primat du politique sur le culturel dans un
continent rongé par des maux politiques tels l’esclavage, la colonisation, la dictature,
le néocolonialisme, etc. On remarque que le précepte socratique, “connais-toi toi-
même”, est à l’œuvre, étant donné que le héros de Musinde semble “se montrer un
homme”, peut-être pas toujours pareil aux autres, mais celui qui n’oublierait pas
facilement ses racines, même au-delà du terroir qui l’a vu naître. TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 Un être multiculturel Josué fait preuve d’une
éducation humaniste dans laquelle la culture gréco-latine occupe une place de choix. Ceci est lisible non seulement à travers des expressions latines traduites dans le texte
“le temps qui ronge tout”(tempus edax rerum) (20), “le temps qui ne nous laisse jamais
les mêmes”(o tempora o mores) (20), mais aussi dans l’évocation de la mythologie grecque
relative à Eurydice et Orphée (21). Le protagoniste du Retour de manivelle est aussi
lecteur de Voltaire et ne cesse de renvoyer le lecteur au personnage de Candide pour
qui “tout est pour le mieux dans le meilleur des mondes”(21, 33). À côté de Candide, on
voit bien que le personnage de Josué a lu des grands classiques et pourrait même
réciter de mémoire la célèbre “Chanson de Roland”, les poèmes de François Villon,
ceux de Ronsard, Du Bellay, de Racine, de La Fontaine, de Corneille, etc. (31) et
pouvait parler avec aisance de Montaigne, Rabelais, Pascal, Descartes, Molière, Musset,
de Vigny, Kafka, Hopkins, Shakespeare, Baudelaire, etc. (32). L’évocation de tous ces
auteurs démontre que Josué est un humaniste dont la culture multidimensionnelle
va au-delà de son village d’origine. Cependant le souvenir du rite de la corne du buffle et du vin de palme, rite
accompli avant de quitter sa terre natale, mérite d’être interprété comme un facteur
déterminant qui lie le héros au temple de son terroir (la nature) comme le ferait le
cordon ombilical de l’embryon au placenta maternel. Ce symbolisme traduit une
spiritualité dont la force se veut supérieure à toute tentative d’usurpation externe. L’on pourrait donc penser à une transmission de force surnaturelle (par cette cérémonie)
qui fait que la personne ainsi admise, à la manière d’un baptême religieux, possède
d’immenses pouvoirs immatériels qui la font accéder à la caste des initiés détenteurs
d’un savoir ésotérique considérable. Cela revient à dire que Musinde, dans Retour de
manivelle, présente au lecteur un héros dont le caractère est invincible. C’est justement
cette invincibilité (il faut au métis culturel, comme au métis biologique, une rare force
de caractère, qui lui permette de dominer et de concilier ses fécondes contradictions
(Senghor 96–97)) qui va faire qu’au lieu de se perdre dans l’universel le héros détienne
plusieurs cultures qui viennent s’accumuler autour d’une culture fortement et
préalablement enracinée et intériorisée ; et par conséquent, le héros devient “le siège
de plusieurs cultures”(115). Y a-t-il affrontement des cultures ? Un être multiculturel La problématique culturelle fait l’ossature de Retour de manivelle. L’observation la plus
immédiate montre cependant que la vision du monde exprimée dans l’œuvre est loin
de dépeindre et de promouvoir un ghetto culturel dans lequel les sujets sont, de
manière statique et essentialiste, voués à un chauvinisme porté à son paroxysme. On
notera que l’ethos qui, d’ordinaire, est “associé à un personnage typique : griot africain,
quimboiseur antillais ou le conteur oriental” est ici incarné dans le personnage de
Josué (Moura 122). Le choix de ce nom judéo-chrétien, dans un contexte de promotion
de la culture africaine n’est ni neutre ni arbitraire. Si la narration du texte est assurée
par ce personnage portant un nom étranger, contrairement à la culture de son village,
on peut déjà entrevoir l’intention de l’auteur de créer un univers différent. Réfléchissant
sur l’onomastique africaine, Malonga écrit ce qui suit : “Chez les Bantu […] nommer
c’est aussi synonyme de définir l’identité de l’individu nommé en même temps dire
l’état et, parfois, la structure psychologique, sinon la pensée de celui qui nomme […]
le nom est un message qui désigne le porteur durant sa vie.” (Malonga 26–27) Il en résulte que, malgré l’impératif de l’observance de la tradition, Retour de
manivelle est loin de confiner les personnages dans un univers culturellement
autarcique. Malgré l’importance de sa tradition, vivre en vase clos serait suicidaire. L’œuvre de Musinde est une vision du monde, celle de l’intellectuel africain moderne
contraint par la force des choses d’aller vers l’autre comme on peut le lire dans
l’intention du héros : “Je veux aller vers un univers inconnu ou aucun guide fiable
n’est prévu. Et pourtant, il faut que j’y aille.” (13) Quitter sa terre natale c’est aussi un besoin à l’époque des grands contacts où “tout
est à repenser”(57). La rencontre de l’autre devenant une nécessité, partir contribuerait
à engendrer “une civilisation idéale qui ne saurait être que métisse, comme le furent TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 101 les plus grandes civilisations de l’Histoire, celles de Sumer, de l’Égypte, de l’Inde,
voire celles de la Chine et de la Grèce.”(Senghor 96) Musinde met sur scène un personnage qui s’abreuve à d’autres cultures. Le
protagoniste déclare, par exemple, être “partagé entre la pratique externe, la pensée
philosophique et littéraire tirée de ses diverses lectures”(33). Un être multiculturel À première ap-
proximation, le lecteur serait tenté de le croire : “On m’a ainsi introduit dans le
monde occidental. Il nous était interdit de parler en langues locales. Parler une langue
nationale était une raison suffisante pour être exclu de l’école.” (31) Néanmoins, lorsqu’on considère que le héros a été préparé et qu’il est “habité”par
une force permanente “in nobis sine nobis”, nous pensons que l’utilisation des termes TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 102 “je devenais ainsi le siège des plusieurs cultures”serait justifiée dans la mesure où ces
cultures sont intégrées et maîtrisées sans aliénation ni heurts. C’est cette juxtaposition
culturelle fondée sur le respect mutuel que Glissant appelle “créolisation”, un concept
qui dépasse la dimension linguistique et que l’auteur définit comme suit : “La
créolisation exige que les éléments hétérogènes mis en relation ”s’intervalorisent”,
c’est-à-dire qu’il n’y ait pas de dégradation ou de diminution de l’être, soit de l’intérieur,
soit de l’extérieur, dans ce contact et dans ce mélange.” (Glissant 19) Le héros de Musinde est non seulement un être libre, mais aussi un pour soi dont
le libre arbitre n’est pas à la merci des courants de pensées contradictoires qu’il contrôle
et transcende. En internationaliste, il fait siens ces propos de Senghor :
“L’internationalisme ne s’édifiera qu’à partir des réalités nationales, voire de celles de
la patrie. […] Mais l’internationalisme, ou mieux la civilisation de l’universel, doit
s’édifier en transcendant les nations.” (Biondi 120) Pourtant, on peut lire une inquiétude grandissante dans le chef du héros qui se
croit “vivre dans un monde différent de celui des autres”(45). Cette idée de
“différence”est renforcée par celle du “vide”: “je regarde autour de moi, un vide”(45). Et ce “vide”est à son tour renforcé par le sentiment de “solitude”(45) qui hante le
protagoniste. Comment alors comprendre ce vide, ou mieux cette peur du vide ? Dans un contexte multiculturel, l’intellectuel africain vivant en Occident, comme
le héros de Retour de manivelle, sent le besoin d’aller vers l’autre (l’Occidental) et de lui
apporter sa contribution. Cependant lorsqu’il est rejeté par l’autre, il se sent seul, et
c’est l’éternelle viduité, aussi bien psychologique que matérielle, qui vient s’accaparer
de sa conscience. Josué affirme “ne pas encore sentir cette viduité parce que sa terre
d’asile actuelle (l’Europe) continue d’utiliser son savoir”(122). TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 Qui suis-je ? Réfléchissant sur la relation entre la pensée philosophique et l’écriture littéraire, Albert
Camus estimait qu’”un roman n’est jamais qu’une philosophie mise en images”, et
que “dans un bon roman toute la philosophie est passée dans les images”(1417). La
lecture de Retour de manivelle expose la récurrence de la question existentielle ”qui
suis-je ?”, notamment aux pages 48, 63, etc. D’aucuns pourraient en faire des analyses
philosophiques qui pourraient déboucher sur des trouvailles ontologiques
multidimensionnelles. Cependant nous voudrions proposer ici une analyse
métaphorique autour des images majeures utilisées par le héros pour se définir. Un être multiculturel (114–15) D’après cet extrait et l’ensemble du texte, le héros sent le “devoir de chercher
nécessairement à se faire comprendre”(54) pour combler le vide et vivre parmi les autres. Le voyage ayant permis au héros de faire un premier pas vers l’autre, il appartient
donc à l’autre de se montrer disponible en sortant d’un “occidentalisme-ghetto”dans
lequel son éducation et sa culture le contraignent de vivre. Ce qui a été dit aux Africains
du roman de Ngal reste encore valable pour les Occidentaux de l’œuvre de Musinde : D’après cet extrait et l’ensemble du texte, le héros sent le “devoir de chercher
nécessairement à se faire comprendre”(54) pour combler le vide et vivre parmi les autres. Le voyage ayant permis au héros de faire un premier pas vers l’autre, il appartient
donc à l’autre de se montrer disponible en sortant d’un “occidentalisme-ghetto”dans
lequel son éducation et sa culture le contraignent de vivre. Ce qui a été dit aux Africains
du roman de Ngal reste encore valable pour les Occidentaux de l’œuvre de Musinde :
Ce qu’un camarade a appelé tout à l’heure “attentat contre notre sécurité”n’est rien
d’autre que “attentat contre notre spécificité, contre notre repli sur nous-mêmes. Mais n’oublions pas qu’une “spécificité”prépare sa propre asphyxie dans la mesure
où elle ne reçoit pas l’oxygène de l’extérieur. Les cultures ne survivent que par
l’ouverture à d’autres cultures qui les libèrent de leur tendance au narcissisme
collectif. (Ngal 112) Visiblement, le protagoniste de Retour de manivelle est un artisan du développement
créatif dans la mesure où il appartient à plusieurs cultures sans aliéner aucune d’elles. (Biondi 124) Un être multiculturel Ces propos suggèrent
que la valeur d’un homme serait définie par la reconnaissance de son mérite par la
société à laquelle il appartient. Ceci relèverait donc de la tradition africaine selon
laquelle “l’homme n’existe que par la communauté”comme le dit ce proverbe zoulou :
umuntu ngumuntu ngabantu. Si le sentiment du “vide”est entrainé par le rejet de la société, la question du motif
pour lequel le héros est rejeté reste entière. Il est évident que l’œuvre de Musinde
mérite d’être lu à la lumière d’autres œuvres, notamment Terre des hommes où une
question similaire est posée : “Pourquoi nous haïr ? Nous sommes solidaires, emportés
sur la même planète, équipage d’un même navire. Et s’il est bon que des civilisations
s’opposent pour favoriser des synthèses nouvelles, il est monstrueux qu’elles
s’entredévorent.” (Saint-Exupéry 233) Par ailleurs, Josué n’est pas un personnage amoral qui serait l’égal de Meursault
du roman L’Étranger d’Albert Camus qui ignorerait les lois sociales,2 ni même ce
personnage immoraliste, à l’instar de Michel du roman de Gide du même nom,3 qui
substituerait ses propres lois aux lois de la nouvelle société. Josué est par contre ce
personnage rationnel et légaliste qui comprend les lois de sa nouvelle société mais TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 103 qui est pourtant jugé durement lorsqu’il tente d’y apporter les lois de sa société
d’origine auxquelles il croit fermement : Un jour, ne pouvant pas supporter ma solitude, j’ai frappé à la porte de ma voisine
pour faire sa connaissance. Cette dame m’a brutalement répondu en menaçant de
faire venir la police. Quand j’ai parlé à des amis qui avaient assuré mon accueil à
Vouillé, ils se sont moqués de moi en me recommandant de ne plus tenter cette
expérience dangereuse. Qu’y a-t-il d’anormal à prendre contact avec des voisins
lorsque ma culture l’autorise ? (114–15) Un jour, ne pouvant pas supporter ma solitude, j’ai frappé à la porte de ma voisine
pour faire sa connaissance. Cette dame m’a brutalement répondu en menaçant de
faire venir la police. Quand j’ai parlé à des amis qui avaient assuré mon accueil à
Vouillé, ils se sont moqués de moi en me recommandant de ne plus tenter cette
expérience dangereuse. Qu’y a-t-il d’anormal à prendre contact avec des voisins
lorsque ma culture l’autorise ? La bête Dans Retour de manivelle, Musinde revient à la problématique de la définition
ontologique de l’homme. Examinons ces propos tenus au héros du roman lors de la TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 104 soutenance de sa thèse de doctorat à l’Université de l’Ubal : “Ce que tu as fait, aucune
bête ne l’aurait fait, a conclu un des membres du jury, se référant à une phrase d’Antoine
de Saint-Exupéry tirée de Terre des hommes ou de Vol de nuit.” (81) Dans cet extrait, Musinde est évasif autour de l’œuvre de Saint-Exupéry d’où ces
propos ont été tirés. Il s’agit bien des propos tenus par Henri Guillaumet repris dans
Terre des hommes de Saint-Exupéry de la sorte : “Ce que j’ai fait, je te le jure, jamais
aucune bête ne l’aurait fait”(45, 52). Même si ces propos repris indifféremment au compte
de Saint-Exupéry mettent en exergue la propriété de l’endurance physique propre à
l’animal, l’image de la bête montre que la première définition de l’homme part de
l’essence animale avant l’ajout de tout autre attribut. L’œuvre renvoie, sans l’exprimer
explicitement, à l’ontologie platonicienne selon laquelle “l’homme est un animal
bipède sans plumes”. Cette considération basée sur les caractéristiques physiques est
renforcée dans le texte par la réflexion du personnage d’Huguette, future épouse de
Josué, sur le sens de la vie : “j’agis, je parle, je ne sais rien et pourtant j’existe”(63). Cela
reviendrait à dire qu’on peut exister sans tout savoir sur le sens de la vie, c’est-à-dire
sans réflexion critique autour de soi. Dans ce sens, l’existence même serait absurde car
elle échappe à toute appréhension philosophique. C’est l’image de la bête. À la
différence d’Huguette qui ne sait rien de son existence Josué “pense”car ce qu’il “a
fait” (une excellente thèse de doctorat) est de l’ordre du rationnel ; et “aucune bête ne
l’aurait fait”suggère que Josué est une bête hors du commun et, par déduction, une
bête dotée, en plus de son physique, d’un intellect exceptionnellement doué. S’agit-il d’une référence implicite au fameux roseau de Blaise Pascal, “l’homme
n’est qu’un roseau, le plus faible de la nature ; mais c’est un roseau pensant”(130) ? Le lecteur pourrait établir cette équivalence dès lors que le protagoniste de Musinde
affirme avoir lu Pascal (32). Le roseau pensant de Pascal équivaut à la bête
intellectuellement douée de Musinde. TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 La bête Aussi l’auteur tente-t-il de situer l’homme ou
l’intellectuel africain, par le fait de son effort de penser, au-delà de tous les autres qui
ont encore beaucoup de similarités avec le commun des mortels. Cette perception du
monde est une première partie de la réponse que Musinde semble apporter à la
question “qui suis-je”. Étant donné qu’il a été démontré que le héros Josué est un
humaniste qui s’abreuve à toutes les cultures, on peut affirmer que son intérêt à la
pensée platonicienne et voltairienne, par exemple, l’aurait poussé à définir l’homme
à partir de l’attribut animal. Dans Candide, qui semble être l’une des sources
d’inspiration de Musinde, le personnage de Pangloss pose la question suivante à un
derviche turc : “Maître, nous venons vous prier de nous dire pourquoi un aussi
étrange animal que l’homme a été formé”(Voltaire 241). Ainsi l’image de la bête est
ressuscitée dans un monde où elle ne serait plus à la mode pour exhumer le débat sur
l’essence animale de l’homme. TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 105 La silhouette Le mot “silhouette”est employé plusieurs fois dans le roman, notamment aux pages
35, 64, etc. Dans les propos “je marche derrière ma silhouette”(35). La propriété d’une
silhouette n’est pas de trainer l’objet qui la crée, mais plutôt de dépendre de la lumière
et des mouvements de cet objet. Si le héros affirme pouvoir “marcher derrière sa
silhouette”, on pourrait à la limite comprendre cette image comme étant la faculté
qu’a l’intellectuel africain de se définir en tant qu’être ; le moment de son hésitation,
comme il en est dans le texte (35), n’arrête cependant pas le déroulement des actions
susceptibles d’influencer son destin. Loin de constituer le destin de l’homme, l’image
de la silhouette qui traine son auteur serait l’inaction de l’homme dans son
incompréhension du mouvement et de l’action du temps. La réalité échappe donc à
l’homme qui n’agit pas. Si “la vie est un champ de bataille où les armées des idées se
confrontent”(35), un homme indécis ou sans actions subirait tout le temps un sort
imposé et serait toujours derrière sa silhouette qui serait “créée”sans lui. Cependant
l’intention du héros est de créer des silhouettes, de les “multiplier”(64) car elles sont
ses “doubles”(64). Ceci ne serait possible qu’à condition de considérer le héros comme
“un centre d’intérêt”ou un objet capable de diffuser son identité à profusion. Concrètement, la “lumière”illumine Josué et ce dernier illumine le monde. Josué
détient donc la “lumière”(122) et cette lumière c’est le savoir intellectuel (122). Ainsi
l’intention de Josué de créer plusieurs silhouettes serait à envisager comme le souci de
transformer son entourage par l’action de son savoir. Et la première silhouette du
genre serait donc son amie Huguette qui devrait exister suite à l’existence et à la
lumière de Josué. Par ailleurs, le ton poétique à la page 64 du roman vient suggérer
que cette influence est réciproque étant donné que Josué est incapable de créer des
doubles sans l’apport d’Huguette. On pourrait en déduire qu’Huguette détient
également une lumière si forte qu’elle participe à la génération des doubles dont le
monde a besoin. La rose Josué est une rose. L’image de “la rose qui doit éclore au milieu des ronces”(56) est
aussi une réponse à la quête de l’identité du héros. La rose étant le symbole par
excellence de l’idéal, de la beauté, de la vie et de l’évolution rayonnante, il conviendrait
de lire les “ronces”auxquelles il est fait allusion dans le roman comme étant un grand
ensemble formé des éléments hostiles à l’idéal de transformation harmonieuse et
positive d’une société. Ces forces opposées à “la lumière de la rose”, qui est pourtant
soucieuse de créer des doubles, se retrouvent dans l’environnement immédiat du
héros, non seulement dans la ville d’Ubal mais aussi à l’étranger, à Vouillé. Cet oxymore
“rose-ronces”constitue une appréhension de l’existence contraire à une vision candide
de la société où tout serait “au mieux dans le meilleur des mondes”, vision dans
laquelle sombrait le héros tout au début du roman. TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 106 Le lecteur entrevoit, à travers cette opposition, un antagonisme viscéral entre les
forces du progrès et celles de la déchéance. L’on en déduit que, selon Musinde, l’être
ne se définit, à l’instar de la rose, que par sa capacité de répondre aux défis qui se
placent sur le chemin de son évolution. Dans cette perspective, l’homme de Musinde
serait un Sisyphe qui, reconnaissant bien l’existence du danger à perpétuité (ronces),
s’apprêterait à vivre au présent sans y opposer immédiatement sa propre vision du
monde. La rose de Musinde éclot donc individuellement sans annihiler son souci
d’illuminer l’environnement dans lequel l’être se développe, en dépit de la multiplicité
des embuches. TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 La question de la mort / l’absurde Par exemple, Josué fait de son univers un lieu de prédilection où TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 107 les conceptions les plus antagonistes se rencontrent : d’un côté, le héros rappelle au
lecteur une conception matérialiste de la vie, et de l’autre une vision spiritualiste est
mise en évidence. On peut lire par exemple que “La mort n’est pas à craindre, car elle
ne nous concerne en rien. Tant que nous sommes, la mort n’est pas là, et dès que la
mort est là, nous ne sommes plus. La mort n’est rien ni pour les vivants ni pour les
morts.” (78) Cette conception épicurienne, que Sartre vient par la suite compléter (79), n’est
pas une réponse à la question brûlante de la mort et de la vie. Elle ne peut constituer,
selon le protagoniste, qu’une thérapeutique pour “ceux qu’effraient les châtiments de
l’au-delà et le système de la vie future”(79). Par cette manière de penser, on peut
entrevoir le fait que pour le héros, l’existence humaine ne devrait pas être obscurcie
par la peur de la mort. La nécessité de comprendre le sens de la mort est un impératif
qui contraint le héros à rechercher la réponse à la problématique de la mort au-delà de
l’absurde exprimé précédemment par les matérialistes. C’est cette interprétation qui
pousse le héros de Musinde à rechercher la réponse ailleurs, notamment chez ceux
qui ne considèrent pas la mort, ainsi que nous le ferons voir, comme la fin de tout. En
substance, le héros de Retour de manivelle se refuse d’être comparé à Meursault (80), ce
personnage matérialiste du roman L’étranger de Camus pour qui la question de la
mort importe peu. Au contraire, Josué cherche à aller au-delà des considérations
physiques:”Mais où suis-je ? […] je me vois étendu au bord de la rivière “Ayamo”. Et
là, bien loin, dans l’eau, vers l’autre rive, j’aperçois ma mère, mon père, mon frère aîné,
ma grand-mère maternelle, ma grand-mère paternelle déjà dans l’au-delà.” (123) Ces propos constituent un récit de la mort. Josué est mort pendant un certain
temps et il revient dans le monde sensible avec un témoignage de l’au-delà. S’il va
jusqu’à raconter ce que les habitants de l’au-delà lui donnaient comme instructions à
exécuter sur terre, cela contribue à confirmer sa certitude sur la continuité de la vie
après la mort apparente. La question de la mort / l’absurde Retour de manivelle de Musinde est un univers discursif où le sens profond de l’existence
est recherché. Pour y parvenir, non seulement l’auteur recourt aux métaphores
expliquées précédemment, mais, à travers une approche différente, le sens de l’existence
est également donné moyennant le recours à une conceptualisation du contraire :
chercher à comprendre la mort, par exemple, donnerait un sens à la vie, voire à la
mort elle-même. Le thème de la mort n’est pas abordé dans le roman de manière théorique et
désintéressée. Pour Musinde, la réflexion critique sur la mort nécessite une
subjectivisation au point que, grâce à son individualisation, on aboutisse à des
considérations on ne peut plus pragmatiques. En effet, pour comprendre la mort,
l’auteur fait penser à un être cher qui meurt ou qui est sur le point de mourir. Le héros
de Retour de manivelle se sert de la maladie de sa mère (“une femme généreuse”, 76)
ainsi que de l’émotion qui accompagne le fait de penser à sa mort imminente pour
livrer au lecteur sa réflexion sur le phénomène de la mort. Dans la présentation de sa
vision du monde, comme nous allons le montrer, l’on remarque que le héros a lu
beaucoup d’auteurs et de courants de pensée de tous les temps qui, bien avant Musinde,
s’étaient penchés sur la même question. Il en résulte que la problématique de la mort
devrait franchir le seuil purement dialectique pour devenir une préoccupation
personnelle du héros, une démarche parallèle à la méthodologie classique en la matière. “Qu’est-ce que la mort ?” Telle est la question que le héros se pose à la page 76 du
roman. Étant donné que “seul l’homme a le privilège de savoir en pleine conscience
qu’il mourra un jour”(77), le héros pense qu’il est impérieux de “comprendre ce
phénomène de la mort, ses caractéristiques, son utilité pour ne pas y voir un obstacle
à l’harmonie du monde”(77). Le roman de Musinde dans son ensemble est la
représentation d’une sagesse qui interdit de craindre la mort, en latin ne mortem
timueritis. Dans sa démarche, le protagoniste de Retour de manivelle agit en pédagogue
fidèle au principe de progression didactique allant du simple au complexe et du
connu à l’inconnu. La question de la mort / l’absurde L’univers de Retour de manivelle est donc à interpréter comme
celui dans lequel la mort n’équivaut pas à la fin de la vie, mais plutôt à l’accès au
monde accessible sous d’autres formes ou bien nécessitant des nouveaux organes de
sens. Aussi contraires qu’ils puissent paraître, l’œuvre de Musinde montre que ces
deux mondes se complètent cependant dans leur différence et que le monde immatériel
de l’au-delà est doté u pouvoir d’influencer le dessein du monde sensible. Ceci se
laisse voir surtout lorsque, en cas des difficultés, le héros évoque le nom de sa mère
(déjà décédée) pour qu’elle le protège contre les forces du mal dans le monde
physique : En rentrant à deux heures du matin sur ce parcours de 30 km, surgit devant nous
un être mystérieux et multicolore dont les lumières scintillantes éblouissaient le
chauffeur qui ne distinguait plus la route. […] J’ai pleuré en évoquant le nom de ma
mère […] L’évocation du nom de ma mère a bloqué tout le jeu […] J’ai évoqué à
nouveau le nom de ma mère. L’être mystérieux nous laisse la voie. (89–90) TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 108 Il s’avère par ailleurs que dans le récit de la mort repris précédemment, la frontière
entre les deux mondes est symbolisée par la rivière Ayamo. Cette rivière représente la
difficulté et l’épaisseur qui contraindraient les êtres à ne s’en tenir qu’à un monde à la
fois. L’instruction donnée au héros est de ne pas traverser cette rivière ; mais plutôt
d’avoir une nette conscience du monde matériel en le vivant conformément à la
tradition incarnée dans la “corne du buffle.”(123 –24) De plus, le héros du roman de Musinde évoque avec intérêt cette pensée chrétienne
reprise par André Gide dans son œuvre Si le grain ne meurt (1924) : “Toute productivité
n’est possible que si le grain meurt. Au juste, pour devenir arbre qui porte des fruits
succulents, le grain meurt sans mourir.” (108) Cette pensée de Jésus retrouvable dans La Bible (“En vérité, en vérité, je vous le dis,
si le grain de blé qui est tombé en terre ne meurt, il reste seul ; mais, s’il meurt, il porte
beaucoup de fruits”(Jean XII : 24) montre que Musinde insinue que la mort n’a de
sens que dans la renaissance. La question de la mort / l’absurde Le héros revient à la vie en tant qu’une nouvelle entité
transformée par le séjour dans l’au-delà. Dans cet univers, cet oscillement entre deux
mondes apparemment distincts est comparable à la maturation de la jeunesse dont “le
cheminement est jalonné des moments de négativité et de positivité, c’est-à-dire des
moments de contradictions dialectiques”(108). C’est justement cette allusion au
cheminement de la jeunesse qui place Musinde sur la même veine que Gide dans la
mesure où la mort symbolique renvoie aux vicissitudes de la vie qui, au lieu de
détourner la jeunesse de son objectif de maturation intérieure, constitue plutôt un
déclic pour sa renaissance ou sa prise de conscience. On peut en déduire que la mort
imagée de Josué peut trouver son interprétation dans la pensée gidienne à laquelle
Musinde donne une forme imagée propre à la cosmogonie bantoue : celle du dialogue
et de l’interconnexion cosmique entre l’en-deçà et l’au-delà. Conclusion Bien qu’étant basé sur la vie de l’auteur, que l’on peut voir en filigrane comme témoin
des abus du pouvoir et des conflits politiques les plus meurtriers, l’œuvre de Musinde
s’abstient cependant de décrire les horreurs ; elle est même timide devant les faits
politiques sporadiques qu’elle évoque. Cette attitude permet à l’auteur d’éviter des
sentiers battus et d’exprimer ses fantasmes tout en intégrant à sa plume une vision du
monde subjective à laquelle il mêle sa connaissance cosmopolite dans l’interprétation
de l’identité. La question culturelle occupe la part du lion dans cette œuvre. Néanmoins, cette culture qui se veut à la fois dynamique et métisse, ne trouve son
sens que dans une conception cyclique où le point de départ et celui d’arrivée se
joignent dans un même être à qui l’internationalisme aurait donné le sens
d’accommodation de l’autre. Si “le romancier raconte la vérité, sa vérité ou celle qu’il
croit être”(Pageaux 17), on ne pourrait qu’en déduire que Musinde a créé dans ce TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 109 roman une nouvelle vérité, celle de la tradition de l’avenir. Et dans cette tradition, la
parole oscille entre des identités apparemment antagonistes, aussi bien celles du passé
gréco-latine, franco-anglaise et africaine que celles d’un présent mondialisé où le
rapetissement spatial s’affronte à une spécificité culturelle exclusive. Si la question de
la mort est mise en exergue dans cette œuvre, elle n’a réellement de sens pour l’auteur
que dans la renaissance, le roman tout entier étant conçu comme une apologie du
devenir. Au sujet de l’existence, à l’instar de l’homme absurde (Sisyphe) qui se sert de sa
condition pour transformer sa situation, le héros de Musinde ajoute à cela une
conviction métaphysique inaliénable, celle de l’existence des entités supérieures
invisibles (ancêtres) qui se situent au-delà de toute appréhension empirique et
rationaliste. C’est à ces entités (d’au-delà de la rivière Ayamo) que l’on devrait puiser
la force pour affronter la rigidité d’une quotidienneté malveillante (les ronces) dans
le monde sensible. On peut dire, pour citer Mouralis, que grâce au voyage et à ses
rencontres multiples, le héros de Retour de manivelle a opposé une vision traditionnelle
de l’Afrique à “une vision du monde diversifiée”, celle d’un “témoin particulier ayant
adopté volontiers un mode de narration autobiographique”(Mouralis 24). Notes 1. “Vagissements”(Poèmes) dans Comme des matins éternels. Kinshasa : UEZA, 1984. 2. Meursault est le héros de l’œuvre L’Étranger d’Albert Camus publiée aux Éditions Gallimard en 1942. 3. Michel est le héros du roman L’immoraliste d’André Gide publié en 1902 aux Éditions Gallimard. Conclusion Le mérite
de l’œuvre de Musinde est d’avoir ainsi juxtaposé des identités apparemment
antagonistes dans un sujet dont le parfait équilibre est sous-tendu par des extrêmes. 1. “Vagissements”(Poèmes) dans Comme des matins éternels. Kinshasa : UEZA, 1984. 2. Meursault est le héros de l’œuvre L’Étranger d’Albert Camus publiée aux Éditions Gallimard en 1942.
É Pascal, Blaise. Pensées. Paris : Phidal, 1995.
Saint-Exupéry, Antoine de. Terre des hommes. Paris : Le Livre de Poche, 1938.
Senghor, Léopold Sédar. Liberté 1. Négritude et humanisme. Paris : Seuil, 1964.
Voltaire. Candide ou l’optimisme et autres contes. Paris : Pocket, 1998 [1759]. TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 Pascal, Blaise. Pensées. Paris : Phidal, 1995. Références Baudelaire, Charles. Les Fleurs du Mal. Œuvres Complètes. Paris : Gallimard, 1951. Biondi, Jean-Pierre. Senghor ou la tentation de l’universel. Paris : Denoël, 1993. Camus, Albert. Essais. Paris : Gallimard, 1965. —. Le mythe de Sisyphe. Paris : Gallimard, 1942. —. Le mythe de Sisyphe. Paris : Gallimard, 1942. Gide, André. Si le grain ne meurt. Paris : Gallimard, 1924. Glissant, Édouard. Introduction à une poétique du divers. Paris : Gallimard, 1996. Glissant, Édouard. Introduction à une poétique du divers. Par Kourouma, Amadou. Les soleils des indépendances. Montréal : Presses de l’Université de Montréal, 1968. É Madebe, Georice. Francophonies invisibles. Émergences, invisibilité, hétérogénéité et sémiotique. Paris :
L’Harmattan, 2009. Malonga, Alpha-Noël. Roman congolais. Tendances thématiques et esthétiques. Paris : L’Harmattan, 2007. Maran, René, Un homme pareil aux autres. Paris : Albin Michel, 1962. Moura, Jean-Marc, Littératures francophones et théorie postcoloniale. Paris : PUF, 2005. Mouralis, Bernard. “Le même et l’autre. Réflexions sur la représentation du voyage dans quelques
œuvres africaines”. Littérature postcoloniale et représentations de l’ailleurs. Bessière, Jean et al. Eds. Paris : Honoré Champion, 1999. 11-27. Musinde, Julien Kilanga. Retour de manivelle. Langres : Riveneuve, 2008. Ngal, Mbwil a Mpaang. Giambatista Viko ou le viol du discours africain. Paris : Hatier, 1984. bwil a Mpaang. Giambatista Viko ou le viol du discours africain. Nganang, Patrice. “Le nœud gordien.”Africultures 59 (2004) : 83–89. Pageaux, Daniel-Henri. et al. Formes et imaginaire du roman. Perspectives sur le roman antique, médiéval,
classique, moderne et contemporain. Paris : Honoré Champion, 1998. TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 110 TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 TYDSKRIF VIR LETTERKUNDE • 50 (1) • 2013 111 111
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English
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Learning spike-timings based representations of sensory stimuli with leaky integrate-and-fire neurons
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BMC neuroscience
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BioMed Central BioMed Central Published: 13 July 2009
BMC Neuroscience 2009, 10(Suppl 1):P144
doi:10.1186/1471-2202-10-S1-P144 This abstract is available from: http://www.biomedcentral.com/1471-2202/10/S1/P144
© 2009 Gutmann and Hyvärinen; licensee BioMed Central Ltd. Previous work Assuming models for the encoder-decoder pair, in previ-
ous work, the free parameters in the models have been
learned from natural stimuli, and successfully related to
properties of the early sensory system [1]. Much emphasis
in previous work has been on the principle that guide the
learning process, for example information theoretical
[2,3] or energetic principles [4]. Less attention was given
to the role of the underlying neuron model. Recently, we
have proposed in [5,6] data representation where the
encoding happens with a simple spiking neuron model
(the Spike Response Model SRM_0 [7]) and the spike tim-
ings serve to continuously update the ongoing internal
representation. Results Stimuli from the environment are sensed by a population
of receptor neurons and then carried by spike trains to fur-
ther processing stages in the brain. Our work deals with
the question how, in the early sensory system, rich stimuli
from the natural environment can be represented by spike
trains. In theoretical approaches to this questions, neural
representation is often modeled as an encoder-decoder
system: a sensory stimulus is encoded into neural activity
and a hypothetical decoder indicated how the neural
activity must be read to construct the internal representa-
tion of the stimulus. Here, we first formulate the theory of an encoder-decoder
system for leaky integrate-and-fire neurons and then
extend the previous models of learning spike timings-
based representations. As time is explicitly present in our
framework, the internal representation that is constructed
from the spike timings can be about the stimulus prior to
the spike or an internal prediction of the future stimulus. Here, we first formulate the theory of an encoder-decoder
system for leaky integrate-and-fire neurons and then
extend the previous models of learning spike timings-
based representations. As time is explicitly present in our
framework, the internal representation that is constructed
from the spike timings can be about the stimulus prior to
the spike or an internal prediction of the future stimulus. We present the learning of an encoder-decoder pair where
each spike is used both to update the prediction of the
future stimulus and to correct the representation of the
stimulus before the spike. We present thus the learning of
a prediction-and-correction based internal representation
of the sensory stimulus. We present the learning of an encoder-decoder pair where
each spike is used both to update the prediction of the
future stimulus and to correct the representation of the
stimulus before the spike. We present thus the learning of
a prediction-and-correction based internal representation
of the sensory stimulus. Acknowledgements g
This work was funder by the Academy of Finland (NEURO program and the
Algodam Centre of Excellence). Open A
Poster presentation
Learning spike-timings based representations of sensory stimuli
with leaky integrate-and-fire neurons
Michael Gutmann*1 and Aapo Hyvärinen1,2 Open Access from Eighteenth Annual Computational Neuroscience Meeting: CNS*2009
Berlin, Germany. 18–23 July 2009 Published: 13 July 2009
BMC Neuroscience 2009, 10(Suppl 1):P144
doi:10.1186/1471-2202-10-S1-P144 6.
Gutmann M, Hyvärinen A: Learning reconstruction and predic-
tion of natural stimuli by a population of spiking neurons.
European Symposium on Artificial Neural Networks (ESANN2009) . 2009-
05-12 7.
Gerstner W, Kistler WK: Spiking Neuron Models Cambridge University
Press; 2002. BMC Neuroscience 2009, 10(Suppl 1):P144 6.
Gutmann M, Hyvärinen A: Learning reconstruction and predic-
tion of natural stimuli by a population of spiking neurons.
European Symposium on Artificial Neural Networks (ESANN2009) . 2009-
05-12
7.
Gerstner W, Kistler WK: Spiking Neuron Models Cambridge University
Press; 2002. References 1. Simoncelli E, Olshausen B: Natural image statistics and neural
representation. Annual Review of Neuroscience 2001, 24:1193-1216. p
f
2. Olshausen B, Field D: Emergence of simple-cell receptive field
properties by learning a sparse code for natural images. Nature 1996, 381:607-609. 3. Smith E, Lewicki M: Efficient auditory coding. Nature 2006,
439:978-982. 4. Vincent BT, Baddeley RJ, Troscianko T, Gilchrist ID: Is the early vis-
ual system optimised to be energy efficient? Network: Compu-
tation in Neural Systems 2006, 16:175-190. 4. Vincent BT, Baddeley RJ, Troscianko T, Gilchrist ID: Is the early vis-
ual system optimised to be energy efficient? Network: Compu-
tation in Neural Systems 2006, 16:175-190. y
5. Gutmann M, Hyvärinen A, Aihara K: Learning encoding and
decoding filters for data representation with a spiking neu-
ron. International Joint Conference on Neural Networks (IJCNN2008)
2008. Page 1 of 2
(page number not for citation purposes) Page 1 of 2
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English
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Thrombocytopenia and Physical Activity among Older Adults: The Tenuous Line between Bleeding Preventiona Physical Functional Decline
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Open access journal of gerontology & geriatric medicine
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Abstract While the treatment of cancer has improved significantly in the last decades contributing to an increase in survival rates, it also associated
with a variety of side effects including thrombocytopenia. Although representing a serious condition to all age groups, as low levels of
platelets can cause bleeding, thrombocytopenia may present a threat to older adults´ overall activity levels and mobility if lower cut off points
for the practice of physical activity aren´t properly set. Therefore the objective of this mini review is to examine the literature in the issue of
thrombocytopenia and physical activity among older adults in order to provide guidelines to health care practitioners about the practice of
physical activity and rehabilitation. Results show that very few studies addressed this issue. There isn´t a consensus regarding cut off points
of low platelets for safely performing physical activity. Moreover, there isn´t a low limit cut off point for suspending all types of activity, and
therefore minimizing further physical functional decline among older adults. Keywords: Low platelets; Mobility; Activity guidelines; Cancer; Exercise Lucelia Luna Melo-Diaz* University of Pelotas, Brasil Submission: June 15, 2017; Published: June 27, 2017 *Corresponding author: Brazilian Company of Hospital Services - University Hospital Federal University of Pelotas,Rua Professor Doutor Araújo,
538, Centro, Pelotas, Rio Grande do Sul, Brasil, Tel:
; Email: Abstract OAJ Gerontol & Geriatric Med 1(5): OAJGGM.MS.ID.555571 (2017) Mini Review
Volume 1 Issue 5 - June 2017
DOI: 10.19080/OAJGGM.2017.01.555571
OAJ Gerontol & Geriatric Med
Copyright © All rights are reserved by Lucelia Luna Melo-Diaz
Open Access Journal of
Gerontology & Geriatric Medicine
ISSN: 2575-8543 Mini Review
Volume 1 Issue 5 - June 2017
DOI: 10.19080/OAJGGM.2017.01.555571
OAJ Gerontol & Geriatric Med
Copyright © All rights are reserved by Lucelia Luna Melo-Diaz
Open Access Journal of
Gerontology & Geriatric Medicine
ISSN: 2575-8543 Open Access Journal of
Gerontology & Geriatric Medicine
ISSN: 2575-8543 OAJ Gerontol & Geriatric Med
Copyright © All rights are reserved by Lucelia Luna Melo-Diaz Thrombocytopenia and Physical Activity among
Older Adults: The Tenuous Line between Bleeding
Preventiona Physical Functional Decline Open Access Journal of Gerontology & Geriatric Medicine strength and power [13], and reduction in the speed of nerve
conduction [14]. In addition, bone health [15], and overall
metabolism may be severely affected [16]. These age-related
changes commonly happen in association with other co-
morbidities. 2- Hospitalization may require an extended bed
rest bringing negative consequences for older adults. Mobility
limitation is linked with negative outcomes, such as loss of
independence [17], higher risk for mortality [18], and occurrence
of falls [17]. Therefore, suspending physical activity due to
thrombocytopenia may present detrimental consequences for
older adults. petechiae and ecchymoses) and risk for intracranial hemorrhage
or gastrointestinal bleeding. When platelet levels are above
20,000/L, clinical manifestations are generally mild, such as
easy bruising. The management of thrombocytopenia does not
include performance of physical activity [21]. The American College of Sports Medicine (ACSM) had a
roundtable about exercise guidelines for cancer survivors, but no
specific guidelines were provided regarding exercise for patients
with low platelets [22]. An updated ACSM exercise guidelines
for cancer survivors recommended that exercise should be
performed with caution in patients with thrombocytopenia,
but no specific recommendations were made for types and
intensity of exercise among those with low platelet count, or a
low cut off point of thrombocytopenia to safely suspend exercise
[23]. Recently, the ACSM has made exercise recommendations
for persons with chronic diseases and disabilities with a
consideration about thrombocytopenia. When platelet levels
are between 20,000 and 50,000, patients should be limited
to use elastic bands, stationary cycles, range of motion, and
ambulation. Vigorous exercise is contraindicated when platelet
levels are below 50,000/mm3. However, this recommendation
is made subjectively, as the ACSM acknowledges that there are
no studies in relation to exercise with patients who have low
platelet levels [24]. While bed rest is needed to prevent bleeding, for older adults,
the question remains, what is the tenuous line between resting
to prevent bleeding and performing physical activity in order
to prevent further physical and function decline, particularly
those in a hospital setting? Is there a cut off point in which it
is safe to suspend all physical activity? Therefore, the objective
of this mini review is to examine the literature in this regard,
and to provide guidance to health professionals, especially those
in the physical rehabilitation field, about existing guidelines
on physical activity for older adults with thrombocytopenia in
hopes to guide practice. Summary of the findings In order to better understand the recommended types
of physical activity among patients with thrombocytopenia,
particularly those undergoing chemotherapy, a literature
review was conducted using the following search terms:
thrombocytopenia or low platelets and physical activity or
exercise or rehabilitation, and older adults. Guidelines from
National Cancer agencies were also included in this review. The Leukemia/Bone Marrow Transplant Program of British
Columbia provides exercise recommendations for different
levels of thrombocytopenia. When the blood count is less than
15,000 platelets per microliter, patients are advised to limit all
activity. When platelets levels are 15,0000 to 20,000 patients
may do gentle exercising without resistance, such as exercising
while sitting or standing, gentle stretching or taking an easy
walk. When platelet levels are between 20,000 to 40,000,
patients may use light resistance, such as weights or latex bands
as well as faster walking and stair climbing. At platelet levels of
40,000 to 60,000 patients may perform gentle aerobic activity,
such as stationary cycling. Higher levels of platelets, which are
still considered low, allow patients to perform aerobic exercise,
such as biking and jogging, with use of proper gear and with
caution against injury [19]. The National Cancer Institute states
that platelet levels below 25,000 per microliter of blood are a
life threatening adverse event, but no further recommendation
regarding physical activity performance is provided [20]. The Seattle Cancer Care Alliance provides specific cut off
points of low platelet levels and recommended activities for
each level. For platelet levels below 10,000, only walking around
the room (e.g. going to the bathroom) is allowed. For platelet
levels between 10,000 and 19,999, strength training without
weights and cardiovascular exercise, both without strain are
allowed, if no signs of bleeding are present. For platelet levels
between 20,000 and 49,999 strength training with weights and
cardiovascular exercise are allowed without strain, if the patient
has no signs of bleeding [25]. Introduction similar to cancer symptoms, such as fatigue, nausea, vomiting,
pain, as well as signs and symptoms, such as alopecia, peripheral
neuropathy and thrombocytopenia [10]. According to the World Health Organization estimate, 14.1
million new cases of cancer occurred worldwide in 2012 [1]. It is a disease more prevalent among older adults compared to
younger adults [2,3]. The incidence of cancer tends to increase
among those aged 75 to 90 years of age [4], and it is anticipated
that there will be a 67% increase in incidence among older adults
by 2030 [5]. The disease can severely affect quality of life among
older adults and a variety of physical and psychological symptoms
may occur. Pain, swelling, bleeding, urinary difficulties, fatigue,
weight loss, changes in bowel habits (diarrhea or constipation),
cough, shortness of breath, nausea and vomiting among others
may occur depending on the type and location of the cancer [6]. Thrombocytopenia
is
a
condition
characterized
by
abnormally low platelet levels in the blood. The normal platelet
count among adults is between 150,000 and 450,000 platelets per
microliter of blood [11]. This condition is a common side effect
among patients who undergo chemotherapy. As a consequence,
patients have a higher tendency for bleeding, particularly when
platelet levels drop below 50,000 per microliter of blood [11]. Therefore, patients often have to limit activity levels in order to
avoid bleeding. Although cancer is the second leading cause of death
globally, accounting for 8.8 million deaths in 2015 [7], mortality
rates have shown a slight decline since 1990. Reasons for this
occurrence are improvements in early screening and treatment
[8]. The most common types of treatments are surgery, systemic
therapies, such as chemotherapy, and hormone and immune
therapies [9]. Although these types of treatments can increase
survival rates, they can also cause a variety of side effects that are Although thrombocytopenia represents a serious condition
for all age groups, this issue may be more prominent among
older adults who undergo chemotherapy, particularly those
who are hospitalized, for important reasons: 1- This age group
experiences a series of age-related physical and cognitive
changes. Some of the main changes include a reduction in
cardiorespiratory capacity [12], reduction in neuromuscular
performance partly due to sarcopenia, which affects muscle 001 How to cite this article: Lucelia L M. Thrombocytopenia and Physical Activity among Older Adults: The Tenuous Line between Bleeding Preventiona
Physical Functional Decline. OAJ Gerontol & Geriatric Med. 2017; 1(5): 555571. DOI: 10.19080/OAJGGM.2017.01.555571 Discussion and Practical Implications The objective of this review was to provide guidance to
health professionals about guidelines for physical activity
among older adults with thrombocytopenia, particularly those
undergoing chemotherapy. Based on the findings presented in
this review, very few studies addressed the issue of physical
activity or exercise for older adults with thrombocytopenia. The
existing guidelines are inconsistent and lack scientific rigor and
testing. Additionally, there is no lower limit, under which it is
safe to suspend physical activity. Given the inconsistency of these guidelines, the choice
between physical activity and rest is arbitrary. The health care
practitioner’s decision to suspend all types of physical activity
may prevent excessive and dangerous bleeding, but may also
cause further damage to physical function, and other fitness-
related components, such as strength and endurance. Considering
that older adults are a vulnerable group due to physiological age- Sekhon and Roy [21], in an evaluation and management
paper about thrombocytopenia, state that when platelet
counts are below 10,000/L there is a risk for spontaneous
mucocutaneous bleeding (gingival bleed, epistaxis, menorrhagia, 002 Open Access Journal of Gerontology & Geriatric Medicine featuring increasing incidence of liver cancer. Cancer 122(9): 1312-
1337. related changes, coupled with other co-morbidities, the decision
to suspend all physical activity can pose a serious health threat
to mobility. For those undergoing chemotherapy, and who are
hospitalized, the situation may be even more complex. Blood
count starts to drop after chemotherapy, taking from 7 to 10
days to reach the lowest count, and it can take from 2 to 3 weeks
for the patient to recover [21]. The inexistence of effective
guidelines for physical activity may present a detrimental
effect on older adults’ mobility and autonomy, as rapid physical
reconditioning may occur. As chemotherapy treatment happens
in cycles, thrombocytopenia can occur frequently. 9. National Cancer Institute. Cancer treatment (2017) Retrieved. 10. National Cancer Institute Side effects (2017) Retrieved. 11. National Heart, Lung and Blood Institute What is Thrombocytopenia? (2012) Retrieved. 12. Hollmann W, Struder HK, Tagarakis CVM, King G (2007) Physical
activity and the elderly. European Journal of Preventive Cardiology 14:
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microinfarcts and demyelination significantly affect cognition in brain
aging. Stroke 35: 410-414. Existing guidelines on physical activity among older adults
with thrombocytopenia lack empirical evidence. Future studies
in this area are needed in order to safely establish physical
activity performance by cut off points of thrombocytopenia. Other physical activity measurement parameters, such as
frequency, intensity, type, and duration should also be considered
by each cut off point. There is a particular need for establishing
a low limit of platelet levels in which physical activity must
be suspended. The lower cut off point would avoid dangerous
bleeding, and yet minimize further functional decline among
older adults. 15. Shephard RJ (1997) Aging, physical activity and health. Toronto,
Ontario: Human Kinetics. 16. Hunter GR, McCartthy JP, Bamman MM (2004) Effects of resistance
training on older adults. Sports Medicine 34(5): 329-348. 17. Rubenstein L Z, Josephson KR (2002) The epidemiology of falls and
syncope. Clinics in Geriatric Medicine 18(2): 141-158. 18. Keeler E, Guralnik JM, Tian H, Wallace R B, Reuben DB (2010) The
impact of functional status on life expectancy in older persons. The
Journals of Gerontology Series A Biological Sciences and Medical
Sciences 65(7): 727-733. References 19. The Leukemia/Bone Marrow Transplant Program of British Columbia. Healthy Living. 1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, et al. (2015)
Cancer incidence and mortality worldwide: The GLOBOCAN Project. Int J Cancer 136(5): E359-E386. 20. National Institutes of Health. Common terminology criteria for adverse
events (CTCAE) version 3.0. 2. Canadian Cancer Society (2017) Cancer Statistics at a glance. 21. Sekhon SS, Roy V (2006) Thrombocytopenia in adults: A practical
approach to evaluation and management. Southern Medical Journal
99(5): 491-498. 3. Courneya K, Karvinen K (2007) Exercise, aging, and cancer. Applied
Physiology Nutrition and Metabolism. Physiologie Appliquee Nutrition
Et Metabolisme 32(6): 1001-1007. 22. Schimtz KH, Courneya KS, Matthews C, Demark-Wahnefried W, Galvão
DA, et al. (2010) American College of Sports Medicine roundtable on
exercise guidelines for cancer survivors. Medicine Science in Sports
Exercise 42(7): 1409-1426. 4. Pedersen JK, Engolm G, Skytthe A, Christensen K (2016) Cancer and
aging: Epidemiology and methodological challenges. Acta Oncologica
55(Suppl 1): 7-12. 5. Smith BD, Smith GL, Hurria A, Hortobagyi GN, Buchholz TA (2009)
Future of cancer incidence in the United States: burdens upon an aging,
changing nation. Journal of Clinical Oncology 27(17): 2758-2765. 23. Wolin KY, Schwartz AL, Matthews CE, Courneya KS, Schmitz KH (2012)
Implementing exercise guidelines for cancer survivors. Journal of
Supportive Oncology 110(5): 171-177. 6. Forbes LJL, Warburton F, Richards MA, Ramirez AJ (2014) Risk factors
for delay in symptomatic presentation: a survey of cancer patients. British Journal of Cancer 111(3): 581-588. 24. Moore GE, Durstine JL, Painter PL (2016) ACSM´s Exercise management
for persons with chronic diseases and disabilities, (4th ed.). Human
Kinetics pp: 232-234. 24. Moore GE, Durstine JL, Painter PL (2016) ACSM´s Exercise management
for persons with chronic diseases and disabilities, (4th ed.). Human
Kinetics pp: 232-234. 7. World Health Organization (2017) Retrieved, 11/06, 2017 25. Seattle Cancer Care Alliance, Exercise and blood value precautions
(2014) Retrieved. 25. Seattle Cancer Care Alliance, Exercise and blood value precautions
(2014) Retrieved. 8. Blythe Ryerson A, Eheman CR, Altekruse SF, Ward JW, Jemal A, et al. References (2016) Annual report to the nation on the status of cancer, 1975-2012, Your next submission with Juniper Publishers
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Review of: "Kirchhoff Coupling Generates ATP, the Chemical Energy of Life"
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Qeios, CC-BY 4.0 · Review, March 27, 2023 Qeios ID: ZKPXED · https://doi.org/10.32388/ZKPXED Review of: "Kirchhoff Coupling Generates ATP, the Chemical
Energy of Life" David Lloyd1
1 Cardiff University David Lloyd1 Potential competing interests: No potential competing interests to declare. I found this short paper very interesting for a biochemist as this physical presentation is not to be found in our text-books. I have only 2 comments. 1. For a biochemist the first and second Kirchoff's laws should be stated at the outset as many biochemists who have not
studied advanced level physics will have only a hazy recall... 1. For a biochemist the first and second Kirchoff's laws should be stated at the outset as many biochemists who have not
studied advanced level physics will have only a hazy recall... studied advanced level physics will have only a hazy recall... 2. Fig. 1 is excellent, but Figs 2 and 3 are not explained in the captions or referred to in the text. Qeios ID: ZKPXED · https://doi.org/10.32388/ZKPXED 1/1
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Stay Mindful and Carry on: Mindfulness Neutralizes COVID-19 Stressors on Work Engagement via Sleep Duration
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Frontiers in psychology
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Stay Mindful and Carry on:
Mindfulness Neutralizes COVID-19
Stressors on Work Engagement via
Sleep Duration Michelle Xue Zheng 1, Theodore Charles Masters-Waage 2, Jingxian Yao 3, Yizhen Lu 4,
Noriko Tan 2 and Jayanth Narayanan 4* 1 Department of Organizational Behavior and Human Resource Management, China Europe International Business School
(CEIBS), Shanghai, China, 2 NUS Business School, Singapore Management University, Singapore, Singapore, 3 Católica
Lisbon School of Business and Economics, Catholic University of Portugal, Lisbon, Portugal, 4 NUS Business School,
National University of Singapore, Singapore, Singapore We examine whether mindfulness can neutralize the negative impact of COVID-19 stressors
on employees’ sleep duration and work engagement. In Study 1, we conducted a field
experiment in Wuhan, China during the lockdown between February 20, 2020, and March
2, 2020, in which we induced state mindfulness by randomly assigning participants to either
a daily mindfulness practice or a daily mind-wandering practice. Results showed that the
sleep duration of participants in the mindfulness condition, compared with the control condition,
was less impacted by COVID-19 stressors (i.e., the increase of infections in the community). In Study 2, in a 10-day daily diary study in the United Kingdom between June 8, 2020, and
June 19, 2020, we replicate our results from Study 1 using a subjective measure of COVID-19
stressors and a daily measure of state mindfulness. In addition, we find that mindfulness
buffers the negative effect of COVID-19 stressors on work engagement mediated by sleep
duration. As the COVID-19 pandemic is ongoing and the number of reported cases continues
to rise globally, our findings suggest that mindfulness is an evidence-based practice that can
effectively neutralize the negative effect of COVID-19 stressors on sleep and work outcomes. The findings of the present study contribute to the employee stress and well-being literature
as well as the emerging organizational research on mindfulness. Edited by:
Bahar Tuncgenc,
University of Nottingham,
United Kingdom
Reviewed by:
Seockhoon Chung,
University of Ulsan, South Korea
Calliope Holingue,
Johns Hopkins University,
United States
*Correspondence:
Jayanth Narayanan Edited by:
Bahar Tuncgenc,
University of Nottingham,
United Kingdom Reviewed by:
Seockhoon Chung,
University of Ulsan, South Korea
Calliope Holingue,
Johns Hopkins University,
United States *Correspondence:
Jayanth Narayanan
jayanth.narayanan@nus.edu.sg *Correspondence:
Jayanth Narayanan
jayanth.narayanan@nus.edu.sg Specialty section:
This article was submitted to
Personality and Social Psychology,
a section of the journal
Frontiers in Psychology ORIGINAL RESEARCH
published: 21 December 2020
doi: 10.3389/fpsyg.2020.610156 INTRODUCTION Received: 25 September 2020
Accepted: 26 November 2020
Published: 21 December 2020 Sleep helps employees recover from work and restore their resources (Hülsheger et al., 2014,
2015; Steed et al., 2019). Short sleep duration is associated with detrimental physical health
outcomes such as cardiovascular diseases, coronary heart diseases, and even mortality (see
Itani et al., 2017, for a review). Low levels of sleep also have destructive effects on psychological
outcomes such as cognitive performance (Lim and Dinges, 2010), neurocognitive functioning
(Durmer and Dinges, 2005), and mental health (Benca et al., 1992). Importantly, employees
with insufficient sleep feel depleted in the workplace, are less satisfied with their jobs, exhibit
less organizational citizenship behaviors, and have poor work performance (Kessler et al., 2011;
Barnes et al., 2012, 2013; Lanaj et al., 2014; see Litwiller et al., 2017, for a review). Keywords: mindfulness, COVID-19 stressors, employee sleep, work engagement, organizational behavior COVID-19 STRESSORS, SLEEP
DURATION, AND WORK ENGAGEMENT Exposure to traumatic events is probably the most powerful
non-organizational factor that disturbs employees’ sleep (Lavie,
2001; see Sinha, 2016, for a review). The ongoing COVID-19
pandemic is the defining global crisis of our time. The UN
has referred to it as the greatest challenge humanity has
faced since the II World War (United Nations, 2020). Even
as we prepare this manuscript, the situation is evolving with
cases rising daily in Africa, the United States, and Europe. Scholars have drawn attention to the impacts it could have
on individuals’ psychological well-being and functioning, with
some scholars referring to it as a “collective trauma” (Silver,
2020; Van Bavel et al., 2020; Kniffin et al., In press). Organizational scholars have termed this type of large-scale
traumatic events as acute-extraorganizational stressors (Byron
and Peterson, 2002; Hochwarter et al., 2008). The defining
feature of an acute-extraorganizational stressor is that it is
driven by a sudden or extreme force that is external to
organizations. Unlike intra-organizational stressors (e.g.,
organizational
restructuring
or
high
work
demands),
organizations cannot take active steps to prevent stressors
induced by COVID-19. Scholars have argued that such extra-
organizational stressors can have a more devastating effect
on employees than intra-organizational stressors (Byron and
Peterson, 2002; Hochwarter et al., 2008). Nevertheless,
organizations can take intervening steps to mitigate the negative
effects of acute-extraorganizational stressors on their employees. As such, it is important for organizational scholars to examine
factors that can protect employees from these negative effects
during the crisis. However, to date, research has mainly focused
on intra-organizational stressors, and thus, we have limited Sleep disruption is a prominent feature of individuals’
neurobiological and physiological response to trauma (Sinha,
2016). Trauma generates a stressful response that leads to
physiological hyperarousal, which in turn disrupts sleep (Lavie,
2001; Germain, 2013; Sinha, 2016). The hyperarousal state
occurs at two levels: primary arousal and secondary arousal
(Ong et al., 2012). Primary arousal refers to cognitive activities
that directly impair sleep, such as worrying about the impact
of COVID-19. Secondary or metacognitive arousal refers to
the awareness and judgment of primary arousal (i.e., thinking
about thinking), which includes how negatively individuals
evaluate their thoughts that occurred at the primary level. For
example, people may further ruminate about their stressful
thoughts about COVID-19 and amplify a hyperarousal state. Citation: Extant research has shown that
more engaged employees perform better in the workplace (Rich
et al., 2010; Christian et al., 2011; Bakker et al., 2012; Van
Wingerden et al., 2017). Employees who are highly engaged
in their work activities not only devote their physical effort,
but are also mentally vigilant and emotionally dedicated to
the endeavor, and thereby performing better in their tasks
(Kahn, 1990; Ashforth and Humphrey, 1995; Rich et al., 2010). We build on the metacognitive theory of mindfulness (Ong
et al., 2012; Jankowski and Holas, 2014) and the recovery
literature (Barnes, 2012; Steed et al., 2019) to propose that
mindfulness can neutralize the negative effect of COVID-19
stressors on employee sleep and employee work engagement
(Schaufeli et al., 2008). Specifically, given that exposure to trauma
can stimulate an intense and sustained state of hyperarousal,
which, in turn, disrupts individuals’ sleep (Lavie, 2001; Germain,
2013; Sinha, 2016), we argue that mindfulness – a state in
which individuals become aware of their present moment
experience – can reduce this hyperarousal state and thereby
mitigate the negative effect on sleep duration. Further, given
that sleep duration is a crucial recovery mechanism that leads
to more engaged employees at work (Barnes, 2012; Lanaj et al.,
2014), we propose that state mindfulness may be able to neutralize
the negative effects of the COVID-19 stressors on work
engagement through the mediating role of sleep duration. Frontiers in Psychology | www.frontiersin.org December 2020 | Volume 11 | Article 610156 Citation: Zheng MX, Masters-Waage TC,
Yao J, Lu Y, Tan N and
Narayanan J (2020) Stay Mindful and
Carry on: Mindfulness Neutralizes
COVID-19 Stressors on Work
Engagement via Sleep Duration. Front. Psychol. 11:610156. doi: 10.3389/fpsyg.2020.610156 December 2020 | Volume 11 | Article 610156 1 Frontiers in Psychology | www.frontiersin.org Mindfulness and COVID-19 Zheng et al. A prominent downstream effect of short sleep duration in
the workplace is impaired work engagement (Lanaj et al., 2014;
Litwiller et al., 2017). Work engagement is a powerful predictor
of employees’ job performance. Extant research has shown that
more engaged employees perform better in the workplace (Rich
et al., 2010; Christian et al., 2011; Bakker et al., 2012; Van
Wingerden et al., 2017). Employees who are highly engaged
in their work activities not only devote their physical effort,
but are also mentally vigilant and emotionally dedicated to
the endeavor, and thereby performing better in their tasks
(Kahn, 1990; Ashforth and Humphrey, 1995; Rich et al., 2010). Given that poor sleep takes a serious toll on employees
and organizations, organizational research has identified
organizational antecedents that impede employees’ sleep (Litwiller
et al., 2017). Previous studies have shown that demands such
as occupational stressors (DeArmond and Chen, 2004),
employees’ late night smartphone use for work (Lanaj et al.,
2014), long hours worked per week (Blau, 2011), and work-
family conflict (Barnes et al., 2012; Berkman et al., 2015) are
antecedents of employees’ reduced sleep duration. Although it
is important to understand organizational factors that keep
employees awake at night, sleep as a recovery process may
also be influenced by non-work factors. In fact, it has long
been recognized that organizational scholars should also consider
the potential role of non-organizational factors in studying
employees’ recovery process (Sonnentag, 2003; see Steed et al.,
2019 for a recent review). understanding about extra-organizational stressors (Hochwarter
et al., 2008). Given this dearth of research, scholars have
called for studies that provide theoretical and practical
recommendations for how organizations can help their
employees manage acute-extraorganizational stressors (Byron
and Peterson, 2002; Hochwarter et al., 2008; James, 2011). A prominent downstream effect of short sleep duration in
the workplace is impaired work engagement (Lanaj et al., 2014;
Litwiller et al., 2017). Work engagement is a powerful predictor
of employees’ job performance. COVID-19 STRESSORS, SLEEP
DURATION, AND WORK ENGAGEMENT They may become more attentive to and obsessed with the
thoughts that occur at the primary level, which may result in
a vicious cycle of falling and/or staying asleep. Indeed, research
has shown that exposure to traumatic events leads to shorter
sleep duration (Sinha, 2016; Goodwin et al., 2018). Thus, it
is possible that employees will experience sleep disruption in
response to COVID-19 stressors.t Returning to work after a good night’s sleep is critical to
ensure employees have sufficient energy and self-regulatory
resources to work (Barnes, 2012) and helps employees achieve
psychological detachment and physiological recovery (Steed
et al., 2019). Thus, sleep is a crucial recovery mechanism
leading to work engagement (Barnes, 2012; Lanaj et al., 2014). Specifically, work engagement is defined as a cognitive-affective
state characterized as being vigorous, dedicated, and absorbed December 2020 | Volume 11 | Article 610156 Frontiers in Psychology | www.frontiersin.org 2 Zheng et al. Mindfulness and COVID-19 in work (Schaufeli et al., 2002; Schaufeli and Bakker, 2004). Engaged employees have high energy, are intensely involved,
and are enthusiastic and immersed in work activities. In
line with our theorizing, past research has established that
sleep duration is positively associated with work engagement
(Lanaj et al., 2014; Litwiller et al., 2017). As discussed above, sleep is disrupted because metacognitive
arousal amplifies the primary arousal triggered by COVID-19
stressors. Mindfulness can specifically mitigate the metacognitive
arousal by shifting the negative metacognitive process to a
more adaptive stance, in which individuals simply observe
and accept their primary thoughts without judgments (Ong
et al., 2012; Jankowski and Holas, 2014). In other words,
mindfulness may prevent a primary arousal state from
developing into a secondary (metacognitive) arousal state. As a result, mindful employees have less difficulty falling
asleep and, thus, experience a longer sleep duration. Indeed,
previous studies have established that mindfulness effectively
increases sleep quantity (Hülsheger et al., 2015; see Ong and
Smith, 2017, for a review). Therefore, building on the
metacognitive theory of mindfulness and previous studies,
we argue that mindfulness may be effective in buffering the
negative effects of COVID-19 stressors on sleep duration. COVID-19 STRESSORS, SLEEP
DURATION, AND WORK ENGAGEMENT Thus, we propose the following hypothesis, j
Given that employees react to trauma by losing sleep
(Lavie, 2001; Germain, 2013; Sinha, 2016) and that sleep
duration is a crucial recovery mechanism leading to work
engagement (Barnes, 2012; Lanaj et al., 2014; Litwiller et al.,
2017), we argue that COVID-19 stressors may damage
employees’ work engagement via impaired sleep duration. However, previous research has suggested that these stressors
do not universally impact employees (Hochwarter et al., 2008). For example, Hochwarter et al. (2008) have found that
employees’ perceived resources interact with the effect of
hurricane induced stressors on job satisfaction such that
hurricane stress reduces job satisfaction for employees with
lower perceived resources while hurricane stress is neutralized
for employees with higher perceived resources. Thus, there
are boundary conditions that determine whether acute-
extraorganizational stressors negatively impact employees. Building on the metacognitive theory of mindfulness which
suggests that mindfulness is effective in reducing hyperarousal
state and improves sleep (Ong et al., 2012; Jankowski and
Holas, 2014), we propose that mindfulness is a boundary
condition that buffers the negative effect of COVID-19 stressors
on sleep duration and in turn work engagement. Hypothesis 1: COVID-19 stressors interacts with
mindfulness to predict sleep duration such that
COVID-19 stressors negatively affects sleep duration
when mindfulness is low while the effect of COVID-19
stressors is buffered when mindfulness is high. Building on the preceding hypothesis that argues that
mindfulness will neutralize the negative effect of COVID-19
stressors on sleep duration and previous evidence that sleep
duration is a key recovery resource leading to work engagement
(Lanaj et al., 2014; Litwiller et al., 2017), we further propose
that the buffering effect of mindfulness on the relationship
between COVID-19 stressors and work engagement is mediated
by sleep duration (see Figure 1). MINDFULNESS NEUTRALIZES THE
NEGATIVE EFFECT OF COVID-19
STRESSORS Hypothesis 2: The interaction effect of COVID-19
stressors and mindfulness on work engagement is
mediated by sleep duration. Mindfulness is defined as a moment-to-moment non-judgmental
awareness of one’s present experience (Brown and Ryan, 2003). Mindfulness can be viewed as a naturally occurring mental
state (measured as a dispositional trait or a transient mental
state) or can be trained through meditation practices (Davidson,
2010). Despite these distinct operationalizations, scholars view
the mindfulness state as a unitary construct across these
measures (Reb and Atkins, 2015; Good et al., 2016). This state
of mind has been linked with numerous positive outcomes,
such as reduced employee stress (for a meta-analysis see Bartlett
et al., 2019), and outcomes more specific to the workplace
(for reviews see Reb and Atkins, 2015; Good et al., 2016; Eby
et al., 2019). Existing research suggests that many of these
benefits are a result of mindfulness, increasing a metacognitive
awareness of one’s experience (Jankowski and Holas, 2014; Kay
and Skarlicki, 2020). Specifically, the metacognitive theory of
mindfulness suggests that a non-judgmental awareness of one’s
present experience facilitates individuals’ capacity to observe
their experience as something separate from themselves. By
generating psychological distance between oneself and one’s
immediate experience, mindfulness supports individuals’ capacity
to observe and to accept their thoughts and experiences without
judgments (Jankowski and Holas, 2014). Frontiers in Psychology | www.frontiersin.org OVERVIEW OF STUDIES We tested our hypotheses in two studies. We tested hypothesis
1 in Study 1 which is a 10-day field experiment conducted
among employees in Wuhan, China during the lockdown
between
February
20,
2020,
and
March
2,
2020. We operationalized COVID-19 stressors as an objective
indicator - the increase of infections in the community as
this was salient to inhabitants in Wuhan at that time. Participants
in this study were randomly assigned to either a daily
mindfulness practice or a daily mind-wandering practice. Study
2 is a 10-day longitudinal survey conducted among employees
in the United Kingdom between June 8, 2020 and June 19,
2020. This study serves two purposes. First, we increased the
ecological validity of our research by replicating the buffering
effect of mindfulness in a different country where the COVID-19
pandemic had spread widely within the country. Second,
we provided additional robustness to our model by adopting December 2020 | Volume 11 | Article 610156 3 Zheng et al. Mindfulness and COVID-19 FIGURE 1 | Conceptual model. [95% mindfulness, 88% mind-wandering, χ2 (1) = 1.78, p = 0.18]. The survey App automatically timestamped the initiation and
the completion of the morning survey. This timestamp was used
to check if participants adhered to their daily practice. Among
participants who completed the 12-day study, one participant
in the mindfulness condition and two participants in the mind-
wandering condition did not practice the exercise in the morning
for at least four consecutive days. In addition, one participant
in the mindfulness condition was not located in Wuhan; since
our study coded for information about new cases in Wuhan,
we dropped this participant from the analysis as well. After
excluding participants who did not meet the study criteria,
we obtained a final sample of 97 with 55 participants remaining
in the mindfulness condition and 42 in the mind-wandering
condition. The sample size in the two conditions is comparable
with previous mindfulness research (Lindsay et al., 2019;
Hafenbrack et al., 2020). The 97 participants (68.04% female)
have an average age of 34.49 years (SD = 5.03), 79.4% have a
college/Bachelor’s degree, and 21.6% have a Masters/PhD degree. Participants in the mindfulness and the mind-wandering conditions
did not significantly differ in terms of their sociodemographic
features. OVERVIEW OF STUDIES Specifically, the distribution of gender in the mindfulness
condition (58.2% female) did not differ from those in the mind-
wandering condition (76.2% female), χ2 (1) = 2.58, p = 0.064. Participants’ age in the mindfulness condition (34.69 years,
SD = 5.66) did not differ from those in the mind-wandering
condition (34.33 years, SD = 4.03), F(1, 96) = 0.39, p = 0.54. There was no difference between mindfulness condition
(27.69 years, SD = 12.42) and mind-wandering condition
(28.36 years, SD = 10.85) in terms of years living in Wuhan,
F(1, 96) = 0.13, p = 0.73. Participants in both conditions share
similar education level (bachelor degree or above 80% vs. 90.5%),
χ2 (1) = 0.71, p = 0.40. In addition, they did not differ in
terms of trait mindfulness (4.82, SD = 0.54 vs. 4.83, SD = 0.83),
F(1, 96) = 0.05, p = 0.83. These participants in the final sample
also did not differ from individuals who initially enrolled in
the study but failed to actually participate in the daily surveys
(gender: χ2 (1) = 0.002, p = 0.97.; age: F(1, 105) = 2.36, p = 0.13,
years living in Wuhan (F(1, 105) = 0.34, p = 0.56, trait
mindfulness F(1, 105) = 0.53, p = 0.47). Although there was
a marginally lower percentage of women in the mindfulness
condition, this was the result of the snowballing technique alternative operationalizations of our primary independent
variables, testing the moderated mediation model by measuring
self-reported state mindfulness as a within-subject individual
difference variable and COVID-19 stressors as employees’
self-reported variable. Frontiers in Psychology | www.frontiersin.org Participants p
A snowballing technique was employed to recruit participants
through an online advertisement posted through the first author’s
personal networks in Wuhan between February 18, 2020 and
February 20, 2020. The ad stated that a study was recruiting
working adults who were experiencing the lockdown in Wuhan
to complete a 12-day study with a compensation of 100 RMB
(approximately USD 14). All procedures in the study were in
accordance with the ethical standards of the institutional review
board at the first author’s institution and with the Declaration
of Helsinki, 1964, and its later amendments or comparable
ethical standards. One day before the daily surveys, participants
completed a consent form online. Subject IDs were assigned
upon submitting the consent form in which a computerized
random number was generated at the end of the form. To
ensure anonymity, we did not ask participants to indicate their
names throughout the study. All participants were blind to
the study condition. Throughout the study, the research assistant
used the subject ID to identify participants. To ensure randomization, the research assistant who managed
the study was blind to the treatment and the hypothesis. Interested
participants scanned a QR on WeChat to enroll in the study. Participants were randomly assigned to one of the two WeChat
anonymous groups. Recruitment stopped when the goal of
enrolling 110 participants was reached. Experimental conditions
were randomly assigned to these two groups by the first author. There are 60 participants in the mindfulness condition and 50
participants in the mind-wandering condition. Three participants
in the mindfulness condition and six participants in the mind-
wandering condition did not complete the daily surveys. Thus,
they were not included in the final analyses. The response rate
of initially enrolled participants to daily surveys did not significantly
differ between the mindfulness and the mind-wandering groups December 2020 | Volume 11 | Article 610156 4 Zheng et al. Mindfulness and COVID-19 FIGURE 2 | This figure visually depicts how daily mindfulness practice mitigated the relationship between COVID-19 stressors and sleep quantity in Study 1. depicts how daily mindfulness practice mitigated the relationship between COVID-19 stressors and sleep quantity in Study 1. State mindfulness induction. As all our participants were
native Chinese speakers, we used audio instructions in Mandarin
that were recorded by a professional mindfulness coach. These
instructions were developed based on well-established English
mindfulness programs (Kiken and Shook, 2011). Participants The audio
instructions have been used in previous research and were
effective in inducing mindfulness and mind-wandering in
Chinese populations (Schuh et al., 2019). These audio instructions
are available on request from the first author.t rather than the planned assignment. As a robustness check,
we controlled for gender in our analyses and found that the
buffering effect of mindfulness on the relationship between
daily confirmed cases and sleep quantity remained significant
(B = 0.06, SE = 0.03, p = 0.02). We found no effect of gender
(B = 0.01 SE = 0.03, p = 0.74). 1http://www.nhc.gov.cn/wjw/xinx/xinxi.shtml Results Specifically, we analyzed
the data with random coefficient modeling (RCMs; Raudenbush
and Bryk, 2002), in which we specified the within-individual-
level relationship between the number of daily confirmed cases
and sleep quantity as a random slope and used the between-
individual-level mindfulness intervention to predict this slope. As shown in Table 2, the mindfulness practice positively
predicted the random slope between daily confirmed cases
and sleep quantity (B = 0.05, SE = 0.03, p = 0.046). To further
probe into the effect of the mindfulness practice, we plotted
the simple slopes for the mindfulness treatment group and
the mind-wandering treatment group, respectively. As shown
in Figure 2, among people assigned to the mind-wandering
group, the number of confirmed cases on a day was negatively
related to their sleep quantity on that day (B = −0.04, SE = 0.01,
p = 0.003). On average, they lost 39 min of sleep with every
thousand confirmed cases reported in the city. In contrast,
among people assigned to the mindfulness practice condition,
their sleep quantity was unaffected by the number of confirmed
cases (B = 0.01, SE = 0.01, p = 0.30). Hypothesis 1 was
thus supported. g
g
Consistent with sleep research, we also controlled for variables
that may influence sleep quantity: sleep quality and daily caffeine
intake (Gellis and Lichstein, 2009; Lanaj et al., 2014). Research
has shown that poor sleep quality on one night can lead to
longer sleep the next night (Banks et al., 2010). Thus, when
predicting sleep quantity, we included sleep quantity and sleep
quality from the previous night (i.e., lagged in time by 1 day)
as control variables. We measured sleep quality with an overall
item on a seven-point Likert scale ranging from 1(very bad)
to 7(very good): “How do you evaluate your night’s sleep?”. We measured daily caffeine intake with one item: “Did you have
beverage that contains caffeine (such as coke, coffee, etc.)?” To check the robustness of our findings, we included sleep
quantity and sleep quality on the previous night (Banks et al.,
2010), trait mindfulness (Hülsheger et al., 2014), daily cumulative
confirmed cases to that date, with daily cases of death reported
as control variables. We found that the positive relationship TABLE 1 | Means, standard deviations, and correlations (Study 1). Results Thus, when
predicting sleep quantity, we included sleep quantity and sleep
quality from the previous night (i.e., lagged in time by 1 day)
as control variables. We measured sleep quality with an overall
item on a seven-point Likert scale ranging from 1(very bad)
to 7(very good): “How do you evaluate your night’s sleep?”. We measured daily caffeine intake with one item: “Did you have
beverage that contains caffeine (such as coke, coffee, etc.)?” Control variables. As a control variable, we measured participants’
trait mindfulness in the baseline survey with a 15-item scale on
a 7-point Likert scale ranging from 1(never) to 7(very often)
(Brown and Ryan, 2003). Fifteen items are “I could be experiencing
some emotion and not be conscious of it until sometime later;
” “I break or spill things because of carelessness, not paying
attention, or thinking of something else;” “I find it difficult to
stay focused on what’s happening in the present”, “I tend to walk
quickly to get to where I’m going without paying attention to
what I experience along the way;” “I tend not to notice feelings
of physical tension or discomfort until they really grab my attention;”
“I forget a person’s name almost as soon as I’ve been told it for
the first time;” “It seems I am “running on automatic” without
much awareness of what I’m doing;” “I rush through activities
without being really attentive to them;” “I get so focused on the
goal I want to achieve that I lose touch with what I am doing
right now to get there;” “I do jobs or tasks automatically, without
being aware of what I’m doing;” “I find myself listening to someone
with one ear, doing something else at the same time;” “I drive
places on “automatic pilot” and then wonder why I went there;”
“I find myself preoccupied with the future or the past;” “I find
myself doing things without paying attention”, and “I snack without
being aware that I’m eating.” All items are reverse coded. (α = 0.79). Given the nested nature of the data (daily observations
nested within individuals), we used a multilevel modeling
approach to test our hypothesis – whether the mindfulness
practice would mitigate the effect of the number of daily
confirmed cases on sleep quantity. Procedure We have also recorded daily death cases (M = 58.36, SD = 36.65)
and cumulative cases (M = 32,658, SD = 3,956.85) between
20 February, 2020 and 1 March, 2020, from the official records. Sleep quantity. We measured sleep quantity in the survey
with the following item taken from the previous studies (Lanaj
et al., 2014). “How many hours of actual sleep did you get
last night?” Recent meta-analytic research has revealed that
the correlation between objective measures of sleep quantity,
such as Actigraph, and self-reported measures of sleep quantity
is high, indicating that self-reported measure is accurate and
reliable (Litwiller et al., 2017). Results Descriptive statistics and bivariate correlations are reported in
Table 1. As a manipulation check, we tested whether the
experimental condition had a significant effect on mindfulness
(Long and Christian, 2015). Participants in the mindfulness
condition reported higher levels of mindfulness than those in
the mind-wandering condition (B = 0.29, SE = 0.12, p = 0.02),
indicating that our manipulation was successful. (
)
Control variables. As a control variable, we measured participants’
trait mindfulness in the baseline survey with a 15-item scale on
a 7-point Likert scale ranging from 1(never) to 7(very often)
(Brown and Ryan, 2003). Fifteen items are “I could be experiencing
some emotion and not be conscious of it until sometime later;
” “I break or spill things because of carelessness, not paying
attention, or thinking of something else;” “I find it difficult to
stay focused on what’s happening in the present”, “I tend to walk
quickly to get to where I’m going without paying attention to
what I experience along the way;” “I tend not to notice feelings
of physical tension or discomfort until they really grab my attention;”
“I forget a person’s name almost as soon as I’ve been told it for
the first time;” “It seems I am “running on automatic” without
much awareness of what I’m doing;” “I rush through activities
without being really attentive to them;” “I get so focused on the
goal I want to achieve that I lose touch with what I am doing
right now to get there;” “I do jobs or tasks automatically, without
being aware of what I’m doing;” “I find myself listening to someone
with one ear, doing something else at the same time;” “I drive
places on “automatic pilot” and then wonder why I went there;”
“I find myself preoccupied with the future or the past;” “I find
myself doing things without paying attention”, and “I snack without
being aware that I’m eating.” All items are reverse coded. (α = 0.79). Consistent with sleep research, we also controlled for variables
that may influence sleep quantity: sleep quality and daily caffeine
intake (Gellis and Lichstein, 2009; Lanaj et al., 2014). Research
has shown that poor sleep quality on one night can lead to
longer sleep the next night (Banks et al., 2010). Procedure Participants completed a baseline assessment on February
20, 2020, that asked for their demographic information and
trait mindfulness a day before the intervention began. Participants in the mindfulness practice condition engaged
in a 10-min mindfulness practice each morning and participants
in the mind-wandering condition engaged in a 10-min mind-
wandering practice for 10 consecutive days from February
21, 2020, (Friday) to March 1, 2020 (Sunday). Each day,
participants in both conditions completed a short morning
survey that was sent via WeChat App in the morning (8 am),
including audio instructions for the practice, a mindfulness
manipulation check, sleep quantity, sleep quality, and caffeine
intake in the previous day. On day 12, participants completed
a brief survey in which they reported their previous night’s
sleep, caffeine intake, and Alipay account. After the completion
of the study, all participants were debriefed and were invited
to a daily group practice at 10 am for a 10-min mindfulness
practice. This was done to ensure that all participants, including
those in the mind-wandering condition, could benefit from
the practice. i
Mindfulness manipulation check. After listening to the
audio clip in the morning, participants rated their momentary
mindfulness on four items on a seven-point Likert scale
(1 = not at all to 7 = Completely) (Long and Christian,
2015). Four items were “I focused on the present,” “I thought
about anything I wanted (reversed coded)”, “I let my mind
wander freely (reversed coded),” and “I was mindful of the
present moment.” COVID-19 stressors. Given that Wuhan is the city that was
seriously affected by the virus before the outbreak in other
cities and countries, the information about daily confirmed
cases was salient to employees in Wuhan. Thus, as a proxy
for the COVID-19 stressors, we recorded the number of increased
cases (M = 384.55, SD = 108.28) in Wuhan between 20 February,
2020 and 1 March, 2020, from the official records of the
Chinese National Health Commission of the People’s Republic
of China.1 December 2020 | Volume 11 | Article 610156 Frontiers in Psychology | www.frontiersin.org 5 Mindfulness and COVID-19 Zheng et al. We have also recorded daily death cases (M = 58.36, SD = 36.65)
and cumulative cases (M = 32,658, SD = 3,956.85) between
20 February, 2020 and 1 March, 2020, from the official records. December 2020 | Volume 11 | Article 610156 n = 97 participants. Gender: 1 = male, 2 = female. Caffeine abstinence: 0 = taking caffeine, 1 = no taking caffeine. Experimental condition: 0
1 = mindfulness intervention group. *p < 0.05; **p < 0.01. Frontiers in Psychology | www.frontiersin.org Measures
COVID 19
t Measures
COVID-19 stressors. Unlike the early outbreak in Wuhan
captured in Study 1, daily case numbers were less prominent
in the UK during the data collection period since the COVID-19
pandemic has widely spread to many countries. Thus, consistent
with previous studies that measure stressors (Wang et al., 2010),
we operationalized COVID-19 stressors as a subjective measure
that directly captures the extent to which people perceive
COVID-19 as a stress that interferes with their work on a
daily basis. To measure this construct, a daily measure of
family-to-work conflict (see Wang et al., 2010) was adapted
by supplanting the terms “home-life” or “family” with “COVID-
19”. Items included “Today at work, how often did COVID-19
interfere with your job or career?”, “Today, how often did
you think about COVID-19 related problems?”, “Today, how
often did COVID-19 interfere with your responsibilities at
work, such as getting to work on time, accomplishing daily
tasks, or working overtime?”, “Today, how often did COVID-19
keep you from spending the amount of time you would like
to spend on job or career-related activities?”, and “Today, how
often did you think about things you need to do related to
COVID-19?” (α = 0.93) In the analyses, this measure was
lagged to represent the previous day’s COVID-19 stressors. between the mindfulness practice and the random slope between
daily confirmed cases and sleep quantity was robust (B = 0.05,
SE = 0.02, p = 0.04) with all these factors controlled for (see
Table 3). In addition, mindfulness practice did not moderate
the relationship between COVID-19 stressors and sleep quality
(B = 0.001, SE = 0.001, p = 0.73). Daily state mindfulness. Individuals’ state mindfulness was
measured on a daily level using an abbreviated version of the
MAAS (Brown and Ryan, 2003) which had been previously
adapted by Liang et al. (2018). Items included, “Today, I rushed
through activities without being attentive to them.”, “Today,
I did things without paying attention.”, “Today, I was preoccupied
with thoughts of the future or the past.”, “Today, I did things
automatically, without being aware of what I was doing.”, and
“Today, I found it difficult to stay focused on what was happening
in the present moment.” (α = 0.89); of note, all items are
reverse-coded. In the analyses, this measure was lagged to
represent the previous day’s mindfulness. Results Mean
SD
1
2
3
4
5
6
7
8
1.Age
34.49
5.03
-
2.Gender
1.67
0.47
0.05
-
3.Years live in Wuhan
27.68
11.79
0.43**
0.05
-
4.Experimental condition
0.57
0.50
0.06
−0.22*
−0.04
-
5.Trait mindfulness
4.81
0.69
0.06
0.03
−0.20*
0.02
-
6.Sleep quantity
460.60
57.66
0.05
−0.07
0.14
0.15
−0.20
-
7.Sleep quality
4.67
0.98
0.07
0.05
0.20*
−0.04
0.00
0.31**
-
8.Caffeine abstinence
0.73
0.36
0.10
−0.07
−0.15
0.08
0.16
0.05
0.01
-
n = 97 participants. Gender: 1 = male, 2 = female. Caffeine abstinence: 0 = taking caffeine, 1 = no taking caffeine. Experimental condition: 0 = mind-wandering control group,
1 = mindfulness intervention group. *p < 0.05; **p < 0.01. n = 97 participants. Gender: 1 = male, 2 = female. Caffeine abstinence: 0 = taking caffeine, 1 = no taking caffeine. Experimental condition: 0 = mind-wandering control group,
1 = mindfulness intervention group. *p < 0.05; **p < 0.01. December 2020 | Volume 11 | Article 610156 6 Mindfulness and COVID-19 Zheng et al. TABLE 2 | Multilevel regression results (Study 1). Predictors
DV = Daily
Sleep quantity
(Minutes)
DV = Slope between
daily confirmed cases
and sleep quantity
Estimate
SE
Estimate
SE
Intercept
324.75**
14.63
−0.01
0.03
Within-individual level
Self-perceived sleep quality
26.41**
2.23
Caffeine abstinence
8.64
6.60
Between-individual level
Experimental condition
17.00
11.67
0.05*
0.03
The effects of self-perceived sleep quality and caffeine abstinence on sleep quantity were
modeled as fixed slopes. Caffeine abstinence was coded as 0 = taking caffeine, 1 = no
taking caffeine. Experimental condition was coded as 0 = mind-wandering control group,
1 = mindfulness intervention group. *p < 0.05; **p < 0.01. TABLE 2 | Multilevel regression results (Study 1). study and (b) working in the United Kingdom. Further, to
ensure data quality, all participants had an approval rating of
95% (or above) for past studies completed on Prolific (Keith
et al., 2017). Based on this, a total sample of 140 participants
(59.3% female) was obtained, with a mean age of 34.1
(SD = 9.10), 75.1% have a college/bachelor degree, and 28.3%
have a Master/PhD degree. Procedureh The study took place over a 2-week period with 10 surveys
sent out on 10 consecutive workdays between June 8, 2020
and June 19, 2020. To be eligible for this study, participants
had to complete a demographic information pre-survey. Each
daily survey was emailed to participants in the evening after
a typical UK workday had ended (5 pm), and this survey
then expired each day at midnight. Participants were paid for
each survey (GBP 1 for 5 min) along with a bonus payment
for completing nine or more surveys (GBP 3). This resulted
in a high completion rate with participants completing 1,302
of the 1,400 daily surveys sent out (93%). TABLE 3 | The robustness check results (Study 1). Predictors
DV = Daily
Sleep quantity
(Minutes)
DV = Confirmed
cases-sleep
quantity slope
Estimate
SE
Estimate
SE
Intercept
225.10
414.61
0.18*
0.08
Within-individual level
Daily cumulative cases
7.80
38.70
Daily cases of death
0.013
0.105
Previous day’s sleep quality
−2.79
2.78
Previous day’s sleep quantity
0.061
0.046
Self-perceived sleep quality
26.68**
2.26
Caffeine abstinence
9.83
6.49
Between-individual level
Trait mindfulness
−0.04*
0.02
Experimental condition
0.05*
0.02
This table shows that the results of the multilevel regression with trait mindfulness, daily
cumulative cases, daily cases of death, and sleep quality and quantity on the previous
day as additional control variables. Caffeine abstinence was coded as 0 = taking
caffeine, 1 = no taking caffeine. Experimental condition was coded as 0 = mind-
wandering control group, 1 = mindfulness intervention group. **p < 0.01; **p < 0.05. TABLE 3 | The robustness check results (Study 1). Participants All participants were recruited through the online platform
Prolific (Palan and Schitter, 2018). Participants were pre-screened
to ensure that they were (a) working full-time throughout the December 2020 | Volume 11 | Article 610156 Frontiers in Psychology | www.frontiersin.org 7 Mindfulness and COVID-19 Zheng et al. TABLE 5 | Multilevel regression results in (Study 2). Predictors
DV = Sleep quantity
Estimate
SE
COVID-19 stressors
−0.007
0.084
State mindfulness
−0.046
0.037
COVID-19 stressors × State mindfulness
0.109*
0.052
*p < 0.05; **p < 0.01. TABLE 5 | Multilevel regression results in (Study 2). Sleep quantity. As in study 1, we measured sleep quantity
with the following item (Lanaj et al., 2014); “How many hours
of actual sleep did you get last night?” Work engagement. Daily work engagement was measured
using an abbreviated 5-item version of the Utrecht Work
Engagement Scale (Schaufeli et al., 2006). Items included, “Today
at work, I felt bursting with energy.”, “Today at work, I felt
strong and vigorous”, “Today, I was enthusiastic about my job”,
“Today, my job inspired me”, and “Today, I was immersed in
my work” (α = 0.89). Control Variables. Consistent with the past-sleep research
(Gellis and Lichstein, 2009; Lanaj et al., 2014), and Study 1,
we measured sleep quality as a control variable. Sleep quality
was measured with an overall item on a 7-point Likert scale
ranging from 1(very bad) to 7(very good): “How would you rate
your sleep quality overall last night?” of COVID-19 stressors on sleep duration; Hypothesis 1 was
thus supported. In addition, results indicated that mindfulness
did not moderate the relationship between COVID-19 stressors
and sleep quality (B = 0.10, SE = 0.06, p = 0.07). p q
y
p
We continued to test a moderated mediation model in which
state mindfulness moderated an indirect effect of COVID-19
stressors on work engagement via sleep quantity (H2). Analyses
revealed that there was a positive relationship between sleep
quantity and work engagement (B = 0.14, SE = 0.03, p < 0.01). With a Monte Carlo simulation method (Preacher et al., 2010),
we found that the indirect effect of COVID-19 stressors on
work engagement via sleep quantity was negative and significant
when state mindfulness was low [estimate = −0.01, 95%
CI = (−0.030, −0.002)] and nonsignificant when state mindfulness
was high [estimate = 0.01, 95% CI = (−0.001, 0.027); difference
in conditional indirect effects = 0.03, 95% CI = (0.002, 0.059)]. Results Descriptive statistics and bivariate correlations for all study
variables are provided in Table 4. As in Study 1, to account for the nested nature of the data,
we used multilevel modeling and centered all predictors around
each participant’s mean score (Hofmann et al., 2000). However,
because we focused on daily state mindfulness (vs. an individual-
level mindfulness practice) in this study and tested only within-
individual (vs. between-individual) effects, we used fixed slope
modeling. The relationships of COVID-19 stressors, state
mindfulness, and their interaction with sleep quantity were
all modeled as fixed slopes. Given that variables such as trait
mindfulness, sleep quality, and daily caffeine intake did not
influence results in any way in Study 1, we did not control
for these variables in our analyses.f Participants Hypothesis 2 was thus supported. GENERAL DISCUSSION We found that induced or measured state mindfulness significantly
buffered the negative effect of COVID-19 stressors on sleep duration
(Studies 1 and 2) and work engagement (Study 2) such that
COVID-19 stressors had negative effects when state mindfulness
was low while negative effects were buffered when state mindfulness
was high. Findings of the present studies contribute to the employee
stress and well-being research as well as the emerging mindfulness
research in the organizational literature. Consistent with Hypothesis 1, the interactive effect of daily
COVID-19 stressors and mindfulness on sleep quantity was
positive and significant (B = 0.11, SE = 0.05, p = 0.04, see
Table 5). Simple slope analyses further revealed that the relationship
between COVID-19 stressors and sleep quantity was negative
when state mindfulness was low (B = −0.11, SE = 0.05, p = 0.03)
and nonsignificant when state mindfulness was high (B = 0.09,
SE = 0.05, p = 0.20); difference in simple slopes = 0.20, SE = 0.10,
p = 0.04; Figure 3). Mindfulness neutralized the negative effect First, our work extends the recovery literature by looking
beyond the widely examined organizational factors and introducing
a non-organization factor that is probably the most powerful
external force that disrupts employees’ sleep. Previous studies
have shown that organizational factors such as occupational
stressors and work-family conflict can harm employees’ sleep
(DeArmond and Chen, 2004; Blau, 2011; Barnes et al., 2012;
Lanaj et al., 2014; Berkman et al., 2015). Understanding these
organizational antecedents can help employees and organizations
achieve better recovery by specifically alleviating these potential
stressors. Unlike organizational factors, traumatic events such
as the COVID-19 pandemic are external shocks that are not
controllable by organizations and employees. Yet, it has detrimental
effects on employees’ sleep (Sinha, 2016). Indeed, a recent review
suggests that “scholars should consider how recovery, an inherently
non-work activity, is impacted by non-work demands and
resources in addition to work-specific demands and resources” TABLE 4 | Means, standard deviations, and correlations (Study 2). Mean
SD
1
2
3
4
5
6
1.Age
34.1
9.10
-
2.Gender
1.62
0.49
−0.20*
-
3.COVID-19
stressors
1.95
0.88
−0.01
0.13
-
4.State
mindfulness
4.86
0.98
0.23**
−0.05
−0.33**
-
5.Sleep
quantity
392.12
53.28
−0.21*
0.21*
−0.16
0.17*
-
6.Work
engagement
3.56
1.04
0.06
−0.12
−0.06
0.44**
0.04
-
n = 140 participants. Gender: 1 = male, 2 = female. *p < 0.05; **p < 0.01. GENERAL DISCUSSION TABLE 4 | Means, standard deviations, and correlations (Study 2). Mean
SD
1
2
3
4
5
6
1.Age
34.1
9.10
-
2.Gender
1.62
0.49
−0.20*
-
3.COVID-19
stressors
1.95
0.88
−0.01
0.13
-
4.State
mindfulness
4.86
0.98
0.23**
−0.05
−0.33**
-
5.Sleep
quantity
392.12
53.28
−0.21*
0.21*
−0.16
0.17*
-
6.Work
engagement
3.56
1.04
0.06
−0.12
−0.06
0.44**
0.04
-
n = 140 participants. Gender: 1 = male, 2 = female. *p < 0.05; **p < 0.01. TABLE 4 | Means, standard deviations, and correlations (Study 2). December 2020 | Volume 11 | Article 610156 8 Zheng et al. Mindfulness and COVID-19 FIGURE 3 | This figure visually depicts how daily state mindfulness mitigated the relationship between COVID-19 stressors and sleep quantity in Study 2. cts how daily state mindfulness mitigated the relationship between COVID-19 stressors and sleep quantity in Study 2. FIGURE 3 | This figure visually depicts how daily state mindfulness mitigated the relationship between COVID-19 stressors and sleep quantity in Study 2. (see Bliese et al., 2017 for a recent review). Although the origins
of the field were stimulated by these events, the majority of
research to date has focused on the effect of intra-organizational
stressors. However, growing evidence has mounted, showing that
acute-extraorganizational stressors play an equally significant role
in impacting employees’ outcomes, e.g., increasing absenteeism
and decreasing job satisfaction, and leading to higher turnover
(Byron and Peterson, 2002; Hochwarter et al., 2008; Dollard
et al., 2013; Ragins et al., 2014; Baruch et al., 2016). Nevertheless,
research on how to tackle these negative effects has lagged
behind (Byron and Peterson, 2002; Hochwarter et al., 2008). Furthermore, the few interventions that have been introduced
in the psychology literature were conducted after the crisis
occurred and focused on “fixing” post-traumatic effects rather
than timely intervening negative effects of the crisis as it unfolds
(Lavie, 2001; Sinha, 2016). This post-hoc approach is at odds
with recent research, suggesting that the early treatment of
trauma-induced stress may be more effective in preventing the
development of post-traumatic negative experiences such as
depression (Sinha, 2016). Therefore, our study is among the
first to examine how the negative impacts of extra-organizational
stressors can be neutralized during a crisis. In doing so,
we identified that mindfulness, both as a state and implemented
as a randomized-controlled intervention, is effective in mitigating
the negative effect of an ongoing crisis on an employee’s well-
being. Frontiers in Psychology | www.frontiersin.org GENERAL DISCUSSION Thus, during the COVID-19 outbreak, organizations
that offered morning meditations to all company employees
(e.g., Google), might have been more effective in managing
the negative impacts of the pandemic on employee engagement. GENERAL DISCUSSION Further, this paper also
contributes to growing research on the role of state mindfulness
in the workplace (e.g., Tuckey et al., 2018; Hafenbrack et al.,
2020), investigating this through a low-dose intervention along
with measuring it as a self-reported state. The synergy of these
results provides promise for future research interested in
examining the daily impacts of mindfulness along with providing
a low-cost (or even free) intervention for organizations to
implement in the light of acute extra-organizational stressors. individuals can benefit and grow from traumatic exposure
(Tedeschi and Blevins, 2015). Specifically, this theory suggests
that mindfulness may facilitate positive reappraisal of the
negative experience. In line with the mindfulness-to-meaning
hypothesis (Garland et al., 2017), this reappraisal process can
in turn make individuals experience growth in aspects such
as personal strength and appreciation of life. Future research
should take a growth perspective and examine the effect of
mindfulness on individuals’ post-traumatic growth.h Furthermore, our study contributes to organizational research
on mindfulness. Past work has found mindfulness can be an
effective intervention in workplace settings providing a myriad
of positive effects on work attitudes and outcomes (for reviews
see Sutcliffe et al., 2016; Kay et al., 2019). Moving beyond
these main effects of mindfulness, this paper identifies
mindfulness as an effective crisis intervention. Specially, this
paper draws on the metacognitive theory of mindfulness and
extends this theory into the context of crisis by showing that
mindfulness can neutralize the negative effects of COVID-19
stressors on work engagement via the mediating role of sleep
duration, a mechanism that is highly vulnerable to the
hyperarousal state triggered by trauma. Further, this paper also
contributes to growing research on the role of state mindfulness
in the workplace (e.g., Tuckey et al., 2018; Hafenbrack et al.,
2020), investigating this through a low-dose intervention along
with measuring it as a self-reported state. The synergy of these
results provides promise for future research interested in
examining the daily impacts of mindfulness along with providing
a low-cost (or even free) intervention for organizations to
implement in the light of acute extra-organizational stressors. Our research also has practical implications. In uncertain
times like the COVID-19 pandemic, how organizations treat
their employees will have a lasting impact on employees’ loyalty,
engagement, and productivity (Carvalho and Areal, 2016). Our
findings suggest that mindfulness practice can be introduced
as an effective employee care program for organizations. Limitations and Future Research
Directions Despite several contributions to the literature, the present study
should be viewed in light of its strengths and weaknesses. First,
we argue that mindfulness can activate a metacognitive process
of observing thoughts without judgment, thus reducing the
secondary arousal related to trauma. While this research
argumentation is consistent with the neuroscience literature on
mindfulness and metacognition (Jankowski and Holas, 2014),
we did not examine the specific psychological mechanisms that
underlie this effect. To date, studies have examined mechanisms
such as cognitive reappraisal, decentering process, and affective
rumination for the effects of mindfulness (Fresco et al., 2007;
Liang et al., 2018; Kay and Skarlicki, 2020). In addition, it is
also possible that COVID-19 stressors are associated with increased
workload, alternative shifts, interpersonal conflict. These are possible
mechanisms of mindfulness on improving sleep duration. Thus,
we suggest that future research could examine the exact mechanisms
in the moderating effects of mindfulness on sleep duration. Furthermore, consistent with previous studies (Barnes, 2012),
our research confirms that sleep quality and sleep quantity
are two distinct concepts. Research suggests that sleep quantity
is more closely related to resources available for work while
sleep quality is more closely related to employees’ perceptions
or emotions (Litwiller et al., 2017). Indeed, we show that sleep
quantity is a crucial mechanism through which mindfulness
exerts a buffering effect on work engagement. This is because
sleep quantity provides resources for employees to be engaged
in the workplace. Future research should take into consideration
other work-related outcomes that are related to perceptions,
such as job satisfaction and negative affect, and examine the
buffering effect of mindfulness on these perceptual outcomes. Frontiers in Psychology | www.frontiersin.org GENERAL DISCUSSION Thus, our work has general theoretical implications for
managing acute-extraorganizational stressors. (Steed et al., 2019; p.24). Our research echoes this view and
highlights the importance of considering public traumatic events
when examining the employees’ recovery process. Second, organizational research on sleep has generally used
one of the two indicators to capture sleep: sleep quality and
sleep quantity (Barnes, 2012; Litwiller et al., 2017). Although
they are conceptually similar and related, they are different
because they tend to be correlated with different variables
(Barclay et al., 2010; Hülsheger et al., 2015; Litwiller et al.,
2017). Consistent with previous studies, our findings that
mindfulness interacts with COVID-19 stressors to influence
sleep quantity, but not quality, further confirms that they are
two distinct concepts (Litwiller et al., 2017). Specifically, compared
to sleep quality, sleep quantity is more closely related to resources
available for work, which have downstream effects on work
engagement (Lanaj et al., 2014). Indeed, we show that sleep
quantity is a crucial mechanism through which mindfulness
exerts a buffering effect on work engagement. Thus, our findings
have important implications for different roles that sleep quality
and quantity may play in work-related outcomes.h Third, our work extends the employee stress and well-being
research by looking beyond the widely examined intra-
organizational stressors and examining the negative effects of
acute-extraorganizational stressors. Research on employee stress
and well-being dates back nearly 100 years, during which traumatic
events such as World War I, the influenza epidemic of 1918,
and the Great Depression have greatly impacted employees December 2020 | Volume 11 | Article 610156 Frontiers in Psychology | www.frontiersin.org 9 Mindfulness and COVID-19 Zheng et al. Furthermore, our study contributes to organizational research
on mindfulness. Past work has found mindfulness can be an
effective intervention in workplace settings providing a myriad
of positive effects on work attitudes and outcomes (for reviews
see Sutcliffe et al., 2016; Kay et al., 2019). Moving beyond
these main effects of mindfulness, this paper identifies
mindfulness as an effective crisis intervention. Specially, this
paper draws on the metacognitive theory of mindfulness and
extends this theory into the context of crisis by showing that
mindfulness can neutralize the negative effects of COVID-19
stressors on work engagement via the mediating role of sleep
duration, a mechanism that is highly vulnerable to the
hyperarousal state triggered by trauma. GENERAL DISCUSSION Importantly, our findings provide further evidence that even
a “low dose” of on-line mindfulness practice is effective (Hülsheger
et al., 2015). Thus, during the COVID-19 outbreak, organizations
that offered morning meditations to all company employees
(e.g., Google), might have been more effective in managing
the negative impacts of the pandemic on employee engagement. p
g
Third, a strength of this study was the dual operationalization
of mindfulness through a randomized-control trial and a daily
self-reported measure. Organizational studies typically focus on
a sole operationalization, despite scholars often referring to
mindfulness state, trait, and trained skill being a unitary construct
(Reb et al., 2020). Therefore, the replication of our results using
two operationalization provides further evidence for the generalized
effect of mindfulness. Nevertheless, the moderation graphs for
Study 1 and 2 have slight discrepancies, suggesting that there
might be fine grained difference between the operationalization. However, despite the discrepancy, it is noteworthy that both
studies support our primary hypothesis that mindfulness will
neutralize the negative effect of COVID-19 stressors, demonstrated
by simple slope analyses, showing that COVID-19 only has a
significant negative effect on sleep quantity when mindfulness
is low. This helps contribute to the literature linking mindfulness
with sleep outcomes (Ong et al., 2012; Ong and Smith, 2017);
nevertheless, there are still important questions for the field to
address. One key question that arises as a result of the current
study is understanding how sleep quantity (and quality) impacts
mindfulness. This study, and the majority of past work (for a
review see Ong and Smith, 2017), has focused on how mindfulness
impacts sleep but given that practicing mindfulness has its own
self-regulatory challenges (Mrazek et al., 2020), it is possible
that a good night’s sleep could increase an individual’s capacity
to engage in, and thus benefit from, mindfulness practice. Building
on the past work, this would suggest a potentially virtuous
cycle in which mindfulness improves sleep and then better sleep
subsequently improves mindfulness. p
g
g
Our research also has practical implications. In uncertain
times like the COVID-19 pandemic, how organizations treat
their employees will have a lasting impact on employees’ loyalty,
engagement, and productivity (Carvalho and Areal, 2016). Our
findings suggest that mindfulness practice can be introduced
as an effective employee care program for organizations. Importantly, our findings provide further evidence that even
a “low dose” of on-line mindfulness practice is effective (Hülsheger
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posttreatment decentering and cognitive reactivity to relapse in major depression. J. FUNDING Study 1 was made possible by funding provided by China
Europe International Business School (CEIBS) to MZ. Study 2
was made possible by the Singapore Ministry of Education
Academic Research Fund Tier 1 (T1 17/2019/115) and National
University of Singapore Research Fund to JN. Study 1 was made possible by funding provided by China
Europe International Business School (CEIBS) to MZ. Study 2
was made possible by the Singapore Ministry of Education
Academic Research Fund Tier 1 (T1 17/2019/115) and National
University of Singapore Research Fund to JN. ETHICS STATEMENT YL collected the data for Study 2. JY, MZ, and TM-W analyzed
the data from both studies. MZ, TM-W, and JN wrote first drafts
of the paper. All authors provide comments and inputs. All authors
contributed to the article and approved the submitted version. The studies involving human participants were reviewed and
approved by Study 1 was approved by the Ethics committee at
China Europe International Business School (CEIBS). Study 2
was approved by the Ethics committee at the National University
of Singapore. The participants provided their written informed
consent to participate in this study. DATA AVAILABILITY STATEMENT f
Second, our research examines the neutralizing of mindfulness
in the relationship between the COVID-19 stressors and
employees’ sleep duration and work engagement. However,
post-traumatic growth theory suggests that it is possible that The raw data supporting the conclusions of this article will
be made available by the authors, without undue reservation. December 2020 | Volume 11 | Article 610156 10 Mindfulness and COVID-19 Zheng et al. AUTHOR CONTRIBUTIONS MZ and JN designed Study 1. MZ collected the data for Study
1. JN, TM-W, YL, and NT designed Study 2. JN, TM-W, and REFERENCES Sleep Med. 32, 246–256. doi: 10.1016/j.sleep.2016.08.006h our understanding of mindfulness in organizations. Organ. Behav. Hum. Decis. Process. 159, 1–7. doi: 10.1016/j.obhdp.2020.04.001 Reb, J., and Atkins, P. W. (Eds.) (2015). Mindfulness in organizations: Founda
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651–660. doi: 10.1016/j.brat.2012.08.001 Conflict of Interest: The authors declare 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: The authors declare that the research was conducted in
the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest. Palan, S., and Schitter, C. (2018). Prolific. Ac-a subject pool for online experiments. J. Behav. Exp. Financ. 17, 22–27. Copyright © 2020 Zheng, Masters-Waage, Yao, Lu, Tan and Narayanan. 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, Mindfulness and COVID-19 Frontiers in Psychology | www.frontiersin.org December 2020 | Volume 11 | Article 610156 Zheng et al. REFERENCES doi: 10.1016/j.jbef.2017.12.004 Frontiers in Psychology | www.frontiersin.org December 2020 | Volume 11 | Article 610156 12 Mindfulness and COVID-19 Zheng et al. provided the original author(s) and the copyright owner(s) 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. December 2020 | Volume 11 | Article 610156 Frontiers in Psychology | www.frontiersin.org 13
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Published: 30 July 2008 This article is available from: http://www.ojrd.com/content/3/1/22 © 2008 Knoers and Levtchenko; 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. Review
Gitelman syndrome
Nine VAM Knoers* and Elena N Levtchenko Open Access Abstract Gitelman syndrome (GS), also referred to as familial hypokalemia-hypomagnesemia, is
characterized by hypokalemic metabolic alkalosis in combination with significant hypomagnesemia
and low urinary calcium excretion. The prevalence is estimated at approximately 1:40,000 and
accordingly, the prevalence of heterozygotes is approximately 1% in Caucasian populations, making
it one of the most frequent inherited renal tubular disorders. In the majority of cases, symptoms
do not appear before the age of six years and the disease is usually diagnosed during adolescence
or adulthood. Transient periods of muscle weakness and tetany, sometimes accompanied by
abdominal pain, vomiting and fever are often seen in GS patients. Paresthesias, especially in the face,
frequently occur. Remarkably, some patients are completely asymptomatic except for the
appearance at adult age of chondrocalcinosis that causes swelling, local heat, and tenderness over
the affected joints. Blood pressure is lower than that in the general population. Sudden cardiac
arrest has been reported occasionally. In general, growth is normal but can be delayed in those GS
patients with severe hypokalemia and hypomagnesemia. GS is transmitted as an autosomal recessive trait. Mutations in the solute carrier family12, member
3 gene, SLC12A3, which encodes the thiazide-sensitive NaCl cotransporter (NCC), are found in the
majority of GS patients. At present, more than 140 different NCC mutations throughout the whole
protein have been identified. In a small minority of GS patients, mutations in the CLCNKB gene,
encoding the chloride channel ClC-Kb have been identified. Diagnosis is based on the clinical symptoms and biochemical abnormalities (hypokalemia, metabolic
alkalosis, hypomagnesemia and hypocalciuria). Bartter syndrome (especially type III) is the most
important genetic disorder to consider in the differential diagnosis of GS. Genetic counseling is
important. Antenatal diagnosis for GS is technically feasible but not advised because of the good
prognosis in the majority of patients. Most asymptomatic patients with GS remain untreated and undergo ambulatory monitoring, once
a year, generally by nephrologists. Lifelong supplementation of magnesium (magnesium-oxide and
magnesium-sulfate) is recommended. Cardiac work-up should be offered to screen for risk factors
of cardiac arrhythmias. All GS patients are encouraged to maintain a high-sodium and high
potassium diet. In general, the long-term prognosis of GS is excellent. Page 1 of 6
(page number not for citation purposes) (page number not for citation purposes) Orphanet Journal of Rare Diseases 2008, 3:22 http://www.ojrd.com/content/3/1/22 http://www.ojrd.com/content/3/1/22 Definition and epidemiology Gitelman syndrome (GS) (OMIM 263800), also referred
to as familial hypokalemia-hypomagnesemia, is an auto-
somal recessive salt-losing renal tubulopathy that is char-
acterized
by
hypomagnesemia,
hypocalciuria
and
secondary aldosteronism, which is responsible for
hypokalemia and metabolic alkalosis [1]. The prevalence
is estimated at ~25 per million and accordingly, the prev-
alence of heterozygotes is approximately 1% in Caucasian
populations, making it one of the most frequent inherited
renal tubular disorders. Gitelman's syndrome Familial hypokalemia-hypomagnesemia Clinical description p
GS patients usually present above six years of age and in
many cases the diagnosis is only made at adult age. Most
patients suffer from tetany, especially during periods of
fever or when extra magnesium is lost due to vomiting or
diarrhea. Paresthesias, especially in the face, frequently
occur. Some patients experience severe fatigue interfering
with daily activities, while others never complain of tired-
ness. The severity of fatigue in GS is not completely related
to the degree of hypokalemia. In contrast to Bartter syn-
drome (a genetically distinct and clinically more severe
tubular transport disorder, which shares the hypokalemic
metabolic alkalosis with GS) polyuria is usually absent or
only mild. In general, growth is normal in GS patients,
however, it can be delayed in patients with severe hypoka-
lemia and hypomagnesemia [2]. Page 2 of 6
(page number not for citation purposes) Disease name and synonyms
Gitelman syndrome Potassium and magnesium depletion prolong the dura-
tion of the action potential of cardiomyocytes and conse-
quently increase the risk for development of ventricular
arrhythmia. Electrocardiograms of patients with Gitelman
syndrome have shown that in about 50% of cases the QT
interval is indeed slightly to moderately prolonged but,
fortunately, is not associated with clinically relevant car-
diac arrhythmias in the far majority of cases [4]. Sudden
cardiac arrest reported in few patients with GS [2,5], war-
rants systematic cardiac screening for identifying other
possible triggering mechanisms or underlying conditions. Blood pressure in GS patients is lower than in the general
population, indicating that even the modest salt wasting
of this disease reduces blood pressure. The results from a
recent study in 35 GS-carriers (with one mutant gene
allele) suggest that GS carriers also have lower blood pres-
sure and may be protected from hypertension [6]. Another study in a large cohort also demonstrated
reduced blood pressure in subjects having SLC12A3 muta-
tions on one allele [7]. These results are distinct from a
previous study in an Amish kindred, in which no reduc-
tion of blood pressure was demonstrated in adult
patients, despite increased Na+ excretion [8]. Thus, further
studies are required to investigate whether the incidence
of cardio-vascular events differs between GS patients or
carries compared to control population. Gitelman's syndrome Etiopathogenesis Page 2 of 6
(page number not for citation purposes) Page 2 of 6
(page number not for citation purposes) http://www.ojrd.com/content/3/1/22 Orphanet Journal of Rare Diseases 2008, 3:22 nature/position of the SLC12A3 mutation combined with
male gender may be a determinant factor in the severity of
GS. Studies in lager cohorts are necessary to confirm this
assumption. sodium will arrive in the collecting duct resulting in mild
volume contraction. The reduced vascular volume acti-
vates the renin-angiotensin-aldosterone system, increas-
ing renin activity and aldosterone levels. The elevated
aldosterone levels give rise to increased electrogenic
sodium reabsorption in the cortical collecting duct via the
epithelial sodium channel (ENaC), defending salt home-
ostasis at the expense of increased secretion of potassium
and hydrogen ions, thus resulting in hypokalemia and
metabolic alkalosis. It has been shown that passive Ca2+
reabsorption in the proximal tubule and reduced abun-
dance of the epithelial Mg2+ channel TRPM6, located in
the DCT explains thiazide-induced hypocalciuria and
hypomagnesemia, respectively [16]. Since thiazides are
known to inhibit NCC, and in view of the phenotypic
resemblance between GS and chronic thiazide-treatment,
it is very likely that similar mechanisms are involved in
the pathogenesis of respectively hypocalciuria and
hypomagnesemia seen in GS. By functional expression studies and results of immuno-
cytochemistry in Xenopus leavis oocytes, it was shown that
most disease-causing NCC mutants are impaired in their
routing to the plasma membrane. Thus, the majority of
mutations belong to the so-called type 2 mutations
which, in contrast to type 1 mutations that impair protein
synthesis, lead to fully synthesized proteins. These type 2
mutant proteins, however, do not traffic appropriately to
the plasma membrane, primarily due to protein misfold-
ing and retention in the endoplasmic reticulum, followed
by rapid proteoasomal degradation. De Jong and co-
workers have shown that NCC misfolding resulting in
defective trafficking in GS is not uniformly complete [11]. Some mutant NCC proteins are only partially retarded in
their trafficking; they do reach the plasma membrane,
albeit to a limited extent, and are partially active. Subse-
quently, it was demonstrated that the intrinsic activity of
these partially retarded mutants is unaffected by the muta-
tion [12,13]. This finding opens the possibility of phar-
macological chaperones, facilitating the routing of
misfolded, trafficking-defective, but otherwise functional
NCCs to the apical membrane, for therapeutic use. Indeed, in an additional study, de Jong et al. Diagnosis, diagnostic methods and differential
diagnosis g
The diagnosis of Gitelman syndrome is based on the clin-
ical symptoms and biochemical abnormalities. The most
typical biochemical abnormalities in GS are hypokalemia,
metabolic alkalosis, hypomagnesemia and hypocalciuria. Serum potassium concentration is comparably low (2.7 ±
0.4 mmol/L) to Bartter syndrome. Serum magnesium con-
centration is low (less than 0.65 mmol/l). In a few GS
patients magnesium concentration is easily maintained in
the normal range early on, which may lead to a false diag-
nosis of Bartter syndrome, and only drops below normal
with time (personal observation). Urinary calcium con-
centration is usually less than 0.2 mmol/mmol creatinine
and rarely exceeds 0.5 mg/kg/day. Hypomagnesemia and
hypocalciuria have always been considered obligate fea-
tures for GS. This assumption has recently been disputed
by Lin et al. [10]. They reported two families with molec-
ularly proven GS, in which male patients had severe
hypokalemia, and were symptomatic with episodes of
paralysis, impaired urinary concentration ability, but with
normal serum magnesium and urinary calcium excretion. Remarkably, female GS patients within these families, car-
rying the same causative mutations as the male patients,
were asymptomatic, had less severe hypokalemia, intact
urine concentration ability, but did have hypomag-
nesemia and hypocalciuria [10]. Although this was a
small study, the authors concluded that gender may affect
phenotypic expression in GS and that hypomagnesemia
and hypocalciuria may not be invariant features of the dis-
order. A minority of patients with the Gitelman phenotype has
been shown to have mutations in the CLCNKB gene,
encoding the renal chloride channel ClC-Kb, located in
basolateral membrane of cells of the thick ascending limb
of Henle's loop (TAL) and the distal tubules. Mutations in
the CLCNKB gene were previously found to be the cause
of classic Bartter syndrome. It is now evident that the clin-
ical phenotype in patients with CLCNKB mutations can
be highly variable, from an antenatal onset of Bartter syn-
drome on one side of the spectrum, to a phenotype closely
resembling Gitelman syndrome at the other side (review
in [9]). Therefore, there is an indication to screen the CLC-
NKB gene in patients with the Gitelman phenotype who
do not have mutations in the SLC12A3 gene. Both loss-of-function mutations in NCC and mutations in
CLC-Kb lead to disruption of NaCl reabsorption in the
DCT (figure 1). Etiopathogenesis found prove
that the transcriptional regulator 4-phenylbutyrate may
be a promising candidate for rescuing partially retarded,
but otherwise functional mutant NCCs [14]. Recently,
another class of mutations in GS was identified by Riveira-
Munoz et al. [15]. This newly identified class includes
mutants which are partly retained in the cell, but in con-
trast to the mutants mentioned above, these mutants do
not show any activity when they reach the cell surface. Etiopathogenesis p
g
In the great majority of cases GS is caused by mutations in
the solute carrier family 12, member 3, SLC12A3 gene,
which encodes the renal thiazide-sensitive sodium-chlo-
ride co-transporter NCC that is specifically expressed in
the apical membrane of cells in the first part of the distal
convoluted tubule (DCT) (reviewed in [9]). NaCl cotrans-
porter (NCC) is a polypeptide of 1021 amino acids and
the 2D-structure is predicted to contain 12 transmem-
brane domains and long intracellular amino- and carbox-
ytermini. At present, more than 140 different, putative
loss-of-function mutations in the SLC12A3 gene have
been identified in GS patients. These mutations include
missense-, nonsense-, frame-shift-, and splice-site muta-
tions and are distributed throughout the whole protein. Some adult GS patients suffer from chondrocalcinosis,
which is assumed to result from chronic hypomag-
nesemia. It causes swelling, local heat, and tenderness
over the affected joints. In earlier clinical reports addi-
tional symptoms, such as ataxia, vertigo, and blurred
vision have been reported. In general, there is extreme inter- and intrafamiliar pheno-
type variability in GS, the latter emphasizing the lack of a
correlation between the severity of symptoms in GS and
the type of mutation in the SLC12A3 gene [10]. Recently,
however, Riviera-Munoz et al. described a small subgroup
of patients with a remarkable severe phenotype, including
an early onset, severe neuromuscular manifestations,
growth retardation and ventricular arrhythmias [2]. The
majority of these patients were male and carried at least
one allele of a splice defect, resulting in a truncating tran-
script, or a non-functional intracellularly retained muta-
tion (see below). They suggested from these data that the Cruz and colleagues have challenged the generally shared
idea that GS is a mild disorder [3]. They evaluated the
symptoms and quality of life (QOL) in 50 adult patients
with molecularly proven GS and compared this cohort of
patients with 25 age- and sex-matched controls. They
found that GS patients had significantly more complaints
than controls, mainly salt craving, musculoskeletal symp-
toms such as tetany and cramps, muscle weakness and
aches, and constitutional symptoms such as fatigue, gen-
eralized weakness and dizziness, and nocturia and poly-
dipsia. In addition, measures of QOL were significantly
lower in GS patients compared to controls. Diagnosis, diagnostic methods and differential
diagnosis When less NaCl is reabsorped, more Prostaglandin excretion is normal and plasma renin activ-
ity and plasma aldosterone concentration are only slightly
elevated compared to Bartter syndrome. Renal functional Page 3 of 6
(page number not for citation purposes) Orphanet Journal of Rare Diseases 2008, 3:22 http://www.ojrd.com/content/3/1/22 A model of transport mechanisms in the DCT
Figure 1
A model of transport mechanisms in the DCT. Sodium-chloride (NaCl) enters the cell via the apical thiazide-sensitive
NCC and leaves the cell through the basolateral Cl- channel (ClC-Kb), and the Na+/K+-ATPase. Indicated also are the recently
identified magnesium channel TRPM6 in the apical membrane, and a putative Na/Mg exchanger in the basolateral membrane. Na+
Cl-
Cl-
2K+
3Na+
ATP
ADP
NCC
Thiazides
Mg2+
Basolateral
Apical
ClC-Kb
Na+/K+-ATPase
TRPM6
Mg2+
Na+
? Barttin
DCT Cell
-10 mV
-65 mV
0 mV Basolateral DCT Cell Na+/K+-ATPase Thiazides A model of transport mechanisms in the DCT
Figure 1
A model of transport mechanisms in the DCT. Sodium-chloride (NaCl) enters the cell via the apical thiazide-sensitive
NCC and leaves the cell through the basolateral Cl- channel (ClC-Kb), and the Na+/K+-ATPase. Indicated also are the recently
identified magnesium channel TRPM6 in the apical membrane, and a putative Na/Mg exchanger in the basolateral membrane. indicating that the defect in GS is located at the level of the
distal tubule. DNA mutation analysis of the gene respon-
sible for GS may confirm the diagnosis. studies have demonstrated normal or slightly decreased
urinary concentrating mechanism, but clearly reduced dis-
tal fractional chloride reabsorption during hypotonic
saline infusion. GS patients have a blunted natriuretic
response to hydrochlorothiazide administration but a
prompt natriuresis after administration of furosemide, Bartter syndrome is the most important genetic disorder
to consider in the differential diagnosis of GS. Especially Page 4 of 6
(page number not for citation purposes) Page 4 of 6
(page number not for citation purposes) Orphanet Journal of Rare Diseases 2008, 3:22 http://www.ojrd.com/content/3/1/22 periods of undercurrent infections especially those
accompanied by vomiting and diarrhea. In case of acute
tetany, 20% MgCl2 should be administered intravenously
(0.1 mmol Mg/kg per dose) and can be repeated every 6
hours. type III Bartter syndrome, which is caused by mutations in
the CLCNKB gene, is clinically and biochemically overlap-
ping with Gitelman syndrome. The other types of Bartter
syndrome usually have an earlier onset and a more severe
phenotype. Abbreviations GS: Gitelman syndrome; QOL: Quality of life; DCT: Distal
convoluted tubule; NCC: Thiazide-sensitive NaCl cotrans-
porter; TAL: Thick ascending limb of Henle's loop; ENaC:
Epithelial sodium channel; TRPM6: Transient receptor
potential channel subfamily M, member 6; NSAID; Non-
steroidal anti-inflammatory drugs. Competing interests The authors declare that they have no competing interests. Genetic counseling g
Genetic counseling is important. Since GS is an autosomal
recessive trait, the recurrence risk for parents with an
affected child is 25%. If the parents already have other
children, who are not obviously affected, it is not abso-
lutely sure that they do not have GS because clinical symp-
toms can appear later in life. If the parents are eager to
know the status of the other child(ren) and in case the
molecular defect in their affected child has been eluci-
dated, DNA-analysis in the other child(ren) may be per-
formed. Adult patients with GS have a low risk of having
children with GS (~1 in 400) unless the patient and his/
her partner are consanguineous. Although technically fea-
sible, antenatal diagnosis for GS is not advised and as yet
has never been asked for because of the good prognosis in
the majority of patients. If symptomatic hypokalemia is not corrected by MgCl2
administration, it can be treated by drugs that antagonize
the activity of aldosterone or block the sodium channel
ENaC in the collecting duct. We prefer the combination of
amiloride (5–10 mg/1.73 m2/day) with KCl (1–3 mmol/
kg/day divided in 3–4 doses). Amiloride should be started
with caution in order to avoid hypotension. Growth and puberty delay in some patients with severe
GS can be corrected by adequate Mg and K supplementa-
tion and a growth-promoting effect of indomethacin was
also reported in GS patients [18]. Cardiac work-up is rec-
ommended to screen for risk factors of cardiac arrhyth-
mias. All patients with GS are encouraged to maintain a
high-sodium and high potassium diet. Prognosis
l g
g
Most asymptomatic patients with GS remain untreated
and undergo ambulatory monitoring (generally by neph-
rologists) with low frequency (1–2 times per year). At
each visit complaints related to hypokalemia (fatigue,
muscle weakness, constipation, cardiac arrhythmias) and
hypomagnesemia (tetany, cramps, paresthesias, joint and
muscle pain) as well as serum levels of K+, bicarbonate
and Mg2+ should be evaluated. In view of the assumption
that chondrocalcinosis is due to magnesium deficiency
(magnesium is a co-factor of various pyrophospatases,
including alkaline phosphatase), there is a clear argument
for lifelong supplementation of magnesium. Normaliza-
tion of serum magnesium is difficult to achieve since high
doses of magnesium cause diarrhea. The bio-availability
of magnesium preparations is different. Magnesium-oxide
and magnesium-sulfate have a significantly lower bio-
availability compared to magnesium-chloride, magne-
sium-lactate and magnesium-aspartate. We recommend
the administration of magnesium-chloride orally to com-
pensate for renal Mg2+ and Cl- losses. Initial daily dose is 3
mmol Mg/m2/24 hrs or 4–5 mg/kg/24 hrs. This dose
should be divided in 3–4 administrations to avoid
diarrhea and has to be adjusted according to serum mag-
nesium levels. The dose usually has to be increased during g
In general, the long-term prognosis of Gitelman syn-
drome is excellent. However, the severity of fatigue may
seriously hamper some patients in their daily activities. Progression to renal insufficiency is extremely rare in GS. As yet, only one patient who developed chronic renal
insufficiency and subsequent progression to end-stage
renal disease has been reported [19]. Diagnosis, diagnostic methods and differential
diagnosis Complaints related to chondrocalcinosis (mainly pseudo-
gout attacks) are caused by the deposition of calcium
pyrophosphate dehydrate crystals in synovium and the
synovial fluid and can be reduced by Mg2+ supplementa-
tion [17]. The symptoms can be controlled by non-steroi-
dal anti-inflammatory drugs (NSAID) and joint surgery is
generally not required. Primary forms of renal hypomagnesemia can be distin-
guished from GS by the absence of hypokalemia. Impor-
tant acquired conditions which should be differentiated
from GS are diuretic and laxative abuse and chronic vom-
iting. The two latter conditions can be confirmed by meas-
uring of low urinary excretion of Cl-. References 1. Gitelman HJ, Graham JB, Welt LG: A new familial disorder char-
acterized by hypokalemia and hypomagnesemia. Trans Assoc
Am Physicians 1966, 79:221-235. 2. Riveira-Munoz E, Chang Q, Godefroid N, Hoenderop JG, Bindels RJ,
Dahan K, Devuyst O: Belgian network for study of gitelman
syndrome. Transcriptional and functional analyses of
SLC12A3 mutations: new clues for the pathogenesis of Gitel-
man syndrome. J Am Soc Nephrol 2007, 1:1271-1283. y
J
p
3. Cruz DN, Shaer AJ, Bia MJ, Lifton RP, Simon DB: Gitelman's syn-
drome revisited: An evaluation of symptoms and health-
related quality of life. Kidney Int 2001, 59:710-717. q
y
y
4. Foglia PEG, Bettineli A, Tosetto C, Cortesi C, Crosazzo L, Edefonti
A, Bianchetti MG: Cardiac work up in primary hypokalemia-
hypomagnesemia (Gitelman syndrome). Nephrol Dial Trans-
plant 2004, 19:1398-1402. p
5. Scognamiglio R, Negut C, Calò LA: Aborted sudden cardiac
death in two patients with Bartter's/Gitelman's syndromes. Clin Nephrol 2007, 67:193-197. p
6. Fava C, Montagnana M, Rosberg L, Burri P, Almgren P, Jönsson A,
Wanby P, Lippi G, Minuz P, Hulthèn LU, Aurell M, Melander O: Sub-
jects heterozygous for genetic loss of function of the thi-
azide-sensitive cotransporter have reduced blood pressure. Hum Mol Genet 2008, 17:413-8. 7. Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-
Cheh C, State MW, Levy D, Lifton RP: Rare independent muta-
tions in renal salt handling genes contribute to blood pres-
sure variation. Nat Genet 2008, 40:592-599. 8. Cruz DN, Simon DB, Nelson-Williams C, Farhi A, Finberg K, Burle-
son L, Gill JR, Lifton RP: Mutations in the Na-Cl cotransporter
reduce blood pressure in humans. Hypertension 2001,
37:1458-1464. 9. Knoers NVAM, Starremans PGJF, Monnens LAH: Hypokalemic
tubular disorders. In Oxford Textbook in Clinical Nephrology Third
edition. Edited by: Davidson AM, Cameron JS, Grunfeld J-P, Ponticelli
C, Ritz E, Winearls CG, van Ypersele C. Oxford University Press;
2005:995-1004. 10. Lin SH, Cheng NL, Hsu YJ, Halperin ML: Intrafamiliar phenotype
variability in patients with Gitelman syndrome having the
same mutations in their thiazide-sensitive sodium/chloride
cotransporter. Am J Kidney Dis 2004, 43:304-312. 11. de Jong JC, Vliet WA van der, Heuvel LPWJ van den, Willems PHGM,
Knoers NVAM, Bindels RJM: Functional expression of mutations
in the human NaCl cotransporter (NCC): evidence for
impaired routing mechanisms in Gitelman's syndrome. J Am
Soc Nephrol 2002, 13:1442-1448. p
12. 19.
Bonfante L, Davis PA, Spinello M, Antonello A, D'Angelo A, Semplicini
A, Galo L: Chronic renal failure, end-stage renal disease, and
peritoneal dialysis in Gitelman's syndrome. Am J Kidney Dis
2001, 38:165-168. References de Jong JC, Willems PHGM, Mooren FJM, Heuvel PWJ van den, Kno-
ers NVAM, Bindels RJM: The structural unit of the thiazide-sen-
sitive NaCl cotransporter (NCC) is a homodimer. J Biol Chem
2003, 278:24302-24307. 13. Sabath E, Meade P, Berkman J, de los Heros P, Moreno E, Bobadilla
NA, Vazquez N, Ellison DH, Gamba G: Pathophysiology of func-
tional mutations of the thiazide-senstivie Na-Cl cotrans-
porter in Gitelman disease. Am J Physiol Renal Physiol 2004,
287:F195-F203. 14. de Jong JC, Willems PHGM, Goossens M, VandeWalle A, Heuvel
LPWJ van den, Knoers NVAM, Bindels RJM: Effects of chemical
chaperones on partially retarded NaCl cotransporter
mutants associated with Gitelman's syndrome in a mouse
cortical collecting duct cell line. Nephrol Dial Transplant 2004,
19:1029-1032. Publish with BioMed Central and every
scientist can read your work free of charge
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disseminating the results of biomedical research in our lifetime."
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Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
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BioMedcentral
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(page number not for citation purposes) Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral Publish with BioMed Central and every
scientist can read your work free of charge 15. Riveira-Munoz E, Chang Q, Bindels RJ, Devuyst O: Gitelman's syn-
drome: towards genoype-phenotype correlations? Pediatr
Nephrol 2007, 22:326-332. p
16. Nijenhuis T, Vallon V, Kemp AWCM van der, Loffing J, Hoenderop
JG, Bindels RJ: Enhanced passive Ca2+ reabsorption and
reduced Mg2+ channel abundance explains thiazide-induced
hypocalciuria and hypomagnesemia. J Clin Invest 2005,
115:1651-1658. 17. Calò L, Punzi L, Semplicini A: Hypomagnesemia and chondrocal-
cinosis in Bartter's and Gitelman's syndrome: review of the
pathogenetic mechanisms. Am J Nephrol 2000, 20:347-350. p
g
J
p
18. Authors' contributions The authors contributed to this review article. They read
and approved the final version of the manuscript. Page 5 of 6
(page number not for citation purposes) Page 5 of 6
(page number not for citation purposes) (page number not for citation purposes) Orphanet Journal of Rare Diseases 2008, 3:22 http://www.ojrd.com/content/3/1/22 References Liaw LC, Banerjee K, Coulthard MG: Dose related growth
response to indometacin in Gitelman syndrome. Arch Dis Child
1999, 81:508-510.
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BREVES NOTAS SOBRE O VIII COLÓQUIO DE GEOCRÍTICA.
|
GEOUSP
| 2,006
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cc-by
| 1,483
|
BREVES NOTAS SOBRE O VIII COLÓQUIO DE GEOCRÍTICA José Aldemir de Oliveira* da lógica de um mundo distante, das
possibilidades ilimitadas, na ilusão de que se
podem reinventar formas espaciais iguais em
qualquer lugar para projetar um mundo que
unifique o ambiente simbólico e atenda
interesses
determinados,
substituindo,
conseqüentemente, a especificidade da cultura
e da história de cada lugar. Foi realizado no final de maio, na cidade
do México, o VII Colóquio Internacional de
Geocrítica, evento que tem acontecido
anualmente em diferentes países ibero-
americanos e encerrará o primeiro ciclo com o X
Colóquio Internacional em 2008 na cidade de
Barcelona
onde ocorreu o primeiro em 1999 q
Barcelona, onde ocorreu o primeiro em 1999. ,
p
O Colóquio reuniu pesquisadores
especialmente
ibero-americanos
que
apresentaram
resultados
de
pesquisas
envolvendo questões atuais da geografia. O
tema, neste VIII - “Geografia Histórica e História
do
Território”
-,
conforme
apontou
o
coordenador do evento, Horácio Capel, na
conferência de abertura, visa lançar um olhar
sobre o passado para corrigir os erros do
presente e apontar os caminhos do futuro,
possibilitando a correção das injustiças sociais,
construindo uma análise crítica acerca de suas
causas. Nesse contexto, o Colóquio busca
reforçar os compromissos dos geógrafos com
os graves problemas sociais que se concretizam
em desigualdades espaciais e na forma pela
qual os diferentes sujeitos se apropriam do
território, construindo um futuro que respeite
as tradições. g
Por fim, Capel apontou que as mudanças
não apresentam o mesmo ritmo: algumas
formas do passado permanecem e outras se
transformam. Em
decorrência,
pode-se
acrescentar que o resgate de modos pretéritos
de produção de formas espaciais chega quase
sempre a nós sem as vozes dos simples e dos
conflitos que são abafados. Todavia, as
especificidades dos lugares explicitam as
contradições e os modos diferenciados de os
sujeitos sociais se relacionarem com o seu
espaço. Neste sentido, a espacialização que se
vai
construindo
com
permanências
e
transformações corresponde às reações
coletivas e aos conflitos que passam pela cultura
e pela memória, ou seja, por ações concretas
dos vários sujeitos sociais que constituem a
resistência
coletiva
à
tendência
homogeneizante que se lhes impõe. *Professor do Programa de Pós-Graduação Sociedade e Cultura na Amazônia – UFAM; participou do evento acima referido com o apoio do
PROAP-CAPES. E-mail: j-aldemir@uol.com.br GEOUSP - Espaço e Tempo, São Paulo, Nº 20, pp. 189 - 191 , 2006 GEOUSP - Espaço e Tempo, São Paulo, Nº 20, pp. 189 - 191 , 2006 -Graduação Sociedade e Cultura na Amazônia – UFAM; participou do evento acima referido com o apoio do
PROAP-CAPES. E-mail: j-aldemir@uol.com.br *Professor do Programa de Pós-Graduação Sociedade e Cultura na Amazônia – UFAM; particip
PROAP-CAPES. E-mail: j-aldemir@uol.com.br 190 - GEOUSP - Espaço e Tempo, São Paulo, Nº 20, 2006 190 - GEOUSP - Espaço e Tempo, São Paulo, Nº 20, 2006 OLIVEIRA , J. A. de Bem ao estilo da geografia crítica,
Robert Herin, destacou em seu discurso,
que, ao mesmo tempo em que é necessário
compreender a realidade com rigor científico,
deve-se ficar comprometido para desvendar
as velhas e as novas máscaras sociais que
se concretizam no espaço. O discurso de
Robert
Herin
pareceu-me
bem
mais
apaixonante do que o texto já publicado,
juntamente com os demais apresentados no
VIII Colóquio, na Revista Scripta Nova. Revista electrónica de geografía y ciencias
sociales, da Universidad de Barcelona,
volume X de agosto de 2006, disponível no
site http://www.ub.es/geocrit/sn/sn-218-
35.htm. passando pela colonização das Américas a temas
atuais ligados à globalização, cujas análises
quase sempre estão ligadas ao patrimônio
material e imaterial. Não, porque alguns
trabalhos trataram o espaço como algo em si
mesmo, como se fosse possível interpretá-lo e
compreendê-lo dissociado da sociedade que o
produz, sem proceder à análise crítica dos
processos, limitando-se ao tratamento dos
dados sem apresentar questionamento da
estrutura social da qual o espaço emerge. Emprestando a frase de uma participante do
evento: “falta gente nestas pesquisas”. Outro
destaque
do
Colóquio:
a
homenagem a um geógrafo pelo conjunto da
obra, a quem foi concedido o Prêmio
Internacional de Geocrítica. Outros importantes
geógrafos já haviam sido homenageados: Elías
Trabulse, Roberto Lobato Corrêa, Thomas F. Glick e Pedro Cunill. Neste VIII Colóquio o
homenageado foi o geógrafo francês Robert
Herin, que realiza trabalhos ligados à geografia
agrária, tratando da migração, despovoamento
e pobreza de áreas rurais da França e da
Espanha. Herin, a partir dessa visão de
Geografia, elabora juntamente com outros
geógrafos na Université de Caen, o conceito de
Geografia Social. Outro aspecto importante do Colóquio
refere-se ao local de sua realização - a
Cidade do México. Uma cidade que vai do
urbanismo caótico à invenção da beleza. Uma cidade que afirma a sua altivez no
desejo de construir em direção ao céu na
verticalidade expressa pelas torres. De modo
contraditório, a cidade criou a beleza
artística
urbana
de
suas
igrejas
e
construções
antigas
e
a
feiúra
dos
subterrâneos e de um trânsito em que
parece não haver lugar para nenhuma
lógica. BREVES NOTAS SOBRE O VIII COLÓQUIO DE GEOCRÍTICA As palavras do coordenador do Colóquio
ressaltaram ainda a perspectiva de romper com
o processo em curso no nosso agora marcado
pelo entendimento dos lugares como se fossem
iguais, numa tendência de produção de formas
espaciais desligadas da história e da cultura do
lugar, tornando os homens e as mulheres reféns Cabe
indagar:
os
trabalhos
apresentados no VIII Colóquio correspondem
a essa expectativa? Dada a variedade e riqueza
de temas abordados, a resposta é sim e não. Sim, do ponto de vista da temporalidade dos
trabalhos que abarcam questões da pré-história, O centro histórico que não se pode
atá-lo apenas ao pedestal de um patrimônio,
como museu, mas como múltiplos centros de
comércio popular, de manifestações políticas
e de gente, muita gente. O centro torna-se
nevrálgico e a cidade parece caminhar na
direção do policentrismo, cuja funcionalidade
só é obtida se articulada à circulação. Por
outro lado, se o centro perde energia, ganha
em prestígio. É que o centro permite, num
relance, ver a cidade pretérita, combinar as
formas e conteúdos do passado. A cidade
cresce em todas as direções, mas o centro
sobreviverá, pela busca do reencontro do
homem consigo e com o seu passado. Se na
cidade antiga havia beleza e a dimensão da
obra, a cidade contemporânea está prestes
a conceber novos encantos em todos os
cantos. Na sua palestra, o Professor Robert
Herin esclareceu que a Geografia Social consiste
na explicação das inter-relações dos fatos
sociais com o espaço para compreender as
desigualdades, a mobilidade e as estruturas de
poder, visando, com isso construir uma geografia
preocupada com o tempo e com o espaço da
vida. Segundo ele, a geografia deve ter
compromisso com o rigor científico, mas esse
rigor não deve ser neutro e por isso cabe ao
geógrafo ter posições críticas e fazer de seu
trabalho um contributo para a interpretação da
realidade
para
além
das
aparências. Acrescentou ainda, que o trabalho do geógrafo
deve ser pautado na ética, na crítica e na luta
pela construção de um mundo melhor com
igualdade
de
direitos,
liberdade
e
fraternidade. Notícias de Encontro: Breves notas sobre o VIII Colóquio de Geocrítica, pp. 189 - 191 Notícias de Encontro: Breves notas sobre o VIII Colóquio de Geocrítica, pp. 189 - 191 Notícias de Encontro: Breves notas sobre o VIII Colóquio de Geocrítica, pp. 189 - 191 191 entre pesquisadores dos países ibero-
americanos e contribuir para a criação de
uma agenda científica para essa parte do
mundo. Projeto científico concebido sempre
como coletivo e integrador, visando explorar
novas interpretações sobre a sociedade e o
espaço. Para tanto, busca estabelecer laços
e redes, fomentando o debate e difundindo
novas idéias capazes de pôr em xeque as
v erdades arraigadas. Para isso, o IX
Colóquio já está marcado para maio e junho
de 2007, na cidade de Porto Alegre no Brasil,
com o tema: “Los problemas del mundo
actual soluciones y alternativas desde la
geografia e las ciencias sociales”. Estejamos
lá para uma análise crítica da geocrítica e
confiando no encontro de pessoas que
acreditam na ciência no geral e na geografia
em particular como contribuição para a
construção de um mundo melhor. Por fim, destaque-se o número de
brasileiros participantes do Colóquio, o que
mostra dois aspectos: O primeiro, a pujança
da Geografia Brasileira com a ampliação de
grupos de pesquisa e a criação de novos
cursos de pós-graduação. O segundo, algo
que parece tabu e por isso fingimos não
reconhecer, que é o papel desempenhado
pelas agências de fomento à pesquisa do
sistema federal e agências estaduais, as
FAPs. Com certeza, poucos brasileiros que
participaram do colóquio o fizeram às suas
expensas, mas com recursos de projetos de
pesquisas. Isso é muito positivo, pois se
trata de uma conquista dos pesquisadores
brasileiros. Por tudo isso, o saldo do Colóquio é
positivo por estar atendendo a um projeto
político no sentido de aprofundar os laços Enviado em agosto de 2006. Aceito em setembro de 2006.
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https://www.surgonco.ru/jour/article/download/316/290
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Technical Features of Robot-Assisted Prostatectomy in Patients with Very Enlarged Prostates
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Kreativnaâ hirurgiâ i onkologiâ
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cc-by
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Оригинальные исследования Оригинальные исследования Оригинальные исследования https://doi.org/10.24060/2076-3093-2018-8-2-33-40 CC BY 4.0 CC BY 4.0 Технические особенности выполнения робот-
ассистированной простатэктомии у пациентов
с выраженным увеличением простаты в объеме А.В. Зырянов1, А.В. Пономарев3, В.О. Смирнов2, А.С. Суриков3 Зырянов Александр
Владимирович —
д.м.н, профессор, проректор
по учебной работе,
е-mail: zav1965@mail.ru,
orcid.org/0000-0001-8105-7233 1 Уральский государственный медицинский университет, Россия, 620028, Екатеринбург, ул. Репина, 3
2 Тюменский государственный медицинский университет, Россия, 625023, Тюмень, ул. Одесская, 54
3 Областной урологический центр АО «Медико-санитарная часть «Нефтяник»,
Россия, 625000, Тюмень, ул. Юрия Семовских, 8/1 1 Уральский государственный медицинский университет, Россия, 620028, Екатеринбург, ул. Репина, 3
2 Тюменский государственный медицинский университет, Россия, 625023, Тюмень, ул. Одесская, 54
3 Областной урологический центр АО «Медико-санитарная часть «Нефтяник»,
Россия, 625000, Тюмень, ул. Юрия Семовских, 8/1
К
l
k
l Пономарев Алексей
Владимирович —
заведующий отделением
онкологии,
е-mail:
ponomarevekb@gmail.com Контакты: Суриков Александр Сергеевич, e-mail: Surikov.a.s@mail.ru Резюме Введение. По данным научных исследований аденокарцинома сочетается с аденоматозной тканью в рамках од-
ной предстательной железы в 10–83,3 % случаев (включая инцидентальный рак). Клиническая ситуация, при
которой аденома достигает внушительных размеров (обычно более 80 см3) и создает значительные трудности
для хирурга, встречается не более чем в 8–10 % всех случаев простатэктомий. Учитывая ограничения дистан-
ционной лучевой терапии, а также брахитерапии, связанные с объемом простаты и неудовлетворительным ка-
чеством мочеиспускания, радикальная простатэктомия у данной группы пациентов остается лечением выбора. Особенности аденомы, такие как большие срединные и боковые доли, наличие цистостомы, а также камней
мочевого пузыря, существенно затрудняют выполнение оперативного пособия. Когда крупный объем проста-
ты является одной из исходных перед оперативным вмешательством, роботическая технология может иметь
определенные преимущества. Смирнов Владислав
Олегович —
аспирант кафедры онколо-
гии с курсом урологии,
e-mail: svo1993@bk.ru Суриков Александр
Сергеевич —
врач-уролог отделения
онкологии,
e-mail: Surikov.a.s@mail.ru,
orcid.org/0000-0003-1238-4761 Суриков Александр
Сергеевич —
врач-уролог отделения
онкологии,
e-mail: Surikov.a.s@mail.ru,
orcid.org/0000-0003-1238-4761 Основной целью данной работы является демонстрация технических преимуществ робот-ассистированной про-
статэктомии, связанных с увеличением простаты. Основной целью данной работы является демонстрация технических преимуществ робот-ассистированной про-
статэктомии, связанных с увеличением простаты. Результаты и обсуждение. Мы попытались описать анатомические ориентиры и возможные хирургические мето-
ды преодоления различных вариаций доброкачественной гиперплазии простаты при робот-ассистированной
простатэктомии. Данная информация особенно полезна хирургам, осваивающим роботическую платформу. Робот-ассистированная простатэктомия может быть эффективно использована при лечении рака предстатель-
ной железы, сопряженного с доброкачественной гиперплазией простаты, а пациенты могут быть уверены в хо-
роших результатах операции. Заключение. При наличии достаточного опыта у хирурга робот-ассистированная простатэктомия может стать
методом выбора в лечении пациентов с большим объемом предстательной железы. Ключевые слова: простаты новообразования, аденокарцинома, робот-ассистированные хирургические опера-
ции, простатэктомия, биопсия Для цитирования: Зырянов А.В., Пономарев А.В., Смирнов В.О., Суриков А.С. Технические особенности выпол-
нения робот-ассистированной простатэктомии у пациентов с выраженным увеличением простаты в объеме. Креативная хирургия и онкология. 2018;8(2):117–124. https://doi.org/10.24060/2076-3093-2018-8-2-33-40 117 117 Креативная хирургия и онкология, том 8, № 2, 2018 Оригинальные исследования For citation: Zyryanov A.V., Ponomarev A.V., Smirnov V.O., Surikov A.S. Technical Features of Robot-Assist-
ed Prostatectomy in Patients with Very Enlarged Prostates. Creative Surgery and Oncology. 2018;8(2):117–124.
https://doi.org/10.24060/2076-3093-2018-8-2-33-40 Creative Surgery and Oncology, Volume 8, No. 2, 2018 Technical Features of Robot-Assisted Prostatectomy
in Patients with Very Enlarged Prostates Aleksandr V. Zyryanov1, Aleksey V. Ponomarev3, Vladislav O. Smirnov2, Aleksandr S. Surikov3 Zyryanov Aleksandr
Vladimirovich —
Doctor of Medical Sciences,
Professor of Urology, Vice-rector
for Academic Affairs,
e-mail: zav1965@mail.ru,
orcid.org/0000-0001-8105-7233 1 Ural State Medical University, 3 Repin str., Yekaterinburg, 620028, Russian Federation
2 Tyumen State Medical University, 54 Odesskaya str., Tyumen, 625023, Russian Federation
3 Regional Urological Center of the Medical Care Unit “Neftyanik”, 8/1 Yuriy Semovskikh str., Tyumen, 625000, Russian Federation
Contacts: Surikov Aleksandr Sergeevich, e-mail: Surikov.a.s@mail.ru for Academic Affairs,
e-mail: zav1965@mail.ru,
orcid.org/0000-0001-8105-7233
Ponomarev Aleksey
Vladimirovich —
Head of the Department of
Oncology,
e-mail:
ponomarevekb@gmail.com
Smirnov Vladislav
Olegovich —
Post-graduate student of the
Department of Oncology with
the Course of Urology,
e-mail: svo1993@bk.ru
Surikov Aleksandr
Sergeevich —
Urologist of the Department of
Oncology,
e-mail: Surikov.a.s@mail.ru,
orcid.org/0000-0003-1238-4761 Ponomarev Aleksey
Vladimirovich —
Head of the Department of
Oncology,
e-mail:
ponomarevekb@gmail.com Zyryanov Aleksandr
Vladimirovich —
Doctor of Medical Sciences,
Professor of Urology, Vice-rector
for Academic Affairs,
e-mail: zav1965@mail.ru,
orcid.org/0000-0001-8105-7233 Ponomarev Aleksey
Vladimirovich —
Head of the Department of
Oncology,
e-mail:
ponomarevekb@gmail.com
Smirnov Vladislav
Olegovich —
Post-graduate student of the
Department of Oncology with
the Course of Urology,
e-mail: svo1993@bk.ru
Surikov Aleksandr
Sergeevich —
Urologist of the Department of
Oncology,
e-mail: Surikov.a.s@mail.ru,
orcid.org/0000-0003-1238-4761 Summary
Introduction y
Introduction. According to scientific studies, adenocarcinoma coincides with adenomatous tissue within the same pros-
tate gland in 10–83.3 % of cases (including incidental cancer). Clinical situations in which the adenoma reaches a signifi-
cant size (typically greater than 80 cm3) and thus creates considerable difficulties for surgeons occurs in not more than in
8–10 % of all cases of prostatectomy. Given the limitations of external beam radiotherapy and brachytherapy related with
prostate volume and poor quality of urination, radical prostatectomy in this group of patients remains the treatment
of choice. Features of adenoma, such as large median and lateral lobes, the presence of cystostomy and bladder stones,
significantly complicate operational benefits. When an enlarged prostate is one of the factors prior to surgery, robotic
technology may have certain advantages. The main goal of this work is to demonstrate the technical advantages of robot-assisted prostatectomy associated with
enlarged prostate. Results and discussion. We describe the anatomical landmarks and possible surgical methods for overcoming different
variations of benign hyperplasia of the prostate using robot-assisted prostatectomy. This information is particularly
useful to surgeons aiming to master robotic surgical platforms. Robot-assisted prostatectomy can be effectively used in
the treatment of prostate cancer associated with benign prostatic hyperplasia, and patients can have confidence in the
results of such an operation. Conclusion. If a surgeon is sufficiently experienced, robot-assisted prostatectomy may become the method of choice in the
treatment of patients with enlarged prostates. Keywords: рrostatic neoplasms, adenocarcinoma, robotic surgical procedures, prostatectomy, biopsy 118 Creative Surgery and Oncology, Volume 8, No. 2, 2018 Оригинальные исследования Оригинальные исследования вмешательств могут привести к проблемам в процессе
проведения РАРП. Например, трансуретральная резек-
ция предстательной железы (ТУРП) увеличивает риск
положительных хирургических краев (ПХК) по резуль-
татам лапароскопической РП (ЛРП) и РАРП [7–10]. С учетом трудностей, связанных с большим объемом про-
статы и, соответственно, с более длительной кривой обу-
чения при РАРП, закономерно возникает необходимость
в работе, раскрывающей основные принципы проведения
простатэктомии у данной сложной группы пациентов [11]. Цель работы: продемонстрировать при выполнении
РАРП последовательно воспроизводимые технические
приемы для преодоления анатомических вариаций,
связанных с увеличением простаты. Введение После введения скрининга на определение уровня про-
стат-специфического антигена (ПСА) большое количе-
ство пациентов с сопутствующей доброкачественной ги-
перплазией простаты (ДГПЖ) попадают в группу риска
и подвергаются биопсии предстательной железы (ПЖ). В настоящее время в практику внедрена таргетная био-
псия простаты под МРТ контролем. При подозрении на
рак предстательной железы (РПЖ) у пациентов с уве-
личенной простатой рекомендуется выполнять таргет-
ную биопсию как первичный метод диагностики [1]. В подавляющем большинстве случаев при таких ус-
ловиях выявляются локализованные формы РПЖ [2]. Учитывая ограничения дистанционной лучевой тера-
пии, а также брахитерапии, связанные с объемом про-
статы и неудовлетворительным качеством мочеиспу-
скания, радикальная простатэктомия (РП) у данной
группы пациентов остается лечением выбора [3, 4]. Технические особенности РАРП Учитывая особенности данной группы пациентов, вы-
сока вероятность развития у них хронической задерж-
ки мочи (ХЗМ) и соответственно, ее осложнений, таких
как конкременты мочевого пузыря и эпицистостомиче-
ские свищи. С момента внедрения рoбoт-aссистирoвaнной рaдикaль-
ной прoстaтэктoмии (РАРП) во Франкфурте в 2000 году
по-прежнему существует значительный интерес как
к технике выполнения операции, так и к ее результатам. Роботизированная система обеспечивает большое коли-
чество преимуществ для хирурга, в их числе: трехмерное
(3D) изображение, улучшенное увеличение, фильтра-
ция дрожания рук и масштабирование движений. Кро-
ме того, технология EndoWrist существенно помогает
в формировании интракорпорального шва при полном
эргономическом комфорте. Остается дискуссионным
вопрос: влияют ли данные преимущества на конечные
результаты оперативного лечения РПЖ [5, 6]? По данным Ю.Г. Аляева и соавт., камни мочевого пу-
зыря у пациентов с РПЖ были выявлены в 1,4 %. Ис-
следователи пришли к выводу, что симультанное уда-
ление камней (цистолитотрипсия) и далее проведенное
радикальное оперативное пособие являются наиболее
оптимальной тактикой лечения [12]. Наличие эпицистостомы также не является непре-
одолимым препятствием для хирурга. При выделении
передней поверхности мочевого пузыря требуется бо-
лее тщательная диссекция с иссечением надлобкового
свища. Следует стремиться к минимизации дефекта
мочевого пузыря с одномоментным его ушиванием
и проверкой МП на герметичность (рис. 1). Особенности ДГПЖ, такие как большие срединные
доли, наличие цистостомы, а также камней мочевого
пузыря, существенно затрудняют выполнение РАРП
у данной группы пациентов. Более того, существует ве-
роятность остаточной медианной доли простаты после
РАРПЭ из-за отсутствия тактильной обратной связи
с роботизированной платформой. Технологические до-
стижения позволяют лечить ДГПЖ различными хирур-
гическими методами. В то же время последствия этих Креативная хирургия и онкология, том 8, № 2, 2018 Техника Montsouris Техника с первичным задним доступом на семен-
ные пузырьки при простатэктомии была предложена
F. Montsouris в 2000 году при освоении ЛРП. При ее
применении первым этапом в пузырно-прямокишеч- Рисунок 1. Диссекция и ушивание зоны эпицистостомичесого свища
Figure 1. Dissection and suturing of fistula area during suprapubic cystostomy 119 Креативная хирургия и онкология, том 8, № 2, 2018 Оригинальные исследования хирург может пройти через передний слой фасции
Денонвилье в ранее выделенный слой без опаски за
соседние органы. ном пространстве выполнялось рассечение брюшины
и выделение семенных пузырьков с дальнейшим ос-
вобождением пространства вдоль задней поверхности
ПЖ. Данный прием абсолютно обоснован при сопут-
ствующей ДГПЖ, учитывая возникающие трудности
при выделении семявыносящих протоков и семенных
пузырьков согласно стандартной методике (рис. 2) [13]. Основные преимущества данной техники: Литературные данные свидетельствуют о том, что зад-
ний доступ к семенным пузырькам для пациентов
с большими простатами является более эффективным
и безопасным [14, 15]. Основные преимущества данной техники: Хирургическая техника и подходы к увеличенной
простате при РАРП • во-первых, хирургу предлагается большая рабочая
зона для выделения семенных пузырьков и семявы-
носящих протоков; После стандартной мобилизации париетальной брю-
шины четвертая рука (Prograsp) натягивает МП в кра-
ниальном направлении, чтобы упростить идентифи-
кацию точки разреза через шейку. Преимущественно
тупое выделение разделяет волокна мочевого пузы-
ря проксимально до идентификации передней части
шейки МП, поскольку она воронкообразно образует
простатический отдел уретры. Акцент на острой и хо-
лодной диссекции ножницами с преимущественным
использованием биполяра минимизирует повреждение
и облегчает дифференцировку текстуры волокон моче-
вого пузыря и простаты. Дым испарения простатиче-
ского секрета указывает на то, что рассечение произво-
дится слишком дистально [16]. • во-вторых, улучшенная визуализация достигается
отсутствием объединенной крови. Для хирургов,
которые выделяют семенные пузырьки только после
отсечения шейки мочевого пузыря, кровь собирает-
ся в ямке, созданной простатой, ее ножками, задней
стенкой МП, и значительно препятствует визуализа-
ции тканей; • в-третьих, безопасная и надежная задняя диссек-
ция шейки мочевого пузыря, а также области апекса
простаты и уретры. Обеспечивая полную мобилиза-
цию предстательной железы по задней поверхности, • в-третьих, безопасная и надежная задняя диссек-
ция шейки мочевого пузыря, а также области апекса
простаты и уретры. Обеспечивая полную мобилиза-
цию предстательной железы по задней поверхности, Рисунок 2. Первичное выделение семявыносящих протоков и семенных пузырьков в пузырно-
прямокишечном пространстве
Figure 2. Primary dissection of vasa deferens and seminal vesicles in the cystic-rectum area Асимметричные боковые доли и/или срединная доля
простаты могут искажать появление вертикальных
волокон шейки мочевого пузыря путем ее смещения
в различных направлениях. Также происходит рас-
тяжение и ослабление шейки МП, что может способ-
ствовать ранней непреднамеренной передней цисто-
томии. При таких условиях шейка мочевого пузыря
может быть сохранена путем ее выделения дистальнее. Крайние дистальные передние цистотомии не ушива-
ются отдельно, предпочтительно включать их дефект
в анастомоз [17]. Отделение переднебоковых отделов мочевого пузыря
от простаты перед отсечением МП сводит к миниму-
му возникновение широкого дефекта в области шейки
МП (рис. 3). После поперечного разреза шейки мочевого пузыря ка-
тетер сдувается и оттягивается назад, чтобы выявить ее
заднюю часть. Слизистая оболочка разделяется холод-
ным рассечением. Далее слизистая оболочка с истон-
ченным мышечным слоем отслаивается от медианной
доли (при ее наличии) с использованием комбинации
тупого и острого рассечения, анатомически сохраняю-
щего шейку мочевого пузыря (рис. 4). Рисунок 2. Первичное выделение семявыносящих протоков и семенных пузырьков в пузырно-
прямокишечном пространстве Figure 2. Primary dissection of vasa deferens and seminal vesicles in the cystic-rectum area Рисунок 3. Асимметричные боковые доли простаты с деформацией шейки мочевого пузыря
Figure 3. Хирургическая техника и подходы к увеличенной
простате при РАРП Asymmetric lateral lobes of the prostate with deformation of the bladder neck При выполнении подслизистой диссекции в ходе выде-
ления внутрипузырной доли хирург определяет «пра-
вильный» слой. Основное последствие роста средней
доли в просвет мочевого пузыря — истончение зад-
ней стенки МП в области треугольника Льето, то есть
в межмочеточниковом пространстве. По факту — по-
сле диссекции средней доли ПЖ по задней полуокруж-
ности шейки МП остаются лишь максимально истон-
ченные мышечный и слизистый слои. Данный факт
важно учитывать при формировании уретро-везикаль-
ного анастомоза (УВА). Рисунок 3. Асимметричные боковые доли простаты с деформацией шейки мочевого пузыря
Figure 3. Asymmetric lateral lobes of the prostate with deformation of the bladder neck Creative Surgery and Oncology, Volume 8, No. 2, 2018 120 Оригинальные исследования После отделения задней поверхности шейки мочевого
пузыря от срединной доли появляется возможность ис-
пользовать Prograsp четвертой руки для ретракции на
медиальной доле. В этот момент ассистент отводит мо-
чевой пузырь, выводя анатомическую плоскость между
задней стенкой МП и основанием простаты. вилье посредством непрерывного шва (Rocco stitch). Данный прием помогает нивелировать напряжение
анастомоза и тем самым снизить риск его несостоя-
тельности (рис. 5) [22]. вилье посредством непрерывного шва (Rocco stitch). Данный прием помогает нивелировать напряжение
анастомоза и тем самым снизить риск его несостоя-
тельности (рис. 5) [22]. По данным B. Rocco и А. Grasso и соавт., применение
задней реконструкции также способствует более ран-
нему (1–3 месяца) восстановлению континентной
функции [23, 24]. После отделения слизистой оболочки мочевого пузыря
потенциальной ловушкой является непреднамеренная
задняя цистотомия при точечной фиксации задней по-
верхности шейки мочевого пузыря. Идентификация
поперечных волокон задней поверхности шейки моче-
вого пузыря, а также визуализация контура слизистой
оболочки МП служат в качестве контрольной точки для
определения плоскости рассечения. Как оговаривалось ранее — выраженное истончение
задней стенки МП после РП может усложнить форми-
рование УВА. В данной клинической ситуации приме-
нение жесткой нити с насечками (V-Loc) может вызвать
прорезывание швов анастомоза и значительно услож-
нить дальнейшую перспективу операции. Наиболее
целесообразно применение гладкой монофиламентной
нити с двумя иглами (рис. 6) [25]. Напротив, рассечение вдоль контура срединной доли
по мере ее приближения к основанию простаты (между
переходной и периферической зонами) приведет к не-
полной резекции предстательной железы [15]. Протягивание швов в таком случае осуществляется
симметрично через уретру с подачей МП в сторону
формирования анастомоза. Протягивание швов в таком случае осуществляется
симметрично через уретру с подачей МП в сторону
формирования анастомоза. Креативная хирургия и онкология, том 8, № 2, 2018 Результаты и обсуждение Стоит изначально отметить, что группа пациентов,
рассматриваемая нами, это абсолютное меньшинство. Несмотря на то что, по данным научных источников,
аденокарцинома в рамках одной ПЖ сочетается с аде-
номатозной тканью в 10–83,3 % случаев (включая инци-
дентальный рак), ситуация, при которой аденома соз-
дает значительные трудности для хирурга, встречается
не более чем в 8–10 % всех РАРП [26–28]. Основные исследования, оценивающие влияние разме-
ра простаты на результаты простатэктомии, проводи-
лись в период с 2003 по 2010 г. Основные исследования, оценивающие влияние разме-
ра простаты на результаты простатэктомии, проводи-
лись в период с 2003 по 2010 г. Хирургическая техника и подходы к увеличенной
простате при РАРП При выполнении приема Montsouris на следующем
этапе семенные протоки и пузырьки достаются сво-
бодно, с выходом на выделенную заднюю поверх-
ность ПЖ. Рисунок 4. Подслизистая диссекция задней полуокружности шейки мочевого пузыря при внутри-
пузырной доле 5,5 см
Figure 4. Submucosal dissection of the back semicircle of the bladder neck with median lobe of 5.5 cm Нервосберегающая операция может быть сложной
ввиду ограничения рабочего пространства в малом
тазу. Эффект массы затрудняет заднее апикальное
рассечение простаты от фасции Денонвилье. При
определении заднего контура предстательной же-
лезы требуется большее вращение, следовательно,
задняя периферическая апикальная диссекция про-
изводится на более позднем этапе. Сосудисто-нерв-
ные пучки (СНП) в таком случае часто смещены бо-
лее кзади. Это наряду с эффектом массы вызывает
большие трудности в процессе визуализации СНП,
особенно на верхушке ПЖ. Экспозиция улучшается
после отделения детрузора и дорзального венозного
комплекса (ДВК). Это позволяет снизить апикальную
нагрузку без чрезмерной медиальной тяги при вра-
щении ПЖ, чтобы компенсировать неблагоприятное
воздействие, вторичное по сравнению с воздействи-
ем массы ПЖ [18, 19]. Рисунок 4. Подслизистая диссекция задней полуокружности шейки мочевого пузыря при внутри-
пузырной доле 5,5 см
Figure 4. Submucosal dissection of the back semicircle of the bladder neck with median lobe of 5.5 cm Рисунок 4. Подслизистая диссекция задней полуокружности шейки мочевого пузыря при внутри-
пузырной доле 5,5 см Figure 4. Submucosal dissection of the back semicircle of the bladder neck with median lobe of 5.5 cm При относительно сохраненной шейке мочевого пузы-
ря отверстия мочеточника остаются вне поля зрения
и проксимальнее, что позволяет не использовать инди-
гокармин или метиленовый синий рутинно. При нало-
жении швов анастомоза берется приблизительно 1 см
отступа на мочевом пузыре, чтобы избежать поврежде-
ния мочеточниковых устьев. Кроме того, для широкой
шейки мочевого пузыря с целью избежать травмиро-
вания мочеточников может выполняться классическая
реконструкция [17, 20, 21]. Рисунок 5. Восстановление фасции Денонвилье (шов Рокко)
Figure 5. Reconstruction of Denonville fascia (Rocco stitch) Одной из основных проблем является широкий раз-
рыв между мочевым пузырем и уретрой, который
остается после удаления большой простаты. Различ-
ные действия могут выполняться для преодоления
чрезмерного напряжения в анастомозе, в том числе
меньший наклон Тренделенбурга или дальнейшая мо-
билизация мочевого пузыря. Однако наиболее логич-
ным представляется восстановление фасции Денон- Рисунок 5. Восстановление фасции Денонвилье (шов Рокко)
Figure 5. Хирургическая техника и подходы к увеличенной
простате при РАРП Reconstruction of Denonville fascia (Rocco stitch) Креативная хирургия и онкология, том 8, № 2, 2018 121 Оригинальные исследования стота наличия положительного хирургического края
при больших простатах приводит к лучшей выживае-
мости без биохимического рецидива. Рисунок 6. Везикоуретральный анастомоз гладкой монофиламентной нитью с двумя иглами
Figure 6. Vesicourethral anastomosis with a smooth monofilament thread and two needles Данные исследования имеют ряд ограничений, таких
как метод РП и/или количество пациентов в исследо-
вании и т.д. Однако основные выводы обладают доста-
точной степенью достоверности. Все авторы сходятся
в одном: РП может быть эффективно использована
у мужчин с большой простатой, а пациенты могут быть
уверены в хороших результатах операции [22, 29–31]. Данные исследования имеют ряд ограничений, таких
как метод РП и/или количество пациентов в исследо-
вании и т.д. Однако основные выводы обладают доста-
точной степенью достоверности. Все авторы сходятся
в одном: РП может быть эффективно использована
у мужчин с большой простатой, а пациенты могут быть
уверены в хороших результатах операции [22, 29–31]. Все больше исследователей приходят к выводу о том,
что методики РП, ЛРП и РАРП являются равноэффек-
тивными, а непосредственные результаты операции во
многом зависят от квалификации и опыта хирурга, вы-
полняющего вмешательство. В клинической ситуации,
при которой крупный объем простаты является одной
из исходных, роботическая технология может иметь
определенные преимущества [32–34]. Рисунок 6. Везикоуретральный анастомоз гладкой монофиламентной нитью с двумя иглами
Figure 6. Vesicourethral anastomosis with a smooth monofilament thread and two needles Процесс обучения роботической хирургии даже
у опытных хирургов занимает значительное время. По данным исследований, кривая обучения РАРП ва-
рьируется от 150 до 600 случаев. Хирурги, зависимые
от тактильного ощущения во время открытой РП,
должны настраиваться на визуальные сигналы. Осо-
бенно сложным этапом РАРП становится диссекция
шейки МП. Опыт предшествующей ЛРП является по-
ложительным фактором при освоении роботической
простатэктомии, особенно в осложненных условиях
[11, 16, 17, 22]. Creative Surgery and Oncology, Volume 8, No. 2, 2018 Заключение Мы попытались описать анатомические ориентиры
и возможные хирургические методы преодоления раз-
личных вариаций ДГПЖ при РАРП. Данная информа-
ция особенно полезна хирургам, осваивающим роботи-
ческую платформу. По их результатам можно сформулировать основные
положения. РП при большом размере простаты ассоциируется: • с большей потерей крови; • с более длительным оперативным временем. • с более длительным оперативным временем. Не влияет на: РАРП может быть эффективно использована при ле-
чении РПЖ, сопряженного с ДГПЖ, а пациенты могут
быть уверены в хороших результатах операции. • продолжительность катетеризации МП и длитель-
ность пребывания в стационаре; При наличии достаточного опыта у хирурга РАРП мо-
жет стать методом выбора в лечении пациентов с боль-
шим объемом предстательной железы. • количество осложнений; • качество удержания мочи; • частоту ранних биохимических рецидивов. Также
отмечена более низкая частота наличия положи-
тельного хирургического края, которая наблюдалась
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journal.pone.0131167 3
Yahya N., Ebert M.A., Bulsara M., Haworth A., Kennedy A., Jo-
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Molinari A.L., Simonelli G., De Concilio B., Porcaro A.B., Del
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transurethral approach during robot-assisted radical prostatectomy
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wjmh.2015.33.3.182 22
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and long-term urinary continence recovery. BMC Urol. 2017;17(1):119. DOI: 10.1186/s12894-017-0308-1 34
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radical prostatectomy: Critical appraisal of the anatomic landmarks for 123 Креативная хирургия и онкология, том 8, № 2, 2018 Оригинальные исследования References DOI: 10.1200/JCO.2013.53.5096 18
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Proximal Fibular Osteotomy Vs Proximal Fibular Osteotomy with Intra-Articular Hyaluronic Acid: Difference in Terms of Pain Relief and Functional Outcome
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Pakistan journal of medicine and dentistry
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OPEN ACCESS OPEN ACCESS ORIGINAL ARTICLE ORIGINAL ARTICLE
OPEN ACCESS
Proximal Fibular Osteotomy Vs Proximal
Fibular Osteotomy with Intra-Articular
Hyaluronic Acid: Difference in Terms of Pain
Relief and Functional Outcome Naseem Munshi1, Muhammad Khalid2, Muhammad Naseem3, Khadijah Abid4
1Orthopedics Department, OPD Ziauddin Medical University, 2Orthopedic Department, Bantva Memon Hospital, 3Orthopedics
Department, Karachi Medical and Dental College, 4Public Health Department, Faculty of Life Sciences, SZABIST, Karachi ABSTRACT Background: Knee osteoarthritis (OA) is a complex, progressive illness marked by cartilage
deterioration and adjacent bone enlargement, and management is challenging due to poor
connective tissue self-regeneration. This study compared the effects of proximal fibular
osteotomy (PFO) alone versus PFO with intra-articular hyaluronic acid injections (IAHA) among
patients presenting with knee OA. Methods: A quasi-experimental study was done at Ziauddin Hospital's orthopedics department from
March 2020 to March 2021. Patients with medial compartment knee joint OA requiring surgery aged
≥40 years and BMI of <30kg/m2 were included. All participants were assigned into two groups, PFO
was done alone in group A (n=30) and with IAHA in group B (n=30). Both groups had their medial
joint spaces measured and documented pre- and post-operatively, as well as their Oxford knee
score and visual analog scale (VAS). SPSS version 25 was used and the normality of data was
assessed using Shapiro-Wilk’s test. The mean difference (post-pre) was compared using the
Mann-Whitney U test. A p≤0.05 was statistically significant. Results: A total of 60 patients were included with a mean age of 51.30±4.87 in Group A and
50.83±6.17 in Group B. Outcomes in both groups were improved considerably with a significant
p<0.0001 in groups, in terms of pain and function determined using Oxford knee score, medial joint
space measurement, and VAS score. Conclusion: PFO alone and PFO with IAHA show significantly improved results in terms of functional
outcomes, but the use of IAHA in combination with PFO, has considerably better results in terms of
pain relief. Keywords: Intra-Articular Injection, Knee Osteoarthritis, Osteoarthritis, Osteotomy METHODS It was a quasi-experimental study conducted at the
orthopedics department of Dr. Ziauddin University
Hospital, Karachi over 1 year from March 2020 to
March 2021. A sample size of 24≈to 30 in each group
was estimated on the Open Epi sample size calcula-
tor. Statistics of mean function sub scores as 67.63 ±
13.65 in the PFO group and 54.10±10.29 in the PFO
plus IAHA group (considering 20% difference
between groups), 99% confidence level, and 90%
power of test were considered for sample size
estimation13. Previously, Total Knee Arthroplasty (TKA) was the
only choice for orthopedic surgeons. Even though it
provides pain relief and deformity repair, it is not the
therapy of choice for the young population5. Uni-compartment knee arthroplasty (UKA) is another
option for TKA. Although this treatment is connected
to a faster recovery time and fewer problems, it is
also linked to a greater risk of reoperation6. High
tibial osteotomy (HTO). is another popular proce-
dure, especially among young patients7. HTO is a
technically difficult operation that can result in prob-
lems such as neurovascular damage, iatrogenic
fracture, and non-union. Furthermore, many individ-
uals may require TKA correction in the future 8, 9. Patients with medial compartment knee joint OA
who needed surgery, were age≥40 years and had a
BMI of less than 30 kg/m2 were included in the study. Patients in whom conservative management was
unsuccessful were also included in this study as they
were good candidates for surgery. Whereas
patients with morbid obesity, tri-compartmental or
bi-compartmental OA, valgus knee deformity,
inflammatory joint disease, or any infection in the
knee joint were not included in this study. A non-ran-
dom consecutive sampling technique was applied
for sample selection. Ethical review approval of Ziauddin Hospital
Nazimabad was attained for the study (ERC
#3092020). All the patients, included in this study,
underwent clinical and radiological evaluation. PFO
alone (group A) or with IAHA (group B) was
performed after receiving written informed consent. Proximal Fibular Osteotomy (PFO) has gained popular-
ity in recent years as a treatment option for individuals
with medial compartment knee OA. It is far more
popular in the East (China and India) than in the
West.10 Its appeal stems from the fact that it is a simpler,
less costly treatment with superior functional results. Corresponding author:
Khadijah Abid j
Public Health Department,
Faculty of Life Sciences,
SZABIST,
Karachi, Pakistan. Email: khadijahabid@gmail.com
doi: https://doi.org/10.36283/PJMD13-1/017 j
Public Health Department,
Faculty of Life Sciences,
SZABIST,
Karachi, Pakistan. Email: khadijahabid@gmail.com
doi: https://doi.org/10.36283/PJMD13-1/017 How to cite: Munshi N, Khalid M, Naseem M, Abid K. Proximal Fibular Osteotomy Vs Proximal Fibular
Osteotomy with Intra-Articular Hyaluronic Acid: Difference in Terms of Pain Relief and Functional
Outcome. Pak J Med Dent. 2024;13(1): 96-101. Doi: 10.36283/PJMD13-1/017 This is an open-access article distributed under the terms of the CreativeCommons Attribution License (CC BY) 4.0
https://creativecommons.org/licenses/by/4.0/ PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2024, VOL. 13 (01) PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2024, VOL. 13 (01) DOI: https://doi.org/10.36283/PJMD13-1/017 Munshi et al. Munshi et al. the effects of PFO alone versus PFO with IAHA injec-
tions in terms of pain relief and functional outcomes
in this study. the effects of PFO alone versus PFO with IAHA injec-
tions in terms of pain relief and functional outcomes
in this study. INTRODUCTION Knee osteoarthritis (OA) is a complex, progressive
illness marked by cartilage deterioration and adjacent
bone enlargement1, 2. Knee OA is expected to affect
16 percent of the global population and 203/10,000
person-years3. Increased
BMI,
advanced
age,
feminine gender, physical inactivity, trauma, and
misalignment are all associated with it1. Malalignment
can cause uneven load distribution inside the knee
joint, exerting extra stress on a specific area of articular
cartilage. An increase in breakdown products is
caused by a local biochemical process involving
pro-inflammatory cytokines, free radicals, and protein-
ases, affecting articular cartilage homeostasis and
lowering synovial fluid viscosity4. before and after surgery. before and after surgery. Oxford knee score and medial space between both
groups was compared using an independent
sample t-test (as the mean difference in Oxford
knee score and medial joint space across groups
followed normal distribution). The mean difference
(post-pre) in VAS score between both groups was
compared using the Mann-Whitney U test (as the
mean difference in VAS score across groups
followed a non-normal distribution). A p-value≤0.05
was considered statistically significant. The collected data was entered into SPSS version 25. The normality of the numeric data was assessed
using Shapiro-Wilk’s test. Data such as age and pre-
and post-Oxford Knee Score were normally distribut-
ed and therefore presented as mean ± standard
deviation. Whereas pre- and post-VAS scores and
pre- and post-medial joint space followed non-nor-
mal distribution and, therefore, presented as
median
and
interquartile
range. Qualitative
variable such as gender was presented in the form
of frequency and percentage. Change in Oxford
knee score (pre and post) in both groups was
assessed using a Paired t-test. Change in medial
joint space (pre and post) and VAS score (pre and
post) in both groups were assessed using the Wilcox-
on Rank test. The mean difference (post-pre) in RESULTS The sixty patients were between the ages of 40 and
65, with a mean age of 51.30±4.87 years in Group A
and 50.83±6.17 years in Group B. In group A, there
were 18 (60%) females and 12 (40%) males, whereas
in group B, there were 16 (53.3%) females and 14
(46.7%) males. (Table 1). Table 1: Baseline characteristics of both groups. Variables
Group A
Group B
Age (years)
51.30±4.87
50.83±6.17
Gender
Male
12 (40%)
14 (46.7%)
Female
18 (60%)
16 (53.3%) Table 1: Baseline characteristics of both groups. Variables
Group A
Group B
Age (years)
51.30±4.87
50.83±6.17
Gender
Male
12 (40%)
14 (46.7%)
Female
18 (60%)
16 (53.3%) Table 1: Baseline characteristics of both groups. Significant pain relief and improved joint function
were observed in all patients using the Oxford knee
scoring system at 1 year post-operatively, with an
average increase of 14.78-15.20 points (as com-
pared to pre-operative evaluation), with a score of
26.23±2.75 pre-operatively improving to 41.01±2.61
post-operatively in group A (p=0.001), and a score
of
27.47±4.10
pre-operatively
improving
to
42.70±2.57
post-operatively
(p=0.001). Similarly,
weight-bearing lower-limb X-rays revealed an increase in the median medial knee joint space
from 3.81 mm pre-operatively to 5.14 mm post-oper-
atively in group A and from 3.79 mm pre-operatively
to 5.21 mm post-operatively in group B, both with a
significant p-value of ≤0.05. In group A, the median
pain score fell from 8 pre-operatively to 4 post-oper-
atively, while in group B, the median pain score
decreased from 8 pre-operatively to 3 post-opera-
tively, with a significant p-value of ≤0.05 in both
groups. METHODS It is
more likely to be used than alternative procedures like
UKA, HTO, and TKA in younger populations and
patients with multiple co-morbidities11. A study
revealed that pain scores dramatically decreased
from 6.4 (baseline) to 2.1 (postoperatively after 1 year)
among 30 patients treated with PFO10. PFO was performed by cutting a single lateral
incision in the fibula that measured 3-5 cm in length. Between the peroneus and soleus muscles, fascia
was incised along the septum. Using an oscillating
saw and an osteotome, a 2-3 cm portion of the
fibula was removed 6 to 10 cm below the caput
fibulae. The surgical site was irrigated with a large
amount of normal saline, and the muscles, fascia,
and skin were then sutured in layers. Intra-articular hyaluronic acid (IAHA) injections have
recently gained popularity as a therapy for knee
OA.12 Restoration of the elastic and viscous charac-
teristics of the synovial fluid, anti-inflammatory and
anti-nociceptive actions, and regulated hyaluronic
acid production are all advantages of IAHA injec-
tions8, 9. A review of 8 meta-analyses reported, that
patients with knee OA treated with IAHA treatment
showed substantial improvement in stiffness, func-
tion, and pain for up to 26 weeks when compared to
controls12. For IAHA injection, the patient is placed in a supine
position with the knee flexed slightly by placing a
towel roll in the popliteal space. The needle insertion
site was marked, a lateral suprapatellar approach
was chosen, and the site of injection was marked
one finger breadth above the patellar edge and
one finger breadth lateral to the patellar edge, and
the drug was injected. Pre and postoperatively, a
weight-bearing x-ray of the diseased knee was
taken in anteroposterior (AP) and lateral perspec-
tives and medial and lateral joint spaces were mea-
sured and documented. At monthly intervals, VAS
and Oxford knee score values were recorded PFO and IAHA, both techniques, might theoretically
be employed together to generate synergistic
benefits for restoring normal architecture and delay-
ing disease progression. As a result, we examined 97 97 PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2024, VOL. 13 (01) DOI: https://doi.org/10.36283/PJMD13-1/017 my Vs Proximal Fibular Osteotomy with Intra-Articular Hyaluronic Acid: Difference in Terms of Pain Relief and Functional Outcome oximal Fibular Osteotomy Vs Proximal Fibular Osteotomy with Intra-Articular Hyaluronic Acid: Difference in Terms of Pain Relief and Func DISCUSSION
O i b
d current study, we have compared the effects of
PFO alone with PFO with IAHA injections in terms of
pain relief and functional outcome. Our research
demonstrates that while both PFO alone and PFO +
IAHA
have
dramatically
improved
functional
outcomes, the use of IAHA in conjunction with PFO
has much superior pain reduction results. In individu-
als with knee OA, IAHA also lowers the requirement
for additional analgesics such as NSAIDs, corticoste-
roids, and opioids, according to recent research20. PFO is based on the idea that non-uniform settling
causes knee OA and accelerates its development14. The lateral section of the tibial plateau is supported
by the fibula and associated soft tissues, but the
medial side is exclusively supported by the medial
tibial cortex. In light of this, the medial side experi-
ences increased axial stress, resulting in non-uniform
settlement and tibial plateau degeneration15. PFO is
used to decrease the lateral fibular support, which
subsequently shifts the stress from the medial to the
lateral side, causing the varus deformity to be
corrected and thereby impacting disease develop-
ment16. Retrospective database analyses show a 0.6–2.2-year
delay in the need for TKR with 5 or more doses of IAHA,
and up to 3.6 years with 5 or more doses of IAHA21-23. In
a prospective comparative study of PFO and HTO for
unilateral varus knee OA, Zou et al. found that the PFO
group had a significantly lower operating time,
peri-operative hemorrhage, time to full weight-bear-
ing, VAS score, and post-operative complications24. PFO improved both the radiological look and function
of varus knee OA, as well as long-term pain alleviation,
according to Yang et al., who performed it in 156
patients with medial compartment knee OA25. Intra-articular drug administration, on the other
hand, which is a concentrated therapeutic dosage
that is dispersed through the joint capsule, may be
regarded as an optimal route of drug delivery in
OA17. Intra-articular drug administration provides
several advantages over systemic drug delivery,
including higher local bioavailability, less systemic
exposure to some medications, fewer side effects,
and a lower cost10, 18, 19. Both above-mentioned treatments give consider-
able advantages on their own, but when used
together, they produce better results, particularly in
terms of pain reduction. RESULTS (Table 2) Table 2: Comparative analysis of pre and post of Oxford knee score, medial joint space, and VAS score in both groups
T1=Pre-operatively, T2=post-operatively at one year, Data presented as Mean±SD or Median (IQR), $ Paired t-test was
applied, ^ Wilcoxon Rank test was applied *Significant at 5% level of significance
Groups
Oxford Knee Score$
p-value
Medial Space (mm)^
p-value
VAS score^
p-value
T1
T2
T1
T2
T1
T2
A
26.23±2.75
41.01±2.61
0.001*
3.81 (3.60-3.91)
5.14 (5.03-5.36)
0.001*
8 (7-9)
4 (4-5)
0.001*
B
27.47±4.10
42.70±2.57
0.001*
3.79 (3.30-3.89)
5.21 (5.08-5.36)
0.001*
8 (7-9)
3 (2-4)
0.001* T1=Pre-operatively, T2=post-operatively at one year, Data presented as Mean±SD or Median (IQR), $ Paired t-test was
applied, ^ Wilcoxon Rank test was applied *Significant at 5% level of significance The mean difference in Oxford knee score and
medial joint space was identical in both groups,
according to an independent t-test. (Table 3) The mean difference in Oxford knee score and
medial joint space was identical in both groups, 98
PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2024, VOL. 13 (01)
DOI: https://doi.org/10.36283/PJMD13-1/017 PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2024, VOL. 13 (01) DOI: https://doi.org/10.36283/PJMD13-1/017 Munshi et al. Munshi et al. Mann-Whitney U test revealed that the change in
VAS score was significantly different between both
groups (U=186, p=0.001). (Figure 1)
*An independent t-test was applied. Table 3: Comparative analysis of mean difference in Oxford knee score and medial joint space between both groups. Figure 1: Comparison of change in VAS score between both groups. Groups
Oxford Knee Score*
p-value
Medial Space (mm)*
p-value
Mean difference
Mean difference
A
-0.466
0.664
-0.11
0.303
B arative analysis of mean difference in Oxford knee score and medial joint space between both groups. Table 3: Comparative analysis of mean difference in Oxford knee score and medial joint space betwee Mann-Whitney U test revealed that the change in
VAS score was significantly different between both
groups (U=186, p=0.001). (Figure 1) Figure 1: Comparison of change in VAS score between both groups. ETHICAL APPROVAL 10. Kakumanu R, Kunadharaju R, Ganesh C. Prospec-
tive study of functional and radiological outcome of
proximal fibular osteotomy for medial compartment
osteoarthritis in elderly. Journal of Orthopaedic Diseas-
es and Traumatology. 2021;4(3):86-91 DOI: 10.4103/-
jodp.jodp_13_21 Ethical review approval of Ziauddin Hospital
Nazimabad was attained for the study (ERC
#3092020). Ethical review approval of Ziauddin Hospital
Nazimabad was attained for the study (ERC
#3092020). ACKNOWLEDGEMENTS
None 9. Rai A, Saurabh A, Shekhar S, Kunwar A, Verma V. Proximal fibular osteotomy for pain relief and func-
tional improvement in patients of osteoarthritis of
knee. International Surgery Journal. 2019;6:2368 DOI:
10.18203/2349-2902.isj20192957 PATIENT CONSENT All the patients, included in this study, underwent
clinical and radiological evaluation. PFO alone
(group A) or with IAHA (group B) was performed
after receiving written informed consent. 11. Vaish A, Kumar Kathiriya Y, Vaishya R. A Critical
Review of Proximal Fibular Osteotomy for Knee
Osteoarthritis. The archives of bone and joint
surgery. 2019;7(5):453-462 12. Bhandari M, Bannuru RR, Babins EM, Martel-Pel-
letier J, Khan M, Raynauld JP, et al. Intra-articular
hyaluronic acid in the treatment of knee osteoarthri-
tis: a Canadian evidence-based perspective. Ther
Adv
Musculoskelet
Dis. 2017;9(9):231-246
DOI:
10.1177/1759720x17729641 DISCUSSION
O i b
d Varacallo M, Luo TD, Johanson NA. Total Knee
Arthroplasty Techniques. In: StatPearls. Treasure
Island (FL): StatPearls Publishing; 2023. 6. Beckmann J, Meier MK, Benignus C, Hecker A, Thien-
pont E. Contemporary knee arthroplasty: one fits all or
time
for
diversity? Arch
Orthop
Trauma
Surg. 2021;141(12):2185-2194 DOI: 10.1007/s00402-021-04042-4
7. Laprade RF, Spiridonov SI, Nystrom LM, Jansson KS. Prospective outcomes of young and middle-aged
adults with medial compartment osteoarthritis treat-
ed with a proximal tibial opening wedge osteotomy. Arthroscopy: The Journal of Arthroscopic & related
Surgery: official publication of the Arthroscopy Asso-
ciation of North America and the International
Arthroscopy Association. 2012;28(3):354-364 DOI:
10.1016/j.arthro.2011.08.310 CONFLICT OF INTEREST There is no conflict of interest. AUTHORS CONTRIBUTIONS NM Conceptualization, Methodology design, Manu-
script writing and editing, and manuscript review. MK, MN Data collection and validation, manuscript
drafting and revision, data entry and management. KA Data analysis and interpretation, manuscript
review, and final approval. 13. Wang X, Wei L, Lv Z, Zhao B, Duan Z, Wu W, et al. Proximal fibular osteotomy: a new surgery for pain relief
and improvement of joint function in patients with knee
osteoarthritis. J Int Med Res. 2017;45(1):282-289 DOI:
10.1177/0300060516676630 DISCUSSION
O i b
d Some of the limitations were
that assessing the results in the treatment of medial There are numerous studies and ample data about
the benefits and use of PFO and IAHA individually,
but there is no study has been conducted on the
use of PFO and IAHA in combination. Hence, in the 99 99 DOI: https://doi.org/10.36283/PJMD13-1/017 PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2024, VOL. 13 (01) Proximal Fibular Osteotomy Vs Proximal Fibular Osteotomy with Intra-Articular Hyaluronic Acid: Difference in Terms of Pain Relief and Functional Outcome Proximal Fibular Osteotomy Vs Proximal Fibular Osteotomy with Intra-Articular Hyaluronic Acid: Difference in Terms of Pain Relief and tional Morphology and Kinesiology. 2019;4(2):30
5. Varacallo M, Luo TD, Johanson NA. Total Knee
Arthroplasty Techniques. In: StatPearls. Treasure
Island (FL): StatPearls Publishing; 2023. 6. Beckmann J, Meier MK, Benignus C, Hecker A, Thien-
pont E. Contemporary knee arthroplasty: one fits all or
time
for
diversity? Arch
Orthop
Trauma
Surg. 2021;141(12):2185-2194 DOI: 10.1007/s00402-021-04042-4
7. Laprade RF, Spiridonov SI, Nystrom LM, Jansson KS. Prospective outcomes of young and middle-aged
adults with medial compartment osteoarthritis treat- compartment
osteoarthritis
required
a
bigger
sample size and a longer follow-up time. Another
flaw was the lack of a control group. Because the
patients were assigned at random, it is impossible to
say which treatment approach will provide better
results in whatever grade of OA. More research
should be done in the future to assess how success-
ful and safe these treatments are on a broader
sample of persons with knee OA. This would help to
broaden the therapeutic applicability of these
methods. tional Morphology and Kinesiology. 2019;4(2):30
5. Varacallo M, Luo TD, Johanson NA. Total Knee
Arthroplasty Techniques. In: StatPearls. Treasure
Island (FL): StatPearls Publishing; 2023. 6. Beckmann J, Meier MK, Benignus C, Hecker A, Thien-
pont E. Contemporary knee arthroplasty: one fits all or
time
for
diversity? Arch
Orthop
Trauma
Surg. 2021;141(12):2185-2194 DOI: 10.1007/s00402-021-04042-4
7. Laprade RF, Spiridonov SI, Nystrom LM, Jansson KS. Prospective outcomes of young and middle-aged
adults with medial compartment osteoarthritis treat-
ed with a proximal tibial opening wedge osteotomy. Arthroscopy: The Journal of Arthroscopic & related
Surgery: official publication of the Arthroscopy Asso-
ciation of North America and the International
Arthroscopy Association. 2012;28(3):354-364 DOI:
10.1016/j.arthro.2011.08.310 tional Morphology and Kinesiology. 2019;4(2):30
5. Varacallo M, Luo TD, Johanson NA. Total Knee
Arthroplasty Techniques. In: StatPearls. Treasure
Island (FL): StatPearls Publishing; 2023. tional Morphology and Kinesiology. 2019;4(2):30
5. CONCLUSION In conclusion, our study highlights the efficacy of
PFO and PFO + IAHA in enhancing functional
outcomes. Notably, the addition of the IAHA with
PFO yields superior pain relief. These findings advo-
cate for a nuanced approach in clinical interven-
tions, emphasizing the potential synergies for
improved patient outcomes in pain management. j
8. Seo SS, Nha KW, Kim TY, Shin YS. Survival of total
knee arthroplasty after high tibial osteotomy versus
primary total knee arthroplasty: A meta-analysis. Medicine (Baltimore). 2019;98(30):e16609 DOI: 10.10
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tti S A li
A V
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M 14. Yang Z-Y, Chen W, Li C-X, Wang J, Shao D-C, Hou
Z-Y, et al. Medial compartment decompression by
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Musumeci G. An Overview of the Pathogenesis and
Treatment of Elbow Osteoarthritis. Journal of Func- 16. Shanmugasundaram S, Kambhampati SB, Saseendar S. Proximal fibular osteotomy in the treatment of medial
osteoarthritis of the knee–A narrative review of literature. PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2024, VOL. 13 (01) DOI: https://doi.org/10.36283/PJMD13-1/017 Munshi et al. ment patients with osteoarthritis. Revista Española
de Cirugía Ortopédica y Traumatología (English
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Ramani T, et al. Toxicology evaluation of drugs admin-
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al journal of toxicology. 2018;37(1):4-27 22. Delbarre A, Amor B, Bardoulat I, Tetafort A,
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24. Zou G, Lan W, Zeng Y, Xie J, Chen S, Qiu Y. Early
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osteoarthritis. Biomedical Research (0970-938X). 2017;28(21) 23. Altman RD, Bedi A, Karlsson J, Sancheti P, Schemitsch
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clinical effect of proximal fibular osteotomy on knee
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10.3928/01477447-20151120-08 21. Mar J, Jurado MR, Arrospide A, Fidalgo AE, Lopez
BS. Cost-analysis of viscosupplementation treatment
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101
DOI: https://doi.org/10.36283/PJMD13-1/017 PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2024, VOL. 13 (01) DOI: https://doi.org/10.36283/PJMD13-1/017
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Protective role of rosemary extract against Etoposide induced liver toxicity, injury and KI67 alterations in rats
|
Journal of Bioscience and Applied Research/Journal of Bioscience and Applied Research
| 2,019
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cc-by-sa
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Abstract Etoposide is chemotherapeutic drugs that inhibit topoisomerase II activity and long been used for the
treatment of human malignancies. The present study was designed to investigate the possible protective effect of
rosemary extract against Etoposide-induced liver toxicity, injury, and KI67 alterations in rats. A total of 40 male Wister
albino rats were divided randomly into four groups (1st group was control; 2nd group was treated with rosemary, 3rd group
was received Etoposide, and 4th group was treated with both rosemary and Etoposide. The administration of Etoposide
significantly caused elevation in ALT, AST, ALP, and liver damage while albumin, total proteins, and KI67 expressions
were significantly decreased when compared with the control group. Co-treated rats with rosemary and Etoposide
maintained the levels of the measured parameters. Finally, it could be concluded that rosemary has a promising role and
it worth to be considered as a natural substance for protecting the liver toxicity and injury induced by Etoposide
chemotherapy. Keywords: Etoposide; Rosemary; Liver functions, KI67 immunohistochemistry; rat. associated with the treatment of several ill-fated
diseases including liver toxicity (Saggu et al., 2014;
Elmasry et al., 2017, 2018; Abd Eldaim et al., 2018;
Akela et al., 2018; Oyouni et al., 2018; Tousson et
al., 2018b). associated with the treatment of several ill-fated
diseases including liver toxicity (Saggu et al., 2014;
Elmasry et al., 2017, 2018; Abd Eldaim et al., 2018;
Akela et al., 2018; Oyouni et al., 2018; Tousson et
al., 2018b). pISSN: 2356 - 9174, eISSN: 2356 –9182 pISSN: 2356 - 9174, eISSN: 2356 –9182 Journal of Bioscience and Applied Research, 2019, Vol.5, No. 1, P.1 -7 1 BioBacta
Journal of Bioscience and Applied Research
www.jbaar.org
Protective role of rosemary extract against Etoposide induced liver toxicity,
injury and KI67 alterations in rats
*Ehab Tousson, Ahmed Masoud, Ezar Hafez, Majd Almakhatreh
Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt
*Corresponding author: toussonehab@yahoo.com BioBacta
Journal of Bioscience and Applied Research
www.jbaar.org BioBacta Received date: December 10, 2018. Accepted: January 13, 2019
DOI: 10.21608/JBAAR.2019.105884 2.2. Experimental groups The 1st group was the control group included rats
received no treatment while the 2nd group was star
anise group included rats received star anise
powdered suspension in distilled water (SA; 100
mg/kg BW) daily by oral gavages for four weeks. The 3rd group was the etoposide group included rats
that injected interprotinally with Etoposide (1mg/kg
B.W/2 day) orally for four weeks (Tousson et al.,
2018a). The 4th group was the group which treated
with star anise plus Etoposide for four weeks. 3.2. Liver histopathology Liver sections of control and rosemary groups
exhibited the normal architecture of the hepatocytes
with prominent round, vesicular basophilic nuclei,
and eosinophilic cytoplasm, and few spaced hepatic
sinusoids arranged in-between the hepatic cords
(Figure 1A&1B). Liver sections of Etoposide group exhibited
moderate
atrophied
vacuolar
degeneration
hepatocytes, some apoptotic cells with deeply
eosinophilic cytoplasm and small deeply stained
pyknotic or fragmented nuclei, marked cellular
infiltrations and marked dilation or congestion in
central and portal veins, vacuolated hepatocytes
(Figure 1C) Liver sections in co-treated Etoposide 2. Materials and methods The expression of Ki67 immunoreactivity (Ki67-
ir) in the liver was detected using the avidin Biotin
Complex (ABC) method according to Tousson et al. (2015). The sections were incubated with anti-
mouse Ki67 monoclonal antibody (dilution 1:50,
DAKO Japan Co, Ltd, Tokyo, Japan) for 1-2 hours
at room temperature. 2.3. Determination of Serum Enzymes At the end of the experimental period, animals
have fasted overnight and for clinical chemistry,
blood samples were individually collected from the
inferior vena cava of each rat in non-heparinized
glass and allowed to stand for 30 min at room
temperature to clot before being centrifuged at 3000
rpm for 15 min. On the other hand; total protein and albumin
levels were significantly decreased in the serum of
the Etoposide treated group as compared to the
control group (P < 0.05) while rats co-treatment
rosemary with Etoposide showed a significant
increase in total protein and albumin levels as
compared to Etoposide treated groups. Both alanine transaminase (ALT/GPT) and
aspartate transaminase (AST/GOT) activities in
serum were assayed by using a commercial kit that
was supplied by Humann (Germany) according to
the method of Schumann and Klauke (2013);
alkaline phosphatase (ALP) activity in serum was
assayed by using a commercial kit that was supplied
by Humann (Germany) according to the method of
Moss and Henderson (1999). Albumin concentration
in serum was assayed by using commercial kit that
was supplied by Diamond (Egypt) according to the
method of Doumas et al. (1971). 1 Introduction Chemotherapy drugs cannot distinguish between
the cancerous cells and the normal cells, which leads
to the side effects (Tousson et al., 2014a-d, 2016,
2018a; Basuony et al., 2015; Bayomy et al., 2017; Al-
Rasheed et al., 2017, 2018). Rosemary (Rosemarinus officinalis) is one of the
household herbs that contains a number of
phytochemicals, including rosmarinic acid, camphor,
caffeic acid, ursolic acid, betulinic acid, and the
antioxidants carnosic acid (Akela et al., 2018). Star
anise fruits are traditionally used in Chinese medicine
to treat stomach aches, vomiting, insomnia, dermal
inflammation, and rheumatic pain as well as a
common spice usage (El-Atrash et al., 2017). Extracts of rosemary leaves possess a variety of
bioactivities
in
vitro
including
antioxidant,
antibacterial,
anti-tumor,
antinociceptive, Etoposide is chemotherapeutic drugs that inhibit
topoisomerase II activity and long been used for the
treatment of human malignancies, where it is a semi-
synthetic
compound
derived
from
the
plant
podophyllum pelltatum (Bayomy et al., 2017; Shin et
al., 2016; Tousson et al., 2018a). Etoposide is
commonly used alone or in combination with other
anticancer agents in the treatment of lung or stomach
cancer, Hodgkin's lymphoma, and AID's. Many plant extracts and their products have been
shown to have significant antioxidant activity which
may be an important property of medicinal plants antiulcerogenic,
antidiuretic,
antidiabetic,
anti-
inflammatory
and
antithrombotic
agents antiulcerogenic,
antidiuretic,
antidiabetic,
anti-
inflammatory
and
antithrombotic
agents pISSN: 2356 - 9174, eISSN: 2356 –9182 pISSN: 2356 - 9174, eISSN: 2356 –9182 Journal of Bioscience and Applied Research, 2019, Vol.5, No. 1, P.1 -7 2 2 (Habtemariam, 2016). Therefore; the present study
was conducted to examine the possible modifying
effects of rosemary aqueous extract against liver
toxicity, liver injury, and Ki67 alterations induced by
Etoposide in male rats. thickness was cut, mounted on clean slides and
stained
with
Ehrlich's
haematoxylin
and
counterstained with eosin as a routine method after
Bancroft and Stevens (1990). 3.1. Serum markers of liver damage Data presented in Table (1) showed that; GPT,
GOT and ALP activities were significantly increased
in serum of rats treated with Etoposide as compared
to the control group (P < 0.05). A significant
increase with co-treatment rosemary with Etoposide
as compared with Etoposide treated group (Table 1). 2.1. Animals The experiments were performed on 40 male rats
weighing 130±10g and 10 week’s age. The rats were
kept in our Faculty animal house for one week
before the experimental work and maintained on a
standard rodent diet and water available ad libitum. After one week of acclimation, rats were equally
divided into four groups. 2.6. Statistical Analysis Data were expressed as mean values ± SE and
statistical analysis was performed using one way
ANOVA to assess significant differences among
treatment groups. The criterion for statistical
significance was set at p<0.01 for the biochemical
data. All statistical analyses were performed using
SPSS statistical version 16 software package
(SPSS® Inc., USA). 3.3. Ki67 expressions in the kidney: On the other hand; Moderate positive reactions
for Ki67-ir (grade 3) were observed in liver sections
of co-treated Etoposide with rosemary (Figure 2D). The
detection
and
distribution
in
Ki67
immunoreactivity (Ki67-ir) in liver sections in the
different groups under study were revealed in
Figures 2A-2D. Table 1: Changes in liver function parameters in different groups. Parameters
Control
Rosemary
Etoposide
Rosemary+Etoposide
GPT (U/l)
24.9±0.87b
22.85±1.55b
51.3±2.06a
44.4±1.96ab
GOT (U/l)
127±3.55b
124±2.96b
216±7.28a
195±6.17a
ALP (U/l)
135±7.02b
121±5.47b
203±6.05a
195±5.42a
Albumin (g/dl)
3.81±0.15b
3.91±0.23b
2.99±0.30a
3.43±0.31ab
Total protein (g/dl)
7.45±0.60b
7.52±0.33b
4.65±0.21a
4.85±0.33a
Data are expressed as mean ± SE of 10 observations. Superscripts of different letters differ significantly (p<0.01) from
each other. bSignificantly different from Etoposide group. aSignificantly different from control group. Table 1: Changes in liver function parameters in different groups. Data are expressed as mean ± SE of 10 observations. Superscripts of different letters differ significantly (p<0.01) from
each other. bSignificantly different from Etoposide group. aSignificantly different from control group. decreased in the treated group with rosemary. Elevated levels of serum AST and ALT enzymes are
indicative of cellular leakage and loss of functional
integrity of cell membranes in the liver (El-Moghazy
et al., 2014; Saggu et al., 2014). 2.4. Histopathological evaluation Immediately
after
decapitation
rats
were
dissected, liver from different groups was quickly
removed, washed in 0.9 saline solutions, and fixed
in 10 % neutral buffered formalin. After fixation,
specimens were dehydrated, cleared, and embedded
in molten paraffin. Kidney sections of 7 microns Journal of Bioscience and Applied Research, 2019, Vol.5, No. 1, P.1 -7 3 pISSN: 2356 - 9174, eISSN: 2356 –9182 with rosemary, group exhibited a mild degree of
improvement in hepatocytes that exhibits moderate
vacuolated
hepatocytes
with
mild
cellular
infiltrations were observed, in addition to mild to
moderate congestion in central and portal veins
(Figure 1D). The liver section in control and rosemary groups
shows a strong positive reaction for Ki67-ir (grade
4) in hepatocyte nuclei (Figures 2A&2B). In contrast
mild to faint positive reactions were detected for
Ki67-ir (grade 1) in the liver sections in the
Etoposide rats group (Figure 2C). 4. Discussion Chemotherapy involves the use of chemical
agents to stop the growth and eliminate cancer cells
even at distant sites from the origin of the primary
tumor (Basuony et al., 2015; Hafez et al., 2017). However, it does not distinguish between cancer and
normal cells, and eliminates not only the fast-
growing cancer cells but also other fast-growing
cells in the body, including, hair and blood cells. The current study aimed to study the possible
modifying effects of rosemary extract against liver
toxicity induced by Etoposide in male albino rats. This result is in harmony with Saleh et al. (2014)
and Basuony et al. (2015) who reported that,
Cisplatin increased during treatments. Also; this
current result is in harmony with Tousson er al. (2014b, 2014c) who reported that; methotrexate-
induced hepatic and renal toxicity in male rats and
the increased in liver function associated with free
radicals trigger cell damage through binding to
cellular macromolecules. Chemotherapy-induced
hepatotoxicity
is
a
common cause of abnormal liver function tests in
patients, this hepatotoxicity is usually begins with
vague clinical symptoms such as fatigue, anorexia,
nausea, dark urine, right upper quadrant discomfort,
and jaundice. Similar findings were reported by Abouzeinab
(2013); Nasr (2013); Abdel-Wahhab et al. (2014)
and Basuony et al. (2015) who reported that;
cisplatin-Induced hepatotoxicity in male rats. The
current results agreed with Juma (1984) and
McDonald et al. (2003) who reported that;
cyclophosphamide-induced toxicity in the human
liver. Also; these results are in agreement with the
previous findings of Kamboj and Sandhir (2011) and
Valentovic et al. (2014) who observed a significant In the current study; a significant increase in
GPT, GOT, and ALP in the Etoposide group
however, this elevation decreased in the treated
group with rosemary. On the other hand; a
significant decrease in albumin, and total protein in
the Etoposide group however, this depletion Journal of Bioscience and Applied Research, 2019, Vol.5, No. 1, P.1 -7 pISSN: 2356 - 9174, eISSN: 2356 –9182 pISSN: 2356 - 9174, eISSN: 2356 –9182 4 decline in serum biochemical parameters due to
cisplatin treatments. In the current study a moderate atrophied
vacuolar degeneration hepatocyte, some apoptotic
cells with deeply eosinophilic cytoplasm and small
deeply stained pyknotic or fragmented nuclei,
marked cellular infiltrations and marked dilation or
congestion in central and portal veins were observed
in liver after treatment with Etoposide. In the current study, our histopathological and
immunohistochemical results showed that treatment
of rats with aqueous extract of rosemary showed
moderate to a good degree of improvement in the
liver structure and functions as compared with
Etoposide group. Figures 1A-1D: Photomicrographs of rat liver sections in the different experimental groups stained with
Haematoxylin & Eosin. A&B: Liver sections in control and rosemary groups revealed normal liver structure
with hepatocytes (Hp). C: Liver sections in Etoposide group revealed exhibited moderate atrophied vacuolar
degeneration hepatocytes (White arrows), some apoptotic cells with deep eosinophilic cytoplasm, marked
cellular infiltrations (arrow heads) and marked dilation or congestion in central and portal veins. D: Liver
sections of co-treated Etoposide with rosemary revealed moderate vacuolated hepatocytes (White arrows) with
mild cellular infiltrations. Figures 1A-1D: Photomicrographs of rat liver sections in the different experimental groups stained with
Haematoxylin & Eosin. A&B: Liver sections in control and rosemary groups revealed normal liver structure
with hepatocytes (Hp). C: Liver sections in Etoposide group revealed exhibited moderate atrophied vacuolar
degeneration hepatocytes (White arrows), some apoptotic cells with deep eosinophilic cytoplasm, marked
cellular infiltrations (arrow heads) and marked dilation or congestion in central and portal veins. D: Liver
sections of co-treated Etoposide with rosemary revealed moderate vacuolated hepatocytes (White arrows) with
mild cellular infiltrations. pISSN: 2356 - 9174, eISSN: 2356 –9182 Journal of Bioscience and Applied Research, 2019, Vol.5, No. 1, P.1 -7 5 Figures 2A-2D: Photomicrographs of rat liver sections in the different experimental groups stained with
Ki67-ir. A&B: Liver sections in control and rosemary groups show moderate positive reaction for Ki67-ir
(arrows) in hepatocytes. C: Liver sections in Etoposide rats group showed mild positive reactions in
hepatocytes were detected for Ki67-ir. D: Moderate positive reactions for Ki67-ir were observed in
hepatocytes in liver sections of co-treated Etoposide with rosemary (arrows). Figures 2A-2D: Photomicrographs of rat liver sections in the different experimental groups stained with
Ki67-ir. A&B: Liver sections in control and rosemary groups show moderate positive reaction for Ki67-ir
(arrows) in hepatocytes. pISSN: 2356 - 9174, eISSN: 2356 –9182 C: Liver sections in Etoposide rats group showed mild positive reactions in
hepatocytes were detected for Ki67-ir. D: Moderate positive reactions for Ki67-ir were observed in
hepatocytes in liver sections of co-treated Etoposide with rosemary (arrows). Abdel-Wahhab, M.A., El-Denshary,
E.S.,
El-
Nekeety, A.A., Hassan, N.S., Abu-Salem,
F.M., Sarhan, N.A., Rihn, B.H. (2014). Impact of soy isoflavones on aflatoxin-
induced oxidative stress and hepatotoxicity
in rats. General Health and Medical
Sciences, 1(2): 9-14 . Our results agreed with Tousson et al. (2014a,b) who
find that ginseng extract acts as a protective and
ameliorated effect on MTX-induced hepatic and
renal toxicity. Our results agreed with Tousson et al. (2014a,b) who
find that ginseng extract acts as a protective and
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renal toxicity. Proliferating cell nuclear antigen (PCNA) or
Ki-67 known as cyclin, is a non-histone nuclear
protein whose level of synthesis correlates directly
with rates of cellular proliferation and DNA
synthesis (Tousson et al., 2011). Antibodies to the
cell-cycle-associated Ki-67 protein have been widely
used for more than a decade as markers of
proliferative cells. In the current study; a significant
decrease in KI67 expression in liver sections after
treatment with Etoposide when compared with
control. In contrast, co-treatment Etoposide with
rosemary extract increases the depletion of Ki67
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POR UMA HISTÓRIA QUE NÃO SEJA ÚNICA: COLONIZAÇÃO E IDENTIDADE NO CONTO “A HISTORIADORA OBSTINADA”, DE CHIMAMANDA NGOZI ADICHIE
|
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Luana Miranda Graduada em Letras e em História pela Universidade Estadual do Centro-oeste (Guarapuava/Brasil). E-mail: luanamiranda291@yahoo.com Recebido em: 9 de setembro de 2020
Aprovado em: 26 de novembro de 2020
Sistema de Avaliação: Double Blind Review
RPR | a. 18 | n. 1 | p. 137-150 | jan./abr. 2021
DOI: https://doi.org/10.25112/rpr.v1i0.2396 Priscila Finger do Prado g
Doutora em Letras pela Universidade Federal do Paraná (Curitiba/Brasil). Docente no Departamento de Letras da Universidade Estadual do Centro-oeste (Guarapuava/Brasil). E-mail: priscilletras@yahoo.com.br p-ISSN: 1807-1112
e-ISSN: 2448-1939
POR UMA HISTÓRIA PLURAL: COLONIZAÇÃO
E IDENTIDADE NO CONTO “A HISTORIADORA
OBSTINADA”, DE CHIMAMANDA NGOZI ADICHIE
FOR A PLURAL HISTORY: COLONIZATION AND IDENTITY IN “THE
HEADSRONG HISTORIAN”, BY CHIMAMANDA NGOZI ADICHIE Priscila Finger do Prado RESUMO O presente artigo busca refletir sobre relações entre literatura e história, ao analisar aspectos sobre
colonização e identidade no conto A historiadora obstinada, do livro No seu Pescoço, da escritora nigeriana
Chimamanda Ngozi Adichie. Na leitura do conto, percebe-se a representação do choque cultural entre o
colonizador e o colonizado, bem como a forma como a identidade cultural africana é alterada, até que haja
uma contemporânea desmistificação da história oficial do colonizador pelo olhar da personagem Grace. O conto, publicado em 2017, aponta para a discussão sobre a necessidade de trabalhar outro olhar sobre
a colonização de países africanos como a Nigéria, desta vez a partir da visão do colonizado. O estudo foi
norteado pela relação entre literatura e história na perspectiva teórica de Linda Hutcheon, com o livro
Poética do Pós-Modernismos, e de Roger Chartier, no livro A História Cultural Entre Práticas e Representações. Sobre a identidade cultural, fez-se uso do trabalho de Stuart Hall no livro A Identidade Cultural da Pós-
Modernidade. Por fim, sobre a problemática do sujeito colonizado foi apresentada a leitura do livro O Retrato
do colonizador precedido pelo Colonizado, de Albert Memmi. O presente artigo busca refletir sobre relações entre literatura e história, ao analisar aspectos sobre
colonização e identidade no conto A historiadora obstinada, do livro No seu Pescoço, da escritora nigeriana
Chimamanda Ngozi Adichie. Na leitura do conto, percebe-se a representação do choque cultural entre o
colonizador e o colonizado, bem como a forma como a identidade cultural africana é alterada, até que haja
uma contemporânea desmistificação da história oficial do colonizador pelo olhar da personagem Grace. uma contemporânea desmistificação da história oficial do colonizador pelo olhar da personagem Grace. O conto, publicado em 2017, aponta para a discussão sobre a necessidade de trabalhar outro olhar sobre
a colonização de países africanos como a Nigéria, desta vez a partir da visão do colonizado. O estudo foi
norteado pela relação entre literatura e história na perspectiva teórica de Linda Hutcheon, com o livro
Poética do Pós-Modernismos, e de Roger Chartier, no livro A História Cultural Entre Práticas e Representações. Sobre a identidade cultural, fez-se uso do trabalho de Stuart Hall no livro A Identidade Cultural da Pós-
Modernidade. Por fim, sobre a problemática do sujeito colonizado foi apresentada a leitura do livro O Retrato
do colonizador precedido pelo Colonizado, de Albert Memmi. Palavras-chave: Chimamanda Adichie. Identidade. Colonização. Literatura e história. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 p-ISSN: 1807-1112
e-ISSN: 2448-1939 ABSTRACT This article aims to analyze relationship between literature and history in the short story “The headstrong
historian”, in the book The thing around your neck, by Chimamanda Ngozi Adichie. We find to understand
how colonization and identity are built in the text. In short story’s lecture, we realize the cultural shock’s
representation between colonizer and colonized, as well as how African cultural identity is changed. We
observe in the text too a contemporary point of view on the character Grace, that demystifies colonizer
official story. The short story was published in 2017, and your plot aims to discuss about other views of
African countries as Nigeria, emphasizing colonized point of view. The study was guided by literature and
history’s relationship proposed by Linda Hutcheon, with the book A Poetics of Posmodernism: History, Theory,
Fiction, and by Roger Chartier, with the book Cultural History: between practices and representations. About
cultural identity, we use Stuart Hall’s study, in the book The question of cultural identity. At least, we present
the lecture of the book The colonizer and the colonized, by Albert Memmi, to think about colonized subject’s
problem. ywords: Chimamanda Adichie. Identity. Colonization. Literature e history. Keywords: Chimamanda Adichie. Identity. Colonization. Literature e history. 1 INTRODUÇÃO No discurso O perigo de uma história única, Chimamanda Ngozi Adichie relata suas experiências com a
leitura em sua vida. Foi uma leitora precoce, começou a ler por volta de quatro anos e começou a escrever
com sete. No entanto, sua leitura e sua escrita estavam voltadas para os livros infantis britânicos e
americanos aos quais ela tinha acesso. Nas histórias que Adichie lia, estavam representados personagens brancos, de olhos azuis,
cabelos lisos, que comiam maçãs, bebiam cerveja de gengibre e brincavam na neve. Ou seja, não havia
representatividade de personagens negros, com cabelos crespos, que comiam manga, inhame, arroz, em
um lugar que não tem necessidade de se preocupar com o nascer do sol. E com essas leituras, ela pensou
que era normal não ser representada nas suas histórias e sim escrever sobre o estrangeiro. A expressão que a escritora utiliza para evidenciar a questão de representatividade é “vulnerável em
face de uma história”, pois sua percepção de literatura mudou, quando conheceu os livros africanos. Ela
percebeu que pessoas como ela “com a pele da cor de chocolate, cujos cabelos crespos que não poderiam
formar rabos-de-cavalo, também podiam existir na literatura”. E essa descoberta possibilitou a quebra de
uma única história sobre os livros e também na produção de suas obras. Agora vamos contextualizar um pouco mais da autora. Adichie nasceu em Enugu, na Nigéria, em
1977. Além da reunião de contos No seu pescoço (2017), é autora também dos romances Meio sol amarelo
(2016), Hibisco roxo (2003), Americanah (2013), os ensaios Sejamos todos feministas (2014) e Para educar
crianças feministas (2017). A autora se autodeclara feminista, assumindo sua identidade de mulher negra
e nigeriana. Suas obras quebram paradigmas que estão postos e institucionalizados em nossa sociedade
hierarquizada, ao questionar a construção social dos papéis de gênero, dando voz aos oprimidos e
silenciados na história e traduzindo aspectos da cultura nigeriana e americana. A obra que iremos trabalhar, No seu pescoço (2017), expõe doze contos: A cela um, Réplica, Uma
experiência privada, Fantasmas, Na segunda-feira da semana passada, Jumping Monkey Hill, No seu pescoço,
A embaixada americana, O tremor, Os casamenteiros, Amanhã é tarde demais, e o último A historiadora
obstinada. Em sua maioria, as protagonistas são mulheres nigerianas. São contos densos de intensa
reflexão sobre a cultura hierarquizada e também sobre a opressão imposta à mulher negra. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 138 p-ISSN: 1807-1112
e-ISSN: 2448-1939 1 INTRODUÇÃO Para atingir o nosso objeto de pesquisa, escolhemos o último conto, A historiadora obstinada, em que a
autora situa a história da personagem Nwamgba, que escolheu seu próprio marido, apesar da contrariedade
dos seus pais. Obierika, seu esposo, pertencia a uma família que carregava consigo uma maldição de
infertilidade. Nwamgba também repete a história, sofre diversos abortos e, por isso, é malvista pela
comunidade. Até que nasce Anikwenwa, o primeiro e único filho do casal. Pouco tempo depois, morre o marido, deixando Nwamgba no meio de uma disputa familiar pelas terras. Os opositores são os primos
do falecido. Nwamgba percebe que precisa colocar o filho em uma escola, para ele aprender inglês e
defender seus direitos nos tribunais dos recém-chegados colonizadores ingleses. Ela opta por deixar seu
filho com os missionários católicos, que ensinavam diretamente a língua inglesa, diferentemente dos
missionários anglicanos que procuravam ensinar primeiro na língua materna. O filho aprendeu não só a
língua como também a cultura do colonizador, consequentemente rejeitou os ensinamentos de sua mãe. Por esse resumo, é possível perceber que aparecem, no conto, questões de identidade e de colonização,
que iremos analisar à luz de teorias sobre os temas. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 139 p-ISSN: 1807-1112
e-ISSN: 2448-1939 afirmação de que tanto a história como a ficção são discursos, construtos humanos,
sistemas de significação, e é a partir dessa identidade que as duas obtêm sua principal
pretensão à verdade [...] (HUTCHEON, 1991, p. 127). afirmação de que tanto a história como a ficção são discursos, construtos humanos,
sistemas de significação, e é a partir dessa identidade que as duas obtêm sua principal
pretensão à verdade [...] (HUTCHEON, 1991, p. 127). Em outras palavras, a metaficção historiográfica tem autoconsciência teórica sobre a história e a
ficção como criações humanas sendo “a base para seu repensar e sua reelaboração das formas e dos
conteúdos do passado” (HUTCHEON, 1991, p. 22). Nessa esteira, é possível ler o conto “A historiadora
obstinada”, de Chimamanda Adichie, que está entrelaçado ao contexto histórico da Nigéria como nação
colonizada. Iremos problematizar adiante essa mistura do histórico com o ficcional, percebendo, como
aponta Hutcheon (1991), a reconstrução/construção histórica do conto e como literatura e história
podem se apoiar mutuamente. O estudo da História também deve ser encarado a partir da questão da retomada da cultura, segundo
Roger Chartier, em A História Cultural Entre Práticas e Representações (1990). A cultura passou a ser o
enfoque de Chartier para compreender melhor as relações humanas, pois os estudos nas décadas de
60 e 70 estavam reduzidos pelas determinações econômicas e sociais. Assim, a historiografia também
começou a beber em outras áreas do conhecimento, como a Antropologia e a Teoria Literária. Nessa linha, iremos analisar um pouco do passado colonial africano, através da leitura do conto,
sendo possível detectar os hábitos e as práticas dos habitantes. Por exemplo, podemos perceber como
era a alimentação, os deuses, a língua, as vestimentas, os papéis sociais desempenhados pelos homens
e pelas mulheres etc., o que alimenta a perspectiva da história cultural. Esses dados são importantes,
especialmente porque o conto apresenta uma passagem temporal que indica alteração dos costumes e
as relações interpessoais, o que contribui para mudanças na identidade cultural das personagens. Sobre a identidade cultural, abordaremos um pouco do trabalho de Stuart Hall, no livro A Identidade
Cultural da Pós-Modernidade (2006). Segundo o estudioso, no primeiro capítulo do livro, intitulado A
identidade em questão, três são as concepções de identidade do ser humano: o sujeito do iluminismo, o
sujeito sociológico e o sujeito pós-moderno. “PÔR SENTIDO AO PASSADO”: HISTÓRIA, LITERATURA, IDENTIDADE E COLONIZA Primeiramente, iremos nortear o estudo da relação entre literatura e história a partir da perspectiva
teórica de Linda Hutcheon, no livro Poética do Pós-Modernismo (1991). Hutcheon (1991) problematiza a
história de forma crítica e contextual, afirmando que tanto a ficção quanto a história são discursos, ou
seja, que: [...] o sentido e a forma não estão nos acontecimentos, mas nos sistemas que transformam
esses “acontecimentos” passados em “fatos” históricos presentes. Isso não é um
“desonesto refúgio para escapar à verdade”, mas um reconhecimento da função de
produção de sentido dos construtos humanos. (HUTCHEON, 1991, p. 122). Dessa maneira, ao mesmo tempo em que o pós-modernismo reinsere os contextos históricos
significativamente, problematiza toda a noção do conhecimento histórico. A autora conclui que não pode
haver um conceito único de história, questionando a autenticidade histórica. Propõe que essa mistura
entre história e ficção (uma adulteração dos fatos) é o meio de fazer com que o leitor se conscientize
sobre a natureza específica do referente histórico (HUTCHEON, 1991, p. 122-123). Hutcheon (1991) esclarece melhor essa relação (História/Literatura), citando Jacques Derrida, que
aponta que a história busca refletir ou construir o passado através dos vestígios do tempo. Em outras
palavras, põe um sentido ao passado e o faz por meio da pressuposição de um final ou uma origem. Também assim procede a ficção. E ela esclarece que a diferença na ficção pós-moderna está em sua
desafiadora autoconsciência sobre aquela imposição que a torna provisória (HUTCHEON, 1991, p. 132). Essa autoconsciência vai ganhar forma no conceito de metaficção historiográfica, que: [...] refuta os métodos naturais, ou de senso comum, para distinguir entre o fato histórico
e a ficção. Ela recusa a visão de que apenas a história tem uma pretensão à verdade,
por meio do questionamento da base dessa pretensão na historiografia e por meio da Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 140 p-ISSN: 1807-1112
e-ISSN: 2448-1939
afirmação de que tanto a história como a ficção são discursos, construtos humanos,
sistemas de significação, e é a partir dessa identidade que as duas obtêm sua principal
pretensão à verdade [...] (HUTCHEON, 1991, p. 127). p-ISSN: 1807-1112
e-ISSN: 2448-1939 A primeira consiste no sujeito do iluminismo, que é o indivíduo
centrado, dotado de capacidades de razão e individualismo (usualmente descrito como masculino); a
segunda é o sujeito sociológico, que não é independente, pois se forma pela relação que estabelece com
os outros, em que o interior (mundo pessoal) e o exterior (mundo público) estão interligados; e a terceira
é o sujeito pós-moderno, que não possui uma identidade única, mas várias, sendo algumas, inclusive,
contraditórias, outras mal resolvidas, promovendo assim um debate em torno da crise de identidade. No conto, iremos analisar como a identidade sociológica da cultura nigeriana sofre alterações com
a presença do colonizador inglês, a partir do caso dos protagonistas do conto. Nossa hipótese é a de que o sujeito ali presente possui características do sujeito pós-moderno, visto que a personagem Grace
percebe os conflitos de identidade com relação à cultura de seus pais (que receberam influencia através
dos ensinamentos do colonizador) e para com a da sua avó (que se manteve resoluta quanto aos seus
costumes). que o sujeito ali presente possui características do sujeito pós-moderno, visto que a personagem Grace
percebe os conflitos de identidade com relação à cultura de seus pais (que receberam influencia através
dos ensinamentos do colonizador) e para com a da sua avó (que se manteve resoluta quanto aos seus
costumes). Por fim, para complementar utilizaremos a problemática do sujeito colonizado, trabalhando com
a leitura do livro O Retrato do colonizador precedido pelo Colonizado, de Albert Memmi (2007). O autor
problematiza a colonização e expõe como o colonizador foi retratado por muito tempo: A alguns às vezes ainda agrada representar o colonizador como um homem de grande
estatura, bronzeado pelo sol, calçado com botinas, apoiado em uma pá – pois ele gosta
de pôr mãos à obra, fixando seu olhar ao longe no horizonte de suas terras; entre duas
ações contra a natureza, ele se devota aos homens, cuida dos doentes e difunde a
cultura, um nobre aventureiro, enfim, um pioneiro (MEMMI, 2007, p. 37). O autor continua argumentando que essa imagem épica talvez correspondeu um dia a alguma realidade
ou apenas se limitou às gravuras das cédulas colônias. Visto que os motivos da empreitada colonial já
foram esclarecidos por todos os historiadores da colonização, ninguém mais acredita na missão cultural
e moral, mas puramente na econômica (MEMMI, 2007, p. 37). E nessa empreitada, não poderíamos deixar de falar em privilégios. Memmi (2007) nos mostra que o
estrangeiro que chega a um país pelo caos da história não consegue criar um espaço para si sem também
tomar o do habitante. Alterando regras, ele as substitui pelas suas. Ele é privilegiado ilegítimo, isto é, um
usurpador não apenas aos olhos do colonizado, mas aos seus próprios olhos. Já o colonizado, segundo Memmi (2007), recebe a representação mítica do colonizador de preguiçoso,
ocioso: “qualquer que seja a função que assuma, qualquer que seja o zelo com a que ele se dedique, nunca
será nada além de preguiçoso” (MEMMI, 2017, 119). E esse argumento serviu para legitimar o trabalho
do colonizador. Também ocorre a desumanização do colonizado e sua mistificação. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 141 p-ISSN: 1807-1112
e-ISSN: 2448-1939 se utilizou da cultura e da língua do colonizador, aliadas à observação dos costumes ancestrais de seu
povo, e essa ambiguidade assumida entre elementos do colonizado e do colonizador, mesmo que em
situação de descolonização, será um fator que propicia a fragmentação do sujeito pós-moderno e, nesse
caso, pós-colonial. 3 A OBSTINAÇÃO DA HISTÓRIADORA: EM BUSCA DE UMA HISTÓRIA QUE NÃO SEJA ÚNICA O conto A historiadora obstinada, como o próprio título menciona, apresenta a obstinação de uma
historiadora em recuperar as vozes silenciadas dos seus antepassados. “Obstinada (o)”, segundo o
Dicionário Aurélio, significa “[Lat. Obstinatu.] adj. 1. Que se aferra a uma ideia, uma opinião, um plano,
etc. 2. Que não deixa dissuadir; irredutível. 3. Que é birrento, teimoso” (FERREIRA, 1975, p. 998). Essa
vontade de recuperar a história tem relação com o próprio projeto literário de Adichie que destaca o perigo
de uma história única. Em verdade, o título vai remeter à terceira geração da família, que é enfocada
na narrativa, com a personagem Grace, que percebe como o período colonial alterou os costumes de
seus antepassados. Através da apresentação do período colonial, podemos observar a descrição do
colonizador na visão do colonizado: No dia em que os homens brancos visitaram seu clã, Nwamgba largou o pote que estava
prestes a colocar no forno, pegou Anikwenwa e as meninas que eram suas aprendizes, e
correu para a praça. A princípio, ficou desapontada em ver a aparência ordinária dos dois
brancos, que pareciam indefesos e eram da cor de albinos, com pernas e braços frágeis
e delgados [...] (ADICHIE, 2017, p. 219). Nesse fragmento, podemos conhecer os aspectos físicos dos missionários europeus que
aparentemente são frágeis e delgados, no entanto o seu papel na colônia foi extremamente vigoroso. Vamos observar melhor como o colonizador foi se adentrando em terras alheias. Através da imposição de
costumes pela predisposição a não enxergar nada de “civilizado” no colonizado, primeiro imbecilizando-
os, depois usando de violência: Ayaju voltou de uma viagem em que fora trocar mercadorias com mais uma história:
as mulheres em Onicha estavam reclamando dos homens brancos. Elas tinham ficado
felizes quando eles construíram um posto de troca, mas agora os brancos estavam
querendo ensiná-las como fazer negócio e, quando os anciãos de Agueke, um clã de
Onicha, se recusaram a colocar os polegares num pedaço de papel, os homens brancos
vieram à noite com os homens normais que os ajudavam e arrasaram a aldeia. Não tinha
sobrado nada. (ADICHIE, 2017, p. 218). Memmi escreve que o
colonizador cria o retrato místico e degradante do colonizado e que é aceito por sua vez. “Ao aceitar essa
ideologia, as classes dominantes confirmam, de certa maneira, o papel que lhes foi atribuído” (MEMMI,
2007, p. 126) de difundir a sua cultura “superior”. A leitura do texto de Memmi (2007) nos ajudará a observar a mudança quanto ao entendimento do
papel do colonizador pelas personagens que representam o colonizado, a partir da passagem temporal
no conto A historiadora obstinada. A partir da forma como o colonizador é representado pelo olhar da
personagem Nwamgba, também é possível desmitificar a história oficial do colonizador. É interessante
destacar, no entanto, que só foi possível à personagem detectar a estrutura da colonização, porque ela Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 142 p-ISSN: 1807-1112
e-ISSN: 2448-1939 Como vislumbramos, as pessoas que viviam na região hoje denominada Nigéria eram organizadas,
possuíam comércio. E com a chegada dos colonizadores, estes não se dão por satisfeitos em apenas fazer
negócios, mas apresentam a intenção de impor suas regras, de modo que, quando encontram resistência
por parte das autoridades de Agueke, partem para a violência com outros grupos nativos. Dessa forma podemos observar o colonizador adentrando no espaço do colonizado, já que impor
suas regras comerciais não seria suficiente para estabelecer seu domínio imperial, era preciso de mais
estratégias políticas, e por isso também construíram tribunais: Semanas depois, Ayaju voltou com outra história: os homens brancos tinham construído
um tribunal em Onicha, onde julgavam disputas locais. Tinham vindo, de fato, para
ficar. Pela primeira vez, Nwamgba duvidou da amiga. Não era possível que o povo de
Onicha não tivesse seu próprio tribunal. O tribunal do clã vizinho ao de Nwamgba, por
exemplo, só fazia sessões durante o festival do inhame novo, de modo que o rancor das
pessoas crescia enquanto elas esperavam por justiça. Um sistema estúpido, na opinião
de Nwamgba, mas, sem dúvida, todos tinham o seu. [...] (ADICHIE, 2017, p. 219-220). A construção de um tribunal (aparelho ideológico do Estado) em terra estrangeira já nos revela que o
colonizador não estava de passagem: tinham vindo, de fato, para ficar. E assim como o colonizado possuía
comércio, eles tinham seus tribunais com suas normas. Vamos observar como o colonizador passou a
julgar em terra estrangeira: [...] Ayaju contou uma história sobre duas pessoas que levaram uma disputa de terra
ao tribunal dos homens brancos; o primeiro homem estava mentindo, mas sabia falar
a língua deles, enquanto o segundo, o verdadeiro dono das terras, não sabia falar a
língua e, por isso, perdeu e foi espancado, preso e obrigado a entregar a propriedade [...]. (ADICHIE, 2017, p. 220). Nesse momento, averiguamos que o colonizador não dita apenas suas regras, mas começa a impor
sua língua ao colonizado. E a linguagem mostra sua força e seu poder oprimindo aqueles que não possuem
seu domínio. Outras duas armas ideológicas que ajudaram a estabelecer o Império Colonial foram a Escola e a
Igreja. Contextualizando um pouco o enredo, observamos a história de Nwamgba, que se vê obrigada
a colocar seu único filho, a luz de seus olhos, para que fosse entregue para os homens brancos por três
motivos. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 143 p-ISSN: 1807-1112
e-ISSN: 2448-1939 Primeiro, lembrando que a personagem pertence à primeira geração, porque após a morte de
seu marido passou a sofrer pressão por parte dos primos do falecido, que ameaçavam tomar suas terras
e, por ser mulher, não conseguia apoio dos anciões do seu clã. Segundo, porque ela percebeu a injustiça feita nos tribunais dos homens brancos por falta de conhecimento da língua estrangeira. E terceiro, por
conta do tráfico de pessoas, que se intensificou com a presença do colonizador: [...] foi a história do menino Iroegbunam, que tinha desaparecido muitos anos atrás e
subitamente reapareceu, um homem adulto, que deixou a mãe viúva em choque ao
contar sua história: um vizinho, com quem seu pai muitas vezes discutia nas reuniões
da aldeia, o raptara quando sua mãe estava no mercado e o levara para os mercadores
de escravos de Aro, que o examinaram e reclamaram que a ferida em sua perna reduziria
seu preço. Então ele e alguns outros foram amarrados pelas mãos, formando uma longa
fila humana, e os homens bateram neles com um pedaço de pau e lhe mandaram andar
mais depressa (ADICHIE, 2017, p. 220-221). Nesse fragmento, podemos perceber que a rivalidade entre as pessoas e os grupos favorecia o tráfico
de escravos. E foi por conta desse medo que a personagem Nwamgba colocou seu filho na escola jesuítica. Primeiro ela foi à missão anglicana: [...] Os alunos ficavam sentados com ripas de madeira no colo, enquanto o professor ficava
de pé diante deles, segurando uma grande bengala, contando uma história sobre um
homem que transformou uma tigela de água em vinho. Nwamgba ficou impressionada
com os óculos do professor e imaginou que o homem da história devia ser um feiticeiro
bastante poderoso para conseguir transformar água em vinho. Mas, quando as meninas
foram separadas dos meninos e uma mulher veio ensiná-las a costurar, Nwamgba
achou uma tolice; em seu clã, as meninas aprendiam a fazer cerâmica e eram os homens
que costuravam tecido. O que a fez desistir completamente da escola, no entanto, foi o
fato de as aulas serem em igbo (ADICHIE, 2017, p. 222). A maneira com que Adichie reflete sobre o cristianismo possibilita a descrição do olhar do colonizado
perante a cultura do colonizador. Notamos também a relação dos papéis sociais desempenhados pelos
homens e pelas mulheres na cultura do colonizado e do colonizador. Diante do exposto, verificamos que
a missão anglicana procurou ensinar as crianças através da língua materna, o que possibilitava maior
rendimento na aquisição da religião cristã. Mas como Nwamgba não estava interessada na cultura e sim
no domino da língua inglesa, optou em colocar seu filho com os missionários católicos. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 144 p-ISSN: 1807-1112
e-ISSN: 2448-1939 E é, nesse momento, que a segunda geração vai se adentrar na cultura do colonizador. O primeiro
passo foi assumir um nome inglês para Anikwenwa, que passou a se chamar Michael. Um nome quase
impronunciável para a mãe, mas que para ela seu filho continuaria a se chamar Anikwenwa, Ani o que
significa deus da terra (ADICHIE, 2017). Alterar o nome significa negar a própria cultura para assumir
outra, e os dois nomes escolhidos fazem referência ao deus que cada denominação religiosa segue. O segundo passo foi assumir as vestimentas do colonizador. O padre Shanahan “deu ao menino uma
camisa e um par de calções, pois o povo de Deus não andava por aí nu” (ADICHIE, 2017, p. 223). No inicio,
Anikwenwa sempre tirava as roupas, “não gostava dos calções e da camisa que o faziam suar, do tecido
que dava coceira ao redor das axilas” (ADICHIE, 2017, p. 224). Mas essa postura com relação às roupas
foi mudando devagar, principalmente quando ele começou a perceber olhares de admiração por parte do
seu clã. E com isso se percebe o quanto a roupa é um elemento estabelecido culturalmente. Perante esse processo de mudança do filho, primeiramente Nwamgba ficou orgulhosa, especialmente
pelo fato de Anikwenwa estar utilizando língua inglesa para poder se defender. Contudo, logo seu orgulho
foi se exaurindo, pois, com o passar do tempo, ela notou que “a curiosidade dos olhos de Anikwenwa havia
diminuído. Havia uma nova gravidade nele, como se subitamente houvesse descoberto que era obrigado
a carregar um mundo pesado demais. Ele passava muito tempo olhando a mesma coisa” (ADICHIE, 2017,
p. 225). Com suas novas roupas, Anikwenwa começou a questionar as de sua mãe, comparando com as da
sua nova cultura: disse a Nwamgba que ela devia amarrar a canga ao redor do peito em vez de ao redor da
cintura, pois sua nudez era pecado. Ela olhou para Anikwenwa, achando graça daquela
seriedade, mas ainda assim preocupada, e perguntou por que ele só notara sua nudez
agora” (ADICHIE, 2017, p. 225). Nesse momento, temos uma fase de contra-identificação com a cultura local e de identificação com
a cultura do colonizador, a esse processo Pierre Bourdieu (1989) denomina de “violência simbólica”. Ela
consiste na violência exercida com a cumplicidade implícita daqueles que a sofrem (classe dominada), e,
também, frequentemente, daqueles que a exercem (classe dominante) na medida em que uns e outros
são inconscientes de a exercer ou a sofrer (BOURDIEU, 1989). Nesse discurso, o colonizador ganha força e
a visão que tem de si e de sua cultura altera a do colonizado. De acordo com Memmi, “[...] o esmagamento
do colonizado está compreendido nos valores colonizadores. Quando o colonizado adota esses valores,
adota também sua própria condenação. Para libertar-se, ao menos acredita nisso, ele aceita destruir-se. [...]” (MEMMI, 2007, p. 164). Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 145 p-ISSN: 1807-1112
e-ISSN: 2448-1939 que o processo de dominação da identidade do menino está quase completo. A maneira como ele rejeita
a comida faz com que percebamos como o colonizador descreve a crença do colonizado: que o processo de dominação da identidade do menino está quase completo. A maneira como ele rejeita
a comida faz com que percebamos como o colonizador descreve a crença do colonizado: Quando chegou a hora de sua cerimônia ima mmuo, Anikwenwa disse que não ia
participar, pois era um costume pagão iniciar os meninos no mundo dos espíritos, um
costume que o padre Shanahan dissera que deveria acabar. Nwamgba puxou a orelha
dele com força e disse-lhe que um albino estrangeiro não podia determinar quando seus
costumes iriam mudar, e assim, até que o próprio clã decidisse que a iniciação iria parar
de acontecer, ou ele participaria ou teria de escolher se era filho dela ou do homem
branco. [...] (p. 225). Pela transformação do personagem Anikwenwa, agora Michael, podemos observar a estratégia do
colonizador, que entrou pacificamente nas terras alheias, ensinando sua cultura para as crianças, mas que
já possuía o objetivo de acabar com a cultura do colonizado, para garantir sua dominação. A personagem
Nwamgba, que sempre esteve ciente desses acontecimentos, tinha, contudo, a esperança de que a sua
história não seria apagada por completo. O casamento também foi pensado com o intuito de perpetuar os ensinamentos do colonizador. Anikwenwa se casou com Mgbeke (nome antes de virar cristã). A cerimônia de casamento para Nwamgba
foi estranha e engraçada, mas ela “suportou-a em silêncio e disse a si mesma que logo ia morrer e se
unir a Obierika, libertando-se de um mundo que cada vez fazia menos sentido” (ADICHIE, 2017, p. 227). A
narrativa do conto, especialmente com essa citação, estabelece relação com a obra O Mundo Se Despedaça
(2019), do autor nigeriano Chinua Achebe, em que também encontramos essa história conflituosa da
constante ameaça do colonizador em apagar as histórias, os costumes e as vidas dos que passam a ser
visto agora simplesmente como colonizados. Com o casamento, Nwamgba acredita que, ao menos, com os seus netos conseguiria manter sua
tradição viva. Mgbeke, antes de receber o nome cristão Agnes, também sofreu vários abortos até dar à
luz a um menino. O padre o batizou com o nome de Peter, mas Nwamgba o chamava de Nnamdi, pois
acreditava que o espírito de Obierika tinha voltado. 1 Contextualizando o conto trata de uma cultura patriarcal, em que as vozes das mulheres não são ouvidas, muito menos res-
peitadas. E nessa passagem Nwamgba esperava um homem para continuar sua história, ter atitude, tomar decisões e enfrentar
o colonizador. No entanto, por mais que tentasse, não sentia o
espírito de seu marido. Até que depois de três abortos, Mgbeke engravidou de uma menina. Nwamgba
achou engraçado o espírito de seu marido vir em forma de uma garota1. O padre batizou-a com o nome Como grande parte do seu tempo foi dedicado a escola inglesa, o menino não mais participava
das atividades e das festas do seu povo, o que contribuiu para gerar o terceiro passo no processo de
colonização, quando Anikwenwa passou a rejeitar o alimento de seus conterrâneos: “parou de comer a
comida da mãe porque, segundo dizia, ela era um sacrifício a falsos ídolos” (ADICHIE, 2017, p. 225). Como
a comida está diretamente ligada à religião, a negação da alimentação por uma questão religiosa significa Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021
146 Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 146 p-ISSN: 1807-1112
e-ISSN: 2448-1939 de Grace, embora Nwamgba a chamasse de Afamefuna, que significa “meu nome não se perderá”. Essa
é a terceira geração. Grace/Afamefuma recebeu a mesma educação do pai, mas seus olhos estavam voltados para as
histórias e poesias de sua avó. Ela foi uma das poucas mulheres que frequentou a University College de
Ibadan e largou Química para fazer História, quando um professor especialista em História do Império
Britânico foi expulso por querer colocar no currículo História Africana. Mais tarde, ela decide mudar seu
nome Grace para Afamefuna. É através dessa jovem que, ao mudar seu nome, assumindo a identidade do seu povo de origem,
que a cultura e a história de sua avó continuarão vivas, mesmo que mescladas à cultura e à história do
colonizador. Grace é a históriadora obstinada que dá nome ao conto. Nos últimos parágrafos do conto, aparece onzes vezes a expressão “foi Grace quem”, para destacar
a importância da tomada de consciência da personagem, bem como para demonstrar quais foram os
elementos que mais sofreram com a colonização: tudo o que Grace percebe e faz: Nos últimos parágrafos do conto, aparece onzes vezes a expressão “foi Grace quem”, para destacar
a importância da tomada de consciência da personagem, bem como para demonstrar quais foram os
elementos que mais sofreram com a colonização: tudo o que Grace percebe e faz: [...] Foi Grace quem começou a repensar tudo o que havia aprendido [...]. Foi Grace
quem começou a repensar tudo o que seu pai havia aprendido [...]. Foi Grace quem, [...],
começaria a ser assombrada pela imagem de uma aldeia destruída, e iria a Londres,
Paris e Onicha, folheando pastas emboloradas em arquivos e reinventando as vidas e
os cheiros do mundo de sua avó para o livro que escreveria, intitulado Pacificando com
balas: uma história recuperada do sul da Nigéria. [...] Foi Grace quem, quando recebia
prêmios da universidade, quando discursava para plateias solenes em conferências
sobre os povos ijaw, ibibio, igbo e efik do sul da Nigéria, quando escrevia relatórios para
organizações internacionais sobre coisas que deviam ser óbvias para qualquer um que
tivesse bom senso, mas pelas quais, mesmo assim, ela recebia remunerações generosas,
imaginava sua avó observando tudo e rindo, muito divertida. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 147 p-ISSN: 1807-1112
e-ISSN: 2448-1939 Foi Grace quem, cercada
por seus prêmios, seus amigos, seu jardim de rosas inigualáveis, mas sentindo-se, sem
saber explicar bem por que, distante de suas raízes no fim da vida, foi a um cartório em
Lagos mudar oficialmente seu primeiro nome de Grace para Afamefuna (p. 230-233). Pela leitura do conto A historiadora obstinada, é possível perceber a relação de autoconsciência destacada
por Hutcheon (1991), quanto à relação entre história e literatura, pois observamos o entrelaçamento
da história da colonização da Nigéria com a leitura do conto, bem como a postura de Chartier, para o
qual notamos a presença de elementos culturais do povo igbo antes da colonização. Também é possível
perceber a diferença no processo de identidade das personagens, já que Nwamgba representa a primeira
geração com traços da identidade sociológica, Anikwenwa representa a segunda geração com traço da
identidade pós-moderna (contraditória), e Afamefuna representa a terceira geração com a identidade pós-moderna (conflituosa). Tal mudança no processo de identificação das personagens tem a ver com o
processo colonial que é relatado no conto, que coincide com o estudado por Memmi, pois observamos a
mudança quanto ao entendimento do papel do colonizador pelas personagens Nwamgba e Afamefuna,
possibilitando a desmistificação da história oficial do colonizador. Por fim, é possível estabelecer relação entre o conto e a fala “O perigo de uma história única” de Adichie,
que usamos para iniciar este trabalho. No conto, acontece a imposição dos costumes dos colonizadores,
e tal situação só é alterada com o processo de tomada de consciência de Grace/Afamefuna, que, ao
buscar maior conhecimento de sua história, percebe que sua história estava sendo suprimida. E com essa
tomada de consciência ela (escritora/personagem) começa a escrever e divulgar o seu conhecimento. 4 CONCLUSÕES Através da leitura do conto A historiadora obstinada, da escritora Chimamanda Ngozi Adichie,
conseguimos perceber a representação do choque cultural entre o colonizador e o colonizado, bem como
a forma como a identidade cultural africana é alterada, até que haja uma contemporânea desmistificação
da história oficial do colonizador pelo olhar da personagem Grace. Conseguimos traçar e vislumbrar as três gerações que parecem no conto: a primeira que consiste na
avó Nwamgba, que carrega consigo a história e a cultura do seu povo e se mostra irresoluta ao negar
a cultura do colonizador. A segunda consiste na transformação de Anikwenwa em Michael, que ao se
inserir na cultura do colonizar, acaba por abraçar essa nova forma de ver o mundo, negando sua própria
identidade original. E, por fim, a terceira geração consiste na história de Grace, que toma consciência
de seu lugar no mundo e se assume como Afamefuna, por ter se dado conta do papel prejudicial da
colonização para com sua cultura e tradição. Trabalhando com história e ficção, entendemos a importância do conhecimento da nossa história
para desconstruir e problematizar discursos que se mostram prontos e acabados na nossa sociedade. Contudo, essa é uma tarefa para alguém obstinado, tal como Nwamgba e Grace, para quem os benefícios
que advém da colonização não podem minimizar o valor da cultura ancestral de seu povo. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 148 p-ISSN: 1807-1112
e-ISSN: 2448-1939 CHARTIER, Roger. A História Cultural entre Práticas e Representações. Lisboa: Difusão Editora, 1990. HALL, Stuart. A identidade cultural na pós-modernidade. Rio de Janeiro: DP&A, 2006. HALL, Stuart. A identidade cultural na pós-modernidade. Rio de Janeiro: DP&A, 2006. HUTCHEON, Linda. Poética do pós-modernismo: história, teoria, ficção. Rio de Janeiro: Imago, 1991. HUTCHEON, Linda. Poética do pós-modernismo: história, teoria, ficção. Rio de Janeiro: Imago, 1991. MEMMI, Albert. Retrato do colonizado precedido de Retrato do colonizador. Rio de Janeiro: Civilização
Brasileira, 2007. MEMMI, Albert. Retrato do colonizado precedido de Retrato do colonizador. Rio de Janeiro: Civilização
Brasileira, 2007. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 149 p-ISSN: 1807-1112
e-ISSN: 2448-1939 . No seu pescoço. São Paulo: ADICHIE, Chimamanda Ngozi. A historiadora obstinada. In: ______. No seu pescoço. São Paulo:
Companhia das Letras, 2017. p. 212-233. ______. “O perigo de uma única história”. Disponível em: http://www.ted.com/talks/lang/por _br/
chimamanda_adichie_the_danger_of_a_single_story.html. Acesso em: 03 maio 2019. ______. “O perigo de uma única história”. Disponível em: http://www.ted.com/talks/lang/por _br/
chimamanda_adichie_the_danger_of_a_single_story.html. Acesso em: 03 maio 2019. BOURDIEU, Pierre. O poder simbólico. Lisboa: Difel, 1989. EU, Pierre. O poder simbólico. Lisboa: Difel, 1989. CHARTIER, Roger. A História Cultural entre Práticas e Representações. Lisboa: Difusão Editora, 1990. REFERÊNCIAS ACHEBE, Chinua. O mundo se despedaça. Tradução: Verra Queiroz da Costa e Silva. São Paulo: Editora
Companhia das Letras, 2009 [1958]. ACHEBE, Chinua. O mundo se despedaça. Tradução: Verra Queiroz da Costa e Silva. São Paulo: Editora
Companhia das Letras, 2009 [1958]. ADICHIE, Chimamanda Ngozi. A historiadora obstinada. In: ______. No seu pescoço. São Paulo:
Companhia das Letras, 2017. p. 212-233. Revista Prâksis | Novo Hamburgo | a. 18 | n. 1 | jan./abr. 2021 150
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Everolimus-Eluting Bioresorbable Scaffolds and Metallic Stents in Diabetic Patients: a Patient-level Pooled Analysis of the Prospective ABSORB DM Benelux Study, TWENTE and DUTCH PEERS
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Hommels et al. Cardiovasc Diabetol (2020) 19:165
https://doi.org/10.1186/s12933-020-01116-2 Hommels et al. Cardiovasc Diabetol (2020) 19:165
https://doi.org/10.1186/s12933-020-01116-2 Cardiovascular Diabetology Abstract Background: Several studies compared everolimus-eluting bioresorbable scaffolds (EE-BRS) with everolimus-eluting
stents (EES), but only few assessed these devices in patients with diabetes mellitus. Aim: To evaluate the safety and efficacy outcomes of all-comer patients with diabetes mellitus up to 2 years after
treatment with EE-BRS or EES. Aim: To evaluate the safety and efficacy outcomes of all-comer patients with diabetes mellitus up to 2 years after
treatment with EE-BRS or EES. Methods: We performed a post hoc pooled analysis of patient-level data in diabetic patients who were treated
with EE-BRS or EES in 3 prospective clinical trials: The ABSORB DM Benelux Study (NTR5447), TWENTE (NTR1256/
NCT01066650) and DUTCH PEERS (NTR2413/NCT01331707). Primary endpoint of the analysis was target lesion failure
(TLF): a composite of cardiac death, target vessel myocardial infarction or clinically driven target lesion revasculariza-
tion. Secondary endpoints included major adverse cardiac events (MACE): a composite of all-cause death, any myo-
cardial infarction or clinically driven target vessel revascularization, as well as definite or probable device thrombosis
(ST). Results: A total of 499 diabetic patients were assessed, of whom 150 received EE-BRS and 249 received EES. Total
available follow-up was 222.6 patient years (PY) in the EE-BRS and 464.9 PY in the EES group. The adverse events rates
were similar in both treatment groups for TLF (7.2 vs. 5.2 events per 100 PY, p = 0.39; adjusted hazard ratio (HR) = 1.48
(95% confidence interval (CI): 0.77–2.87), p = 0.24), MACE (9.1 vs. 8.3 per 100 PY, p = 0.83; adjusted HR = 1.23 (95% CI:
0.70–2.17), p = 0.47), and ST (0.9 vs. 0.6 per 100 PY, p > 0.99). Conclusion: In this patient-level pooled analysis of patients with diabetes mellitus from 3 clinical trials, EE-BRS
showed clinical outcomes that were quite similar to EES. Keywords: Bioresorbable scaffolds, Drug-eluting stents, Diabetes mellitus, Coronary artery disease, Percutaneous
coronary intervention, Device thrombosis *Correspondence: ekedhi@me.com p
5 Department of Cardiology, Hôpital Erasme Université Libre de Bruxelles,
Brussels, Belgium B usse s, Be g u
Full list of author information is available at the end of the article g
Full list of author information is available at the end of the article © The Author(s) 2020. 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://creativeco
mmons.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. Background Diabetes mellitus is a well-established predictor of
adverse clinical and angiographic events following per-
cutaneous coronary intervention (PCI) with metallic *Correspondence: ekedhi@me.com Percutaneous coronary intervention procedure Implanted EE-BRS devices were the bioresorbable poly-
mer drug-eluting scaffold ABSORB BVS system and the
ABSORB GT1 system (Abbott Vascular, Santa Clara, CA,
USA). Both devices are composed of poly-L-lactic acid
with a strut thickness of 150 µm, covered by a polymer
coating of poly-DL-lactic-acid that elutes everolimus. EE-BRS are expected to be completely resorbed within
3 years [12]. The available scaffold diameters ranged from
2.50 to 3.50 mm with lengths of 8–28 mm. The durable
fluoropolymer-coated Xience V EES (Abbott Vascular,
Santa Clara, CA, USA) is a cobalt-chromium EES with
a strut thickness of 81 µm; it was available in diameters
from 2.25 to 4.00 mm with lengths of 8 to 28 mm. The
strut thickness, polymer coating, eluted drug and avail-
able stent diameters of the platinum-chromium-based
Promus Element EES (Boston Scientific, Natick, MA,
USA) were the same as for Xience V EES; the available
stent length ranged from of 8 to 38 mm.i The everolimus-eluting bioresorbable scaffolds (EE-
BRS) were developed to overcome some shortcomings
of the metallic DES [11, 12]. Because of its resorbable
nature and its transient presence after implantation, it
was hypothesized that EE-BRS may be associated with a
more favorable restoration of the treated coronary ves-
sel and a more favorable long-term clinical outcome. In addition, the use of EE-BRS may allow for repeating
PCI in the same target lesion without loss of lumen size
due to the lifelong presence of multiple layers of metal-
lic stents in the coronary vessel. Furthermore, it is likely
to facilitate coronary artery bypass grafting (CABG) that
may be required at a later point in time. Consequently,
patients with diabetes mellitus, who are known to have
an increased risk of repeated PCI and finally surgical
treatment with CABG, may show a particular benefit
from PCI with the utilization of EE-BRS. Angiographic success was defined as a < 50% resid-
ual stenosis of the target lesion after successful device
implantation, as visually assessed. Procedural success was
defined as angiographic success without occurrence of
any adverse cardiac event during index hospitalization. In
general, dual antiplatelet therapy (DAPT) was prescribed
for 12 months after PCI, reflecting contemporary inter-
national guideline recommendations. Nevertheless, data from studies that compare EE-BRS
and DES in an all-diabetic patient population are scarce. © The Author(s) 2020. 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://creativeco
mmons.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. Hommels et al. Cardiovasc Diabetol (2020) 19:165 Page 2 of 10 zotarolimus-eluting stents in an all-comer population. All
available follow-up data up to 2 years after PCI was used. drug-eluting stents (DES) for obstructive coronary artery
disease (CAD) [1–6]. Various disease-related factors con-
tribute to a pro-inflammatory and pro-thrombotic state
that reduce the prognosis of patients with diabetes mel-
litus, such as endothelial dysfunction, changes in plaque
composition, platelet activation and coagulation distur-
bances [7]. Following PCI with stent implantation, the
aforementioned mechanisms may be even aggravated by
the permanent presence of the metallic vascular pros-
thesis that mechanically distorts the arterial geometry,
delays vascular healing and constrains vascular response
in the treated coronary segment [8–10]. Study population For the purpose of this analysis we pooled data of all
patients with diabetes mellitus, who were treated with
EE-BRS in de novo coronary lesions in the prospective
ABSORB DM Benelux Study or who underwent PCI
with EES in de novo coronary lesions in the TWENTE
and DUTCH PEERS trials. The design and outcomes
of the individual studies have been reported previously
[13–16]. The ABSORB DM Benelux Study is a prospec-
tive, international observational study in all-comer
patients with diabetes mellitus who underwent PCI with
EE-BRS. The TWENTE study is a Dutch randomized
trial that examined the performance of second-gener-
ation EES to zotarolimus-eluting stents in all-comers. DUTCH PEERS is a Dutch multicenter randomized trial
that assessed the performance of new-generation EES to Percutaneous coronary intervention procedure For that reason, we pooled patient-level data from 3 pro-
spective clinical trials to compare the clinical outcome
after PCI with EE-BRS and everolimus-eluting stents
(EES) in all-comers with diabetes mellitus. Study endpoints
Th
i
d The primary device-oriented endpoint of target lesion
failure (TLF) is a composite of cardiac death, target vessel
myocardial infarction (TV-MI) or clinically driven tar-
get lesion revascularization (TLR). Secondary endpoints
include the patient-oriented endpoint of major adverse
cardiac events (MACE), which is defined as a compos-
ite of all-cause death, any myocardial infarction (MI) or
clinically driven target vessel revascularization (TVR), as
well as definite or probable device (i.e., scaffold or stent)
thrombosis (ST). All endpoints were defined according to
clinical data standards of the American College of Car-
diology/American Heart Association and the Academic
Research Consortium [17–20]. Statistical analysis P-values < 0.05
were considered statistically significant. Statistical analy-
ses were performed using SPSS version 25 (IBM Corp.,
Armonk, NY, USA). Baseline clinical characteristics of both patient groups
are shown in Table 1. Patients treated with EE-BRS were
younger and had less frequently a family history of cardi-
ovascular disease. Other baseline characteristics, includ-
ing previous ischemic heart disease, the prescription of
insulin and the clinical presentation at the time of the
index PCI did mostly not differ between the groups. The
incidence of chronic kidney failure (serum creatinine lev-
els of ≥ 130 µmol per liter), as determinant risk factor for
cardiovascular disease, was limited and also did not differ
between both study populations. The angiographic characteristics are presented in
Table 2. There was no significant difference in the dis-
tribution of the target vessels, except for left main stem
treatment which was only performed in a few EES-
treated patients. In the EE-BRS group, a few patients with
CABG treatment were included. Although the number of
target lesions per patient was similar in both groups, the
EES group had a greater number of treated vessels, which
resulted in a longer total device length. In addition, the
EES group underwent more often bifurcation treatment. Nevertheless, both treatment groups showed similar
angiographic and procedural results. Statistical analysis Categorical variables are presented as frequencies and
percentages. A 2-sided Pearson’s Chi square test was per-
formed to determine significant differences between the
EE-BRS and EES groups. The Fisher’s exact test was used if
appropriate due to small sample sizes with low frequency
variables. Continuous variables with normal distribution Hommels et al. Cardiovasc Diabetol (2020) 19:165 Hommels et al. Cardiovasc Diabetol (2020) 19:165 Page 3 of 10 Page 3 of 10 are presented as mean and standard deviation, and a
2-sided independent student t-test was used to compare
groups. An equal variance was upheld unless a significant
Levene’s test had been calculated; in that case a t-test with
unequal variances was conducted. The clinical outcomes
of the composite endpoints TLF and MACE were obtained
by means of Kaplan–Meier survival methods with time-to-
event analysis in addition to a log-rank test at 2-year fol-
low-up. To adjust for variable time to follow-up between
trials, all endpoints and the composites of TLF and MACE
are presented in event rates per 100 patient years (PY) with
the Poisson distribution given with 95% confidence inter-
val (CI). In addition, a multivariate Cox regression model
with adjustment for age, insulin-treated diabetes melli-
tus, number of treated vessels, total treated vessel length
and treatment group was built for both TLF and MACE,
reported as hazard ratio (HR) and 95% CI. P-values < 0.05
were considered statistically significant. Statistical analy-
ses were performed using SPSS version 25 (IBM Corp.,
Armonk, NY, USA). are presented as mean and standard deviation, and a
2-sided independent student t-test was used to compare
groups. An equal variance was upheld unless a significant
Levene’s test had been calculated; in that case a t-test with
unequal variances was conducted. The clinical outcomes
of the composite endpoints TLF and MACE were obtained
by means of Kaplan–Meier survival methods with time-to-
event analysis in addition to a log-rank test at 2-year fol-
low-up. To adjust for variable time to follow-up between
trials, all endpoints and the composites of TLF and MACE
are presented in event rates per 100 patient years (PY) with
the Poisson distribution given with 95% confidence inter-
val (CI). In addition, a multivariate Cox regression model
with adjustment for age, insulin-treated diabetes melli-
tus, number of treated vessels, total treated vessel length
and treatment group was built for both TLF and MACE,
reported as hazard ratio (HR) and 95% CI. EE-BRS everolimus-eluting bioresorbable scaffolds, EES everolimus-eluting stents, PCI percutaneous coronary intervention, CABG coronary artery bypass grafting
a Plus–minus values are mean ± standard deviation (SD) and the number in italica represent the known total of which the variable was calculated
b Renal insufficiency was defined as serum creatinine level of ≥ 130 µmol per liter. A p-value < 0.05 was considered as statistically significant Shown are the clinical characteristics at baseline for both patient groups EE-BRS everolimus-eluting bioresorbable scaffolds, EES everolimus-eluting stents, PCI percutaneous coronary intervention, CABG coronary artery bypass grafting
a Plus minus values are mean±standard deviation (SD) and the number in italica represent the known total of which the variable was calculated acteristics at baseline for both patient groups Results Patient-level data were available from 499 diabetic
patients; 150 patients (188 target lesions) were treated
with EE-BRS and 249 (336 target lesions) with EES. Follow-up was available for all but 3 patients who were
lost to follow-up. Total available follow-up was 222.6 PY Follow-up was available for all but 3 patients who were
lost to follow-up. Total available follow-up was 222.6 PY Table 1 Clinical characteristics of the patients at baseline
Shown are the clinical characteristics at baseline for both patient groups
Baseline clinical characteristic
EE-BRS (n = 150)
EES (n = 249)
p-value
Age (years)–mean ± SD
64.3 ± 10.1
67.1 ± 10.2
<0.01
Sex (male)–no.(%)
108 (72.0)
159 (63.9)
0.09
Body-mass index (kg/m2)–mean ± SD; no. 29.5 ± 5.1, 148a
29.3 ± 4.8, 221a
0.63
Risk factors–no.(%)
Insulin-treated diabetes mellitus
52 (34.7)
163 (34.5)
0.98
Arterial hypertension
104 (69.3)
176 (70.7)
0.78
Hypercholesterolemia
100 (66.7)
157 (63.1)
0.47
Family history of cardiovascular disease
59 (39.3)
131 (52.6)
0.01
Current smoker
35 (23.3)
50 (20.1)
0.44
Medical history–no.(%)
Previous acute coronary syndrome
28 (18.7)
59 (23.7)
0.24
Previous PCI
37 (24.7)
54 (21.7)
0.49
Previous CABG
8 (5.3)
24 (9.6)
0.13
Chronic renal failureb
6 (4.0)
12 (4.8)
0.70
Clinical presentation–no.(%)
Acute coronary syndrome
73 (48.7)
120 (48.2)
0.93
Myocardial infarction
47 (31.3)
74 (29.7)
0.73
Unstable angina pectoris
26 (17.3)
46 (18.5)
0.77
Non-acute coronary syndrome
77 (51.3)
129 (51.8)
0.93 Table 1 Clinical characteristics of the patients at baseline Shown are the clinical characteristics at baseline for both patient groups Hommels et al. Cardiovasc Diabetol (2020) 19:165 Page 4 of 10 Table 2 Angiographic characteristics of the patients at baseline
Shown are the angiographic characteristics for both patient groups
a Plus–minus values are mean ± standard deviation (SD) and the numbers in italica represent the known total of which the variable was calculated
b L
th f l
i
d
i
d b ll
d i
illi
t
(
)
th di
t
f th d
i
Baseline angiographic characteristic
EE-BRS
EES
P-value
Patient-level analysis
Number of patients
150
249
Number of treated vessels–mean ± SD
1.1 ± 0.3
1.2 ± 0.4
<0.01
Number of treated target lesions–mean ± SD
1.3 ± 0.5
1.3 ± 0.6
0.22
Total number of devices–mean ± SD
1.4 ± 0.7
1.8 ± 1.1
<0.01
Lesion-level analysis
Number of lesions
188
336
Coronary artery lesion distribution–no. Results A p-value < 0.05 was considered as statistically significant; no significant differences were found between
both treatment groups at follow-up arget lesion failure was defined as a composite of cardiac death, target vessel myocardial infarction and clinically driven target lesion reva Major adverse cardiac events were defined as a composite of all-cause death, any myocardial infarction and clinically driven target vessel re ji
p
,
y
y
y
g
c Device thrombosis was defined as early if observed between 0 and 30 days after index procedure, including a further distinction between acute ≤ 24 h and
subacute > 1–30 days. Device thrombosis was defined as late if ≤ 1 year and as very late if > 1 year
EE-BRS everolimus-eluting bioresorbable scaffolds, EES everolimus-eluting stents, PY patient years, KM Kaplan–Meier, CI confidence interval, TLF target lesion
f il
TV MI t
t
l
di l i f
ti
TLR t
t l
i
l
i
ti
MACE
j
d
di
t
MI
di l i f
ti
TVR t
t
l c Device thrombosis was defined as early if observed between 0 and 30 days after index procedure, including a further distinction between acute ≤ 24 h and
subacute > 1–30 days. Device thrombosis was defined as late if ≤ 1 year and as very late if > 1 year
EE-BRS everolimus-eluting bioresorbable scaffolds, EES everolimus-eluting stents, PY patient years, KM Kaplan–Meier, CI confidence interval, TLF target lesion
failure, TV-MI target vessel myocardial infarction, TLR target lesion revascularization, MACE major adverse cardiac events, MI myocardial infarction, TVR target vessel
l
i
ti
ST d
i
th
b
i EE-BRS everolimus-eluting bioresorbable scaffolds, EES everolimus-eluting stents, PY patient years, KM Kaplan–Meier, CI confidence interval, TLF target lesion
failure, TV-MI target vessel myocardial infarction, TLR target lesion revascularization, MACE major adverse cardiac events, MI myocardial infarction, TVR target vessel
revascularization, ST device thrombosis Comparing EE‑BRS to EES in a wider perspective Comparing EE‑BRS to EES in a wider perspective
Despite advances in interventional therapies and the
implementation of new-generation DES, diabetic patients
still have worse angiographic and clinical outcomes
compared to nondiabetic patients undergoing PCI [21]. Nevertheless, as shown in the EXCEL trial, the relative
30-day and 3-year outcomes of PCI with EES compared
to CABG were consistent in diabetic and nondiabetic
patients with left main disease with low or intermediate
SYNTAX score [22]. Other factors like renal failure and
hemodialysis as well as in-stent restenosis, both occur-
ring more frequently in diabetic patients, might influence
safety outcomes in this group [23]. Results Cardiovasc Diabetol (2020) 19:165 Page 5 of 10 Table 3 Safety and efficacy outcomes at follow-up
Shown are the clinical outcomes represented as endpoints and clinical events. Three patients were lost to follow-up in the everolimus-eluting bioresorbable scaffolds
group. The results are presented by 2-year Kaplan–Meier estimates and are also reported in event rates per 100 patient years with 95% confidence intervals to adjust
for the variable time to follow-up between both groups. A p-value<0.05 was considered as statistically significant; no significant differences were found between
Endpoints and clinical
events
EE-BRS (n = 147)
EES (n = 249)
P-value log
rank
P-value PY
event rate
% (n) KM
100 PY (95% CI)
% (n) KM
100 PY (95% CI)
TLFa
11.7 [16]
7.2 (4.1–11.7)
9.7 [24]
5.2 (3.3–7.7)
0.40
0.39
Cardiac death
3.4 [5]
2.1 (0.7–5.0)
4.4 [11]
2.3 (1.1–4.1)
0.84
>0.99
TV-MI
3.6 [5]
2.2 (0.7–5.1)
2.8 [7]
1.5 (0.6–3.1)
0.69
0.69
TLR
5.5 [7]
3.1 (1.2–6.3)
3.3 [8]
1.7 (0.7–3.3)
0.23
0.36
MACEb
15.2 [20]
9.1 (5.6–14.1)
15.3 [38]
8.3 (5.9–11.4)
0.75
0.83
All-cause death
3.4 [5]
2.1 (0.7–5.0)
6.8 [17]
3.5 (2.1–5.6)
0.35
0.45
Any MI
4.9 [7]
3.1 (1.3–6.4)
3.2 [8]
1.7 (0.7–3.3)
0.40
0.36
TVR
9.3 [11]
4.9 (2.5–8.8)
6.6 [16]
3.4 (2.0–5.5)
0.29
0.46
STc
1.4 [2]
0.9 (0.1–3.1)
1.2 [3]
0.6 (0.1–1.8)
0.90
>0.99
Early
[2]
[2]
Acute
(0)
[2]
Subacute
[2]
(0)
Late
(0)
[1]
Very late
(0)
(0)
Definite
[1]
[1]
Probable
[1]
[2] Table 3 Safety and efficacy outcomes at follow-up Shown are the clinical outcomes represented as endpoints and clinical events. Three patients were lost to follow-up in the everolimus-eluting bioresorbable scaffolds
group. The results are presented by 2-year Kaplan–Meier estimates and are also reported in event rates per 100 patient years with 95% confidence intervals to adjust
for the variable time to follow-up between both groups. A p-value < 0.05 was considered as statistically significant; no significant differences were found between
both treatment groups at follow-up Shown are the clinical outcomes represented as endpoints and clinical events. Three patients were lost to follow-up in the everolimus-eluting bioresorbable scaffolds
group. The results are presented by 2-year Kaplan–Meier estimates and are also reported in event rates per 100 patient years with 95% confidence intervals to adjust
for the variable time to follow-up between both groups. Results (%)
Right coronary artery
57 (30.3)
108 (32.1)
0.67
Left anterior descending artery
89 (47.6)
144 (42.9)
0.42
Circumflex artery
40 (21.3)
82 (24.4)
0.30
Left main
0 (0)
10 (3.0)
0.02
Arterial or venous graft
2 (1.1)
0
0.13
Coronary artery lesion characteristics
Bifurcation or trifurcation–no. (%)
27 (14.4)
77 (22.9)
0.02
Device-level analysis
Number of devices
214
454
Diameter device–mean ± SDb
3.0 ± 0.4
2.9 ± 0.5
<0.01
Highest device inflation–mean ± SD; no.c
14.3 ± 2.6, 211a
15.4 ± 4.3, 446a
<0.01
Total length of devices per lesion–mean ± SDb
23.7 ± 11.7
27.0 ± 15.4
<0.01
Procedure-level analysis
Number of PCI procedures
188
336
Results–no. (%)
Post-procedural TIMI grade 3
186 (100), 186a
334 (99.4)
0.54
Angiographic success
185 (100), 185a
334 (99.4)
0.54
Procedural success
184 (97.9)
327 (97.3)
0.70
Peri-implantation procedures
Predilatation–no. (%)
177 (94.1)
227 (67.6)
<0.01
Predilatation balloon size–mean ± SD; no.b
2.8 ± 0.8, 176*
2.4 ± 0.5
<0.01
Predilatation pressure–mean ± SD; no.c
14.8 ± 4.0, 174a
14.5 ± 4.5
0.51
Postdilatation–no. (%)
142 (75.5)
273 (81.3)
0.12
Postdilatation balloon size–mean ± SDb
3.2 ± 0.5
3.1 ± 0.5
0.34
Postdilatation pressure–mean ± SDc
17.3 ± 4.3
22.5 ± 5.5
<0.01 Table 2 Angiographic characteristics of the patients at baseline p
y
gi
EE-BRS everolimus-eluting bioresorbable scaffolds, EES everolimus-eluting stents, PCI percutaneous coronary intervention, TIMI Thrombolysis in Myocardial Infarction
with grade 3 referenced as completely restored flow for the EE-BRS and 464.9 PY for the EES arm. Clinical
outcomes are reported in Table 3 and Figs. 1 and 2. In
all clinical endpoints, there was no significant difference
between EE-BRS and EES. TLF event rates were 7.2 vs. 5.2 events per 100 PY (p = 0.39) (adjusted HR 1.48, 95%
CI 0.77–2.87; p = 0.24). The rates of MACE were 9.1 vs. 8.3 events per 100 PY (p = 0.83) (adjusted HR 1.23, 95%
CI: 0.70–2.17; p = 0.47). No significant difference was observed in any individual component of these composite
endpoints. ST rates were 0.9 vs. 0.6 per 100 PY (p > 0.99). Table 4 present the results of the multivariate Cox regres-
sion model for TLF and MACE. After correction for age,
insulin-treated diabetes mellitus, multiple vessel treat-
ment and total treated length, we found again no signifi-
cant difference between EE-BRS and EES. Nevertheless,
multivariable analysis revealed borderline non-significant Hommels et al. Fig. 1 Shown are the Kaplan–Meier curves with 95% confidence intervals in function of a target lesion failure free survival and b major adverse
cardiac events free survival at 2-year follow-up. A log-rank test did not prove significant differences between both treatment groups. In a Cox
regression model with adjustment for age, insulin-treated diabetes mellitus, number of treated vessels and total treated length, treatment with
everolimus-eluting bioresorbable scaffolds could not be identified as significant risk factor for both target lesion failure and major adverse cardiac
events. TLF target lesion failure, EE-BRS everolimus-eluting bioresorbable scaffolds, EES everolimus-eluting stents, HR hazard ratio, CI confidence
interval, MACE major adverse cardiac events
(See figure on next page.) relations between insulin-treatment and TLF (p = 0.06)
and between patient age and MACE (p = 0.06). relations between insulin-treatment and TLF (p = 0.06)
and between patient age and MACE (p = 0.06). Discussionh This analysis of pooled patient-level data, compar-
ing EE-BRS with EES for treatment of CAD in patients
with diabetes mellitus, showed similar event rates in
both treatment groups for the device-oriented and the
patient-oriented composite endpoint. Importantly, safety
outcomes were also similar, including cardiac death and
TV-MI. A multivariable analysis for the composite clini-
cal endpoints TLF and MACE confirmed the absence of a
significant difference between both groups. Fig. 1 Shown are the Kaplan–Meier curves with 95% confidence intervals in function of a target lesion failure free survival and b major adverse
cardiac events free survival at 2-year follow-up. A log-rank test did not prove significant differences between both treatment groups. In a Cox
regression model with adjustment for age, insulin-treated diabetes mellitus, number of treated vessels and total treated length, treatment with
everolimus-eluting bioresorbable scaffolds could not be identified as significant risk factor for both target lesion failure and major adverse cardiac
events. TLF target lesion failure, EE-BRS everolimus-eluting bioresorbable scaffolds, EES everolimus-eluting stents, HR hazard ratio, CI confidence
interval, MACE major adverse cardiac events
(i
g
p g ) Hommels et al. Cardiovasc Diabetol (2020) 19:165 Page 6 of 10 Hommels et al. Cardiovasc Diabetol (2020) 19:165 Page 7 of 10 Fig. 2 Shown are the event rates per 100 patient years for the
composite endpoints a target lesion failure and b major adverse
cardiac events for both treatment groups (red and blue bars). In
addition, the 95% confidence intervals are also presented (red and
blue brackets). No significant differences were found between
both treatment groups. TLF target lesion failure, CI confidence
interval, EE-BRS everolimus-eluting bioresorbable scaffolds, EES
everolimus-eluting stents, MACE major adverse cardiac events bioresorbable devices and a modified duration of DAPT
(to correspond with scaffold resorption). Yet, it should
be considered that in these previous trials operator
experience with EE-BRS implantation was limited and
that there was no requirement to follow a formal EE-
BRS implantation protocol such as the pre-dilatation,
sizing, and post-dilatation (PSP) approach, which in
other studies improved safety outcomes [33–35]. Inter-
estingly, a 3-year landmark analysis of the ABSORB
trials showed a significant improvement in composite
safety outcomes beyond the resorption of the EE-BRS
[36, 37]. Nevertheless, previous trials did not focus exclusively
on diabetic patients. Patients with diabetes mellitus rep-
resent a high-risk population that theoretically might
show particular benefit from being treated with biore-
sorbable devices. Discussionh This is because such devices ‘disappear’
over time, which allows for repeated PCI procedures in
the same coronary segment without resulting in multi-
ple metallic mesh layers. Furthermore, the EE-BRS does
not cause an incessant stimulation of the diabetes-related
vascular inflammation, which may be the case in the per-
manent presence of certain durable polymers. Therefore,
it is conceivable that after PCI with EE-BRS long-term
results might be more favorable in patients with diabetes
mellitus (despite short and medium-term results similar
to EES). Fig. 2 Shown are the event rates per 100 patient years for the
composite endpoints a target lesion failure and b major adverse
cardiac events for both treatment groups (red and blue bars). In
addition, the 95% confidence intervals are also presented (red and
blue brackets). No significant differences were found between
both treatment groups. TLF target lesion failure, CI confidence
interval, EE-BRS everolimus-eluting bioresorbable scaffolds, EES
everolimus-eluting stents, MACE major adverse cardiac events Revascularization in insulin‑treated diabetic patients p
In our current all-diabetic patient population, treatment
with insulin showed an, albeit statistically non-signifi-
cant, trend towards prediction of TLF. Such relation was
observed in the diabetes mellitus subgroup analysis of the
ABSORB trials and in a pooled analysis of the SPIRIT and
COMPARE trials; both analyses found insulin-treatment
to be a risk factor for TLF after PCI with either EE-BRS
or EES [6, 24]. The increased event risk in insulin-treated
patients may be attributable to the generally longer his-
tory of diabetes mellitus, during which the diabetes-
induced chronic vascular inflammation stimulates the
progression of atherosclerosis, alters plaque composition
and promotes the development of advanced lesions. It is
plausible that the severity and duration of diabetes mel-
litus is related to the risk of cardiovascular complications
and it appears reasonable to consider this when choosing
a coronary revascularization strategy. In the present analysis, diabetic patients treated with
EES showed clinical event rates similar to those of the
EES-treated diabetic patients in a pooled analysis of the
SPIRIT and COMPARE trials [6]. Furthermore, in our
patients with diabetes mellitus the efficacy and short-
and long-term safety of EE-BRS treatment were favora-
ble and comparable to previous studies [24–27]. The
performance of EE-BRS and EES have been compared
in non-exclusive diabetic populations. Despite some
promising early results, long-term assessment revealed
no advantage for treating all-comers with EE-BRS, but
revealed a higher incidence of TV-MI and ST, and a
greater angiographic late lumen loss [28–32]. The find-
ings of these trials led to the current withdrawal and
utilization of EE-BRS and suggest a need for refined Patient selection for EE‑BRS treatment Younger patients with non-insulin-treated diabetes mel-
litus and CAD of limited extent may be the most suitable
candidates for treatment with EE-BRS (as an alternative
to EES), as they might have the greatest benefit from the
bioresorbable nature of the device. Furthermore, younger Hommels et al. Cardiovasc Diabetol (2020) 19:165 Page 8 of 10 Table 4 Multivariate Cox regression models for target lesion failure and major adverse cardiac events
Section A. Multivariate Cox regression model for target lesion failure adjusted for age, insulin-treated diabetes mellitus, number of treated vessels, total treated length
and destined treatment group
Section B. The same model calculated for major adverse cardiac events. Risk factors are given in hazard ratios with 95% confidence intervals with corresponding
p-values
A p-value < 0.05 was considered as statistically significant. No significant differences between both everolimus-eluting bioresorbable scaffolds and everolimus-eluting
stents treatment groups were ascertained. Insulin-treatment for diabetes mellitus was the only variable that showed a trend as predictor for target lesion failure as
was age for major adverse cardiac events
TLF target lesion failure, CI confidence interval, EE-BRS everolimus-eluting bioresorbable scaffolds, EES everolimus-eluting stents, MACE major adverse cardiac events
A. Variable for outcome TLF
Hazard ratio
95% CI
P-value
Age at device implantation
1.03
0.99–1.06
0.12
Insulin-treated diabetes mellitus
1.82
0.97–3.41
0.06
Number of treated vessels
1.17
0.47–2.91
0.74
Total treated length
1.00
0.99–1.02
0.84
EE-BRS vs. EES
1.48
0.77–2.87
0.24
B. Variable for outcome MACE
Hazard ratio
95% CI
p-value
Age at device implantation
1.03
1.00–1.05
0.06
Insulin-treated diabetes mellitus
1.40
0.82–2.37
0.22
Number of treated vessels
1.57
0.77–3.22
0.22
Total treated length
1.00
0.99–1.01
0.88
EE-BRS vs. EES
1.23
0.70–2.17
0.47 ble 4 Multivariate Cox regression models for target lesion failure and major adverse cardiac events lesion characteristics. Hence, a multivariable analysis was
performed, striving for adjustment of known confounders. Due to the population size, this study was not powered to
detect significant differences in the composites of the end-
points, particularly low-incidence events like mortality
and ST, therefore these results should be interpreted with
necessary caution. As we decided to share our results now
as debate concerning the future of bioresorbable devices
continues, this study does not provide sufficient informa-
tion about differences between these devices in the phase
beyond scaffold resorption in the majority of the EE-BRS
patients. Patient selection for EE‑BRS treatment To correct for the variable time to follow-up,
results were presented in PY which theoretically may
obscure time-to-event related adverse outcomes. To mini-
mize this limitation we also reported time-to-event analy-
sis and the nominal incidence of ST. While the operators
had considerable experience with implanting EE-BRS, the
use of the PSP approach and intracoronary imaging were
not mandatory according to the study protocol, while this
might have improved clinical outcomes. Finally, as the cur-
rent EE-BRS generation has been withdrawn from daily
practice due to the comprised safety outcomes, the clinical
implication of our results should be interpreted in the light
of newer bioresorbable devices which have been intro-
duced recently. patients have a lower bleeding risk than elderly patients,
which is beneficial considering that a prolonged DAPT
regimen may be indicated following PCI with EE-BRS. Nevertheless, further dedicated research is required
to assess the usefulness of EE-BRS in younger patients. Most likely, such studies will test novel thinner-strut
bioresorbable scaffolds that recently became available
for research purposes and these studies should include a
long-term follow-up [38]. Limitationsh The hypothesis-generating findings of the present post hoc
analysis should be interpreted carefully in the light of sev-
eral limitations, including the intrinsic limitations of any
comparison between multiple patient cohorts from dif-
ferent studies. Low cholesterol, and in particular values of
remnant-like particle cholesterol below 0.5 mmol per liter
have shown to be a predictor of freedom from in-stent
restenosis in diabetic patients for all types of stents [39]. In our study detailed patient specific cholesterol values
were unobtainable, however a dichotomic hypercholes-
terolemia (treated) was available and similarly distributed
between both groups. Profound data concerning the sta-
tus of diabetes mellitus and medication prescription were
unavailable for a substantial share of the included patients. Yet, all study participants were treated in the same geo-
graphical region and they received the same concomi-
tant medication reflecting the international guidelines. Although most baseline clinical characteristics were fairly
comparable, there were some differences in angiographic Conclusion In this patient-level pooled analysis of diabetic patients
from 3 prospective clinical trials, EE-BRS showed simi-
lar clinical outcomes to EES. These results encourage Page 9 of 10 Page 9 of 10 Hommels et al. Cardiovasc Diabetol (2020) 19:165 Funding
D Department of Cardiology, Isala Hospital, Zwolle. Unrestricted research grant
from Abbott Vascular and Boston Scientific. 7. Moreno PR, Fuster V. New aspects in the pathogenesis of diabetic athero-
thrombosis. J Am Coll Cardiol. 2004;44:2293–300. 7. Moreno PR, Fuster V. New aspects in the pathogenesis of diabetic athero-
thrombosis. J Am Coll Cardiol. 2004;44:2293–300. 8. Yamaji K, Kimura T, Morimoto T, et al. Very long-term (15 to 20 years) clini-
cal and angiographic outcome after coronary bare metal stent implanta-
tion. Circ Cardiovasc Interv. 2010;3:468–75. 8. Yamaji K, Kimura T, Morimoto T, et al. Very long-term (15 to 20 years) clini-
cal and angiographic outcome after coronary bare metal stent implanta-
tion. Circ Cardiovasc Interv. 2010;3:468–75. Acknowledgements The study team is grateful to P.C. Koopmans, statistician and epidemiologist
from Signidat, Groningen, The Netherlands, for contributing to the statistical
analysis of this trial. 4. Grube E, Chevalier B, Guagliumi G, et al. The SPIRIT V diabetic study: a
randomized clinical evaluation of the XIENCE V everolimus-eluting stent
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novo coronary artery lesions. Am Heart J. 2012;163:867–75. Availability of data and materials The dataset used and/or analyzed in the current manuscript was pooled from
3 individual clinical trials. Consequently, the authors of this manuscript do not
own the underlying patient-level data. Upon reasonable request, data may
be available from the respective organizing centers, but not without formal
permission of the principal investigators and the steering committees of each
of the separate trials. Corresponding author for any data request is E. Kedhi,
the senior corresponding author of this manuscript. 9. Otsaka F, Vorpahl M, Nakano M, et al. Pathology of second-generation
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scaffolds for coronary revascularization. Circulation. 2016;134:168–82.f Competing interests g
E.H. Ploumen and C. von Birgelen indicated that the research department of
Thoraxcentrum Twente has received institutional research grants from Abbott
Vascular, Biotronik, Boston Scientific and Medtronic. The other authors declare
that they have no competing interests. 17. Cutlip DE, Windecker S, Mehran R, et al. Clinical end points in
coronary stent trials: a case for standardized definitions. Circulation. 2007;115:2344–51. 18. Vranckx P, Cutlip DE, Mehran R, et al. Myocardial infarction adjudication in
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Intervention. 2010;5:871–4. References CABG: Coronary artery bypass grafting; CAD: Coronary artery disease; CI: Con-
fidence interval; DAPT: Dual antiplatelet therapy; EE-BRS: Everolimus-eluting
bioresorbable scaffolds; EES: Everolimus-eluting stents; HR: Hazard ratio;
MACE: Major adverse cardiac events; MI: Myocardial infarction; PCI: Percutane-
ous coronary intervention; PSP: Pre-dilatation, sizing, and post-dilatation; PY:
Patient years; ST: Device thrombosis; TLF: Target lesion failure; TLR: Target lesion
revascularization; TV-MI: Target vessel myocardial infarction; TVR: Target vessel
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trial. Circulation. 2004;109:2727–31. 3. Van Belle E, Périé M, Braune D, et al. Effects of coronary stenting on vessel
patency and long-term clinical outcome after percutaneous coronary
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Not applicable. 16. von Birgelen C, Sen H, Lam MK, et al. Third-generation zotarolimus-
eluting and everolimus-eluting stents in all-comer patients requiring
a percutaneous coronary intervention (DUTCH PEERS): a randomised,
single-blind, multicentre, non-inferiority trial. Lancet. 2014;383:413–23. Authors’ contributions This manuscript is an original work and it is not under consideration for publi-
cation elsewhere. EK and TMH designed the study, co-drafted the manuscript
and gave final approval, TMH performed statistical analysis, RSH, BB, EF, GL, EHP
and CB critically revised the manuscript for intellectual content. All authors
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novel bioresorbable scaffolds in this patient population. further prospective long-term follow-up research with
novel bioresorbable scaffolds in this patient population. Received: 28 June 2020 Accepted: 12 September 2020 Received: 28 June 2020 Accepted: 12 September 2020 Ethics approval and consent to participate The ABSORB DM Benelux Study (NTR5447) was performed in concurrence
with the Declaration of Helsinki and was registered at the Netherlands Trial
Register (NTR) acknowledged by the World Health Organization (WHO) and
International Committee of Medical Journal Editors (ICMJE). The TWENTE
(NTR1256/NCT01066650) and DUTCH PEERS (NTR2413/NCT01331707) trials
were both conducted in concurrence with the Declaration of Helsinki and
were registered at the NTR as well as at ClinicalTrials.gov. An approval for
ethical committees was sought in all participating centers and countries. The
clinical investigations did not test any non-approved device. The ABSORB
BVS and the ABSORB GT1 are CE marked products and were used widely in
daily clinical practice all over the world from 2009 to 2017. The Xience V EES
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1 Predictors of long-term adverse
events after Absorb bioresorbable vascular scaffold implantation: a
1,933-patient pooled analysis from international registries. EuroInterven-
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domised comparison between an everolimus-eluting bioresorbable scaf-
fold and an everolimus-eluting metallic stent for the treatment of coro-
nary artery stenosis (ABSORB II trial). EuroIntervention. 2018;13:1561–4. 26. Campos CM, Caixeta A, Franken M, et al. Risk and timing of clinical events
according to diabetic status of patients treated with everolimus-eluting
bioresorbable vascular scaffolds versus everolimus-eluting stent: 2-year
results from a propensity score matched comparison of ABSORB EXTEND
and SPIRIT trials. Catheter Cardiovasc Interv. 2018;91:387–95. 37. Goel S, Pasam RT, Chava S, et al. Three to four years outcomes of the
Absorb bioresorbable vascular scaffold versus second-generation drug-
eluting stent: a meta-analysis. Catheter Cardiovasc Interv. 2020;95:216–23. 38. Seth A, Onuma Y, Chandra P, et al. Three-year clinical and two-year multi-
modality imaging outcomes of a thin-strut sirolimus-eluting bioresorb-
able vascular scaffold: MeRes-1 trial. EuroIntervention. 2019;15:607–14. 27. Anadol R, Schnitzler K, Lorenz L, et al. Three-years outcomes of diabetic
patients treated with coronary bioresorbable scaffolds. BMC Cardiovasc
Disord. 2018;18:92. 39. Qin Z, Zhou K, Li YP, et al. Remnant lipoproteins play an important role of
in-stent restenosis in type 2 diabetes undergoing percutaneous coronary
intervention: a single-centre observational cohort study. Cardiovasc
Diabetol. 2019;18:11. 28. Serruys PW, Chevalier B, Dudek D, et al. A bioresorbable everolimus-elut-
ing scaffold versus a metallic everolimus-eluting stent for ischaemic heart
disease caused by de-novo native coronary artery lesions (ABSORB II): an
interim 1-year analysis of clinical and procedural secondary outcomes
from a randomised controlled trial. Lancet. 2015;385:43–54. Publisher’s Note 29. Ellis SG, Kereiakes DJ, Metzger DC, et al. Everolimus-eluting bioresorbable
scaffolds for coronary artery disease. N Engl J Med. 2015;373:1905–15. 29. Ellis SG, Kereiakes DJ, Metzger DC, et al. Everolimus-eluting bioresorbable
scaffolds for coronary artery disease. N Engl J Med. 2015;373:1905–15. 30. Kereiakes DJ, Ellis SG, Metzger C, et al. 3-Year clinical outcomes
with everolimus-eluting bioresorbable coronary scaffolds: the ABSORB III
trial. J Am Coll Cardiol. 2017;70:2852–62. Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. 30. Kereiakes DJ, Ellis SG, Metzger C, et al. 3-Year clinical outcomes
with everolimus-eluting bioresorbable coronary scaffolds: the ABSORB III
trial. J Am Coll Cardiol. 2017;70:2852–62. 31. Serruys PW, Chevalier B, Sotomi Y, et al. Comparison of an everolimus-
eluting bioresorbable scaffold with an everolimus-eluting metallic
stent for the treatment of coronary artery stenosis (ABSORB II): a 3 year, 31. Serruys PW, Chevalier B, Sotomi Y, et al. Comparison of an everolimus-
eluting bioresorbable scaffold with an everolimus-eluting metallic
stent for the treatment of coronary artery stenosis (ABSORB II): a 3 year, •
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Sensors 2011, 11, 9069-9097; doi:10.3390/s110909069
sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
Article
Geometric Calibration and Radiometric Correction of LiDAR
Data and Their Impact on the Quality of Derived Products
Ayman F. Habib 1,*, Ana P. Kersting 1, Ahmed Shaker 2 and Wai-Yeung Yan 2
1 Department of Geomatics Engineering, University of Calgary, 2500 University Drive NW, Calgary,
Alberta T2N 1N4, Canada; E-Mail: ana.kersting@ucalgary.ca
2 Department of Civil Engineering, Ryerson University, 350 Victoria Street, Toronto, Ontario M5B 2K3,
Canada; E-Mails: ahmed.shaker@ryerson.ca (A.S.); waiyeung.yan@ryerson.ca (W.-Y.Y.)
* Author to whom correspondence should be addressed; E-Mail: ahabib@ucalgary.ca;
Tel.: +1-403-220-7105; Fax: +1-403- 284-1980.
Received: 22 June 2011; in revised form: 1 September 2011 / Accepted: 3 September 2011 /
Published: 21 September 2011
Abstract: LiDAR (Light Detection And Ranging) systems are capable of providing 3D
OPEN ACCESS Sensors 2011, 11, 9069-9097; doi:10.3390/s110909069 sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
OPEN ACCESS sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
OPEN ACCESS Keywords: LiDAR; geometric calibration; radiometric correction 1. Introduction The importance of geometric calibration and radiometric correction of active remote sensing data
has been emphasized for Japan Earth Resources Satellite-1 Synthetic Aperture Radar (JERS-1 SAR) [1],
RADARSAT [2], European Remote-Sensing Satellite Synthetic Aperture Radar (ERS SAR) [3], and
Advanced Land Observation Satellite Phased Array type L-band Synthetic Aperture Radar (ALOS
PALSAR) [4]. Different rigorous geometric and radiometric correction models were developed by
considering the scanning geometry, backscattering mechanism, and terrain induced distortions [5]. It
has been shown that these factors have significant influence on the derived data products. LiDAR
systems have similar operational principles to RADAR and SAR systems. Laser energy is emitted and
the backscattered energy from the object space is recorded by the LiDAR system. The backscattered
energy and the time delay between the signal emission and reception are used to derive a 3D point
cloud, which is represented by the XYZ coordinates and the intensity I representing the peak
backscattered laser energy from the object. Due to systematic errors in the LiDAR system parameters
and measurements, geometric calibration and radiometric correction of the positional and intensity data
are essential to ensure the best accuracy of the delivered products. The geometric calibration of LiDAR systems aims at estimating and removing all the systematic
errors from the point cloud coordinates such that only random errors are left. Systematic errors in the
LiDAR data are mainly caused by biases in the system parameters, e.g., biases in the mounting
parameters relating the system components (lever arm and boresight angles) and biases in the
measured ranges and mirror angles. The elimination/reduction of such systematic errors or their impact
has been the focus of the LiDAR research community in the past few years. The existing approaches
can be classified into two main categories: system-driven (calibration) and data-driven (strip
adjustment) procedures. This categorization is mainly related to the nature of the utilized data and
mathematical model. System-driven (calibration) procedures are based on the physical sensor model
relating the system measurements/parameters to the ground coordinates of the LiDAR points. These
procedures incorporate the system’s raw data or at least the trajectory and time-tagged point cloud for
the estimation of biases in the system parameters with the help of the LiDAR point positioning
equation. Ayman F. Habib 1,*, Ana P. Kersting 1, Ahmed Shaker 2 and Wai-Yeung Yan 2 1 Department of Geomatics Engineering, University of Calgary, 2500 University Drive NW, Calgary,
Alberta T2N 1N4, Canada; E-Mail: ana.kersting@ucalgary.ca
2 Department of Civil Engineering, Ryerson University, 350 Victoria Street, Toronto, Ontario M5B 2K3,
Canada; E-Mails: ahmed.shaker@ryerson.ca (A.S.); waiyeung.yan@ryerson.ca (W.-Y.Y.) 1 Department of Geomatics Engineering, University of Calgary, 2500 University Drive NW, Calgary,
Alberta T2N 1N4, Canada; E-Mail: ana.kersting@ucalgary.ca * Author to whom correspondence should be addressed; E-Mail: ahabib@ucalgary.ca;
Tel.: +1-403-220-7105; Fax: +1-403- 284-1980. * Author to whom correspondence should be addressed; E-Mail: ahabib@ucalgary.ca;
Tel.: +1-403-220-7105; Fax: +1-403- 284-1980. Received: 22 June 2011; in revised form: 1 September 2011 / Accepted: 3 September 2011 /
Published: 21 September 2011 Received: 22 June 2011; in revised form: 1 September 2011 / Accepted: 3 September 2011 /
Published: 21 September 2011 Received: 22 June 2011; in revised form: 1 September 2011 / Accepted: 3 September 2011 /
Published: 21 September 2011 Abstract: LiDAR (Light Detection And Ranging) systems are capable of providing 3D
positional and spectral information (in the utilized spectrum range) of the mapped surface. Due to systematic errors in the system parameters and measurements, LiDAR systems
require geometric calibration and radiometric correction of the intensity data in order to
maximize the benefit from the collected positional and spectral information. This paper
presents a practical approach for the geometric calibration of LiDAR systems and
radiometric correction of collected intensity data while investigating their impact on the
quality of the derived products. The proposed approach includes the use of a quasi-rigorous
geometric calibration and the radar equation for the radiometric correction of intensity
data. The proposed quasi-rigorous calibration procedure requires time-tagged point cloud
and trajectory position data, which are available to most of the data users. The paper
presents a methodology for evaluating the impact of the geometric calibration on the
relative and absolute accuracy of the LiDAR point cloud. Furthermore, the impact of the
geometric calibration and radiometric correction on land cover classification accuracy is
investigated. The feasibility of the proposed methods and their impact on the derived
products are demonstrated through experimental results using real data. Sensors 2011, 11 9070 Sensors 2011, 11 least the trajectory and time-tagged point cloud [13-15]. In spite of the relaxed data requirements
in [13-15], these methods have some shortcomings. For instance, in [13] and [14], only biases in the
boresight angles are estimated during the calibration procedure. Moreover, the parameters describing
the utilized surface in [13] are estimated within the calibration procedure. Thus, the number of
estimated parameters depends on the extent of the area being utilized in the calibration procedure. The
introduced procedure in [14] estimates the boresight angles using observed discrepancies between
conjugate surface elements in overlapping LiDAR strips. The discrepancies are obtained through a
matching procedure that uses interpolated data. The implemented matching procedure does not lead to
accurate estimates of the horizontal discrepancies when compared to the estimated vertical
discrepancy. Therefore, the estimated biases in the boresight pitch and heading angles are of lower
accuracy than the boresight roll angle since the former lead to horizontal discrepancies while the latter
leads to horizontal as well as vertical discrepancies among overlapping strips. In [15], point primitives
are utilized to establish the correspondence between overlapping strips. Due to the irregular nature of
the LiDAR points, the identification of distinct and conjugate points in overlapping strips is quite
difficult and not reliable. Moreover, this method assumes that the true ground coordinates can be
derived by averaging the coordinates of tie points in overlapping strips. The averaging process will not
lead to the correct estimate of the LiDAR point in the presence of some systematic errors. Finally, the
above studies did not elaborate on the required flight configuration and control requirement for a
reliable and economic calibration technique. Radiometric correction of the LiDAR data is a relatively new research area. It aims at converting
the recorded intensity data into the spectral reflectance of an object. The radar equation has been
proposed for radiometric correction of the LiDAR intensity data in [16] and [17]. The radar equation
considers the effects of the measured laser range, angle of reflection, and atmospheric attenuation to
retrieve the surface reflectance in the near infrared red (NIR) spectrum. Radiometric calibration of
LiDAR data is proposed by researchers at the Finnish Geodetic Institute in a number of recent
publications [18-20]. 1. Introduction In this paper, the term “raw data” is used to denote all the quantities present in the LiDAR
point positioning equation (i.e., position and orientation information as well as the measured range and
scan angle for each pulse). Moreover, the utilized sequence of rotation angles, which define the system
attitude and boresight angles, have to be clearly specified. The access to the system raw measurements
is usually restricted to LiDAR system manufacturers. Such a restriction has triggered the development
of methods that only utilize the XYZ coordinates of the LiDAR point cloud. These methods are
classified as data-driven (or strip adjustment) procedures [6-9]. The major drawback of such methods
is the mathematical model employed to relate the LiDAR strips and the reference frame. The effects of
systematic errors in the system parameters are usually modeled by an arbitrary transformation function
between the laser strip and the reference frame coordinate systems. Depending on the nature of the
inherent biases in the LiDAR system parameters, the utilized transformation function might not be
appropriate. As already mentioned, rigorous geometric calibration procedures are based on the physical
mathematical model (i.e., LiDAR point positioning equation) that involves the raw data [10-12] or at 9071 Sensors 2011, 11 2. Geometric System Calibration In this section, the proposed geometric calibration procedure for the estimation of biases in the
system parameters is described. This method, which is denoted as “Quasi-Rigorous” since it only
requires the time-tagged point cloud and trajectory position data, utilizes LiDAR data in overlapping
strips. Biases in the system parameters are estimated by reducing discrepancies between conjugate
surface elements in overlapping strips and control data, if available. The proposed method will be explained in the following subsections. First, the mathematical model
relating conjugate surface elements in overlapping LiDAR strips as well as overlapping LiDAR and
control surfaces in the presence of systematic errors is derived. Based on the analysis of the derived
mathematical model, remarks regarding the necessary flight and control configuration for LiDAR
system calibration are outlined. The established mathematical model for the calibration procedure is
derived based on point primitives (i.e., conjugate points in overlapping point cloud data). However,
point-to-point correspondence cannot be assumed due to the irregular/semi-random nature of the
LiDAR points. In this work, conjugate point and Triangular Irregular Network (TIN) patch pairs are
proposed as primitives. A modification to the traditional Gauss Markov Stochastic model that would
allow for the utilization of these conjugate primitives while using the established point-based
mathematical model is finally described. Sensors 2011, 11 Sensors 2011, 11 assessment with checkpoints acquired from an orthophoto is used to assess and compare the
classification results from the original and the geometrically calibrated and radiometrically corrected
datasets. Sensors 2011, 11 This work is based on the utilization of reference targets (e.g., Spectralon
reference plates) and ground features (e.g., beach sand and asphalt) to calibrate the LiDAR intensity
data by comparing the measured reflectance with in situ measurements from spectroradiometer and
NIR digital camera. The study has established that the homogeneity and the size of the calibration
targets play a critical role in the radiometric calibration. Effects of the system parameters such as the
aperture size [21] and the automatic gain control [22] have been also investigated. Practical methods
were developed to eliminate the effects of these parameters through absolute correction and relative
calibration approaches. Despite the previous research on geometric calibration of LiDAR systems and radiometric
correction of the intensity data, the impact of these approaches on the quality of the derived products is
still an open research area. This paper presents methods for geometric calibration and radiometric
correction of airborne LiDAR data while evaluating their effect on the geo-positional accuracy and
classification of the intensity data. The geometric calibration involves a quasi-rigorous procedure for
the estimation of biases in the system parameters. The geo-positioning accuracy of the adjusted point
cloud is assessed based on quantifying the degree of compatibility between LiDAR and control
surfaces before and after the calibration process. The radiometric correction utilizes the radar equation
to determine the spectral reflectance of objects. Then, land cover classification is conducted. Accuracy 9072 2.1. Mathematical Model The coordinates of the LiDAR points are the result of combining the derived measurements from
each of its system components, as well as the mounting parameters relating such components. The
relationship between the system measurements and parameters is embodied in the LiDAR point
positioning equation [23-24], Equation (1). The position of an object point ܺԦீ is derived through the
summation of three vectors ሺ ܺԦ, ܲሬԦீ, and ݎԦ) after applying the appropriate rotations (ܴఠ,ఝ,, ܴ௱ఠ,௱ఝ,௱,
and ܴௌഀఈ ௌഁఉ) (refer to Figure 1). In this equation, ܺԦ is the vector from the origin of the ground
reference frame to the origin of the Inertial Measurement Unit (IMU) coordinate system, ܲሬԦீ (lever arm
vector) is the offset between the IMU and laser unit coordinate systems (w.r.t. the IMU body frame),
and ݎԦ is the laser range vector whose magnitude is equivalent to the distance from the laser firing point
to its footprint. It should be noted that ܺԦ is derived through the Global Positioning System/ Inertial
Navigation System (GPS/INS) integration process while considering the lever arm vector between the
IMU body frame and the phase centre of the GPS antenna. The term ܴఠ,ఝ, stands for the rotation
matrix relating the ground and IMU coordinate systems—which is derived through the GPS/INS
integration process. The term ܴ௱ఠ,௱ఝ,௱ represents the rotation matrix relating the IMU and laser unit
coordinate systems—which is defined by the boresight pitch ሺ߂߱), roll ሺ߂߮), and heading ሺ߂ߢ) angles
(considering the y-axis of the IMU body frame aligned along the flight direction), while the term
ܴௌഀఈ ௌഁఉ refers to the rotation matrix relating the laser unit and laser beam coordinate systems with
ߙ and ߚ being the mirror scan angles. ܵఈ and ܵఉ are the scale factors of the angles measured by the 9073 Sensors 2011, 11 scanner, while ߂ݎ is a systematic error in the measured range. The involved quantities in the LiDAR
point positioning equation are all measured during the acquisition process except for the mounting
parameters (i.e., the lever arm and boresight angles), the scan angles scale factors, and the range error;
these parameters are usually determined through a calibration procedure. Figure 1. Coordinate systems and involved quantities in the LiDAR point positioning equation. Figure 1. Coordinate systems and involved quantities in the LiDAR point positioning e ܺԦீ= ܺԦܴఠ,ఝ,ܲሬԦீܴఠ,ఝ,ܴ௱ఠ,௱ఝ,௱ܴௌഀఈௌഁఉ
0
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െݏ݅݊݇
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0
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where
zlu
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yb
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YG
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1. Mapping Reference Frame
Object point
Laser beam
3. Laser unit
4. Laser beam
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xlb
ylb
zlb
xlu
ylu ܺԦீ= ܺԦܴఠ,ఝ,ܲሬԦீܴఠ,ఝ,ܴ௱ఠ,௱ఝ,௱ܴௌഀఈௌഁఉ
0
0
െሺݎ߂ݎ)
൩
(1)
zlu
xb
yb
zb
XG
ZG
YG
2. IMU body frame
1. Mapping Reference Frame
Object point
Laser beam
3. Laser unit
4. Laser beam
r
r
G
G
Δ
+
o
X
G
κ
ϕ
ω
,
,
R
G
P
G
κ
Δ
ϕ
Δ
ω
Δ
,
,
R
β
β
α
α
S
SR
G
X
G
xlb
ylb
zlb
xlu
ylu 2. IMU body frame 3. Laser unit zlu 4. Sensors 2011, 11 a given point ܺԦீሺܶݎݑ݁) is derived using the true values for the system parameters ݔ௧ and noise free
measurements ݈. However, if we only have access to biased system parameters ݔ as well as noisy
measurements ݈, the bias/noise contaminated coordinates of a given point ܺԦீሺܾ/݊) can be expressed
by Equation (4). The expression in Equation (3) can be rewritten in the form in Equation (5), where ߜݔ
denotes the biases in the system parameters and ݁ denotes the noise in the measurements: ܺԦீሺܶݎݑ݁) = ݂ሺݔ௧, ݈)
(3)
ܺԦீሺܾ/݊) = ݂ሺݔ, ݈)
(4)
ܺԦீሺܶݎݑ݁) = ݂ሺݔെߜݔ, ݈െ݁)
(5) (3) ܺԦீሺܾ/݊) = ݂ሺݔ, ݈) (4) ܺԦீሺܶݎݑ݁) = ݂ሺݔെߜݔ, ݈െ݁)
(5) (5) Equation (5) can be expanded using Taylor’s series to the form in Equation (6) after ignoring
second and higher order terms. This equation can be rewritten according to the form in Equation (7),
which indicates that the bias/noise contaminated coordinates ܺԦீሺܾ/݊) is equivalent to the summation
of the true coordinates ܺԦீሺܶݎݑ݁) and other terms that describe the impact of biases in the system
parameters and noise in the system measurements. Starting from Equation (2), the mathematical
expression for the impact of biases in the system parameters on the derived ground coordinates ߜܺԦீ is
given in Equation (8): ܺԦீ ሺܶݎݑ݁) ൎ݂ሺݔ, ݈) െ ߲݂߲ݔ
⁄
|௫್,ߜݔെ߲݂߲݈
⁄
|௫್,݁= ܺԦீሺܾ/݊) െ ܣߜݔെܤ݁
(6)
ܺԦீሺܾ/݊) = ܺԦீ ሺܶݎݑ݁) ܣߜݔܤ݁= ܺԦீሺܶݎݑ݁) ߜܺԦீܤ݁
(7) ܺԦீ ሺܶݎݑ݁) ൎ݂ሺݔ, ݈) െ ߲݂߲ݔ
⁄
|௫್,ߜݔെ߲݂߲݈
⁄
|௫್,݁= ܺԦீሺܾ/݊) െ ܣߜݔെܤ݁
(6)
ܺԦீሺܾ/݊) = ܺԦீ ሺܶݎݑ݁) ܣߜݔܤ݁= ܺԦீሺܶݎݑ݁) ߜܺԦீܤ݁
(7)
ߜܺԦீ=
ܿݏߢ ߜ߂ܺെݏ݅݊݇ߜ߂ܻݏ݅݊ߢݖߜ߂߱ܿݏߢݖߜ߂߮
ݏ݅݊ߢ ߜ߂ܺܿݏ݇ߜ߂ܻെܿݏߢݖߜ߂߱ݏ݅݊ߢݖߜ߂߮
ߜ߂ܼെݔ ߜ߂߮
൩
െݏ݅݊ߢ ݔ ߜ߂ߢെܿݏ݇ݏ݅݊ሺܵߚ) ߜ߂ݎܿݏߢ ݖ ߚ ߜܵ
ܿݏ݇ ݔ ߜ߂ߢെݏ݅݊ߢݏ݅݊ሺܵߚ) ߜ߂ݎݏ݅݊ߢݖߚߜܵ
െܿݏሺܵߚ) ߜ߂ݎെݔߚߜܵ
(8) ܺԦீ ሺܶݎݑ݁) ൎ݂ሺݔ, ݈) െ ߲݂߲ݔ
⁄
|௫್,ߜݔെ߲݂߲݈
⁄
|௫್,݁= ܺԦீሺܾ/݊) െ ܣߜݔെܤ݁
(6)
ܺԦீሺܾ/݊) = ܺԦீ ሺܶݎݑ݁) ܣߜݔܤ݁= ܺԦீሺܶݎݑ݁) ߜܺԦீܤ݁
(7)
ߜܺԦீ=
ܿݏߢ ߜ߂ܺെݏ݅݊݇ߜ߂ܻݏ݅݊ߢݖߜ߂߱ܿݏߢݖߜ߂߮
ݏ݅݊ߢ ߜ߂ܺܿݏ݇ߜ߂ܻെܿݏߢݖߜ߂߱ݏ݅݊ߢݖߜ߂߮
ߜ߂ܼെݔ ߜ߂߮
൩
െݏ݅݊ߢ ݔ ߜ߂ߢെܿݏ݇ݏ݅݊ሺܵߚ) ߜ߂ݎܿݏߢ ݖ ߚ ߜܵ
ܿݏ݇ ݔ ߜ߂ߢെݏ݅݊ߢݏ݅݊ሺܵߚ) ߜ߂ݎݏ݅݊ߢݖߚߜܵ
െܿݏሺܵߚ) ߜ߂ݎെݔߚߜܵ
(8) (6) (7) (8) Having two conjugate points in overlapping strips, which will be denoted by subscripts A and B
hereafter, the difference between the bias/noise contaminated coordinates of these points can be
expressed by Equation (9). Since we are dealing with conjugate points in overlapping strips, the true
coordinates of the respective points in strips A and B, i.e., ܺԦீಲሺܶݎݑ݁) ܽ݊݀ ܺԦீಳሺܶݎݑ݁), should be
identical. Sensors 2011, 11 Laser beam κ
ϕ
ω
,
,
R o
X
G 1. Mapping Reference Frame (1) ܺԦீ= ܺԦ
ܿݏߢ
െݏ݅݊݇
0
ݏ݅݊݇
ܿݏ ߢ
0
0
0
1
൩
Δܺ
Δܻ
Δܼ
൩
ܿݏߢ
െݏ݅݊݇
0
ݏ݅݊݇
ܿݏߢ
0
0
0
1
൩
1
െΔߢ
Δ߮
Δߢ
1
െΔ߱
െΔ߮
Δ߱
1
൩
െሺݎΔݎ)ݏ݅݊ሺܵߚ)
0
െሺݎΔݎ)ܿݏሺܵߚ)
൩
= ܺԦ
ܿݏߢ
െݏ݅݊݇
0
ݏ݅݊݇
ܿݏ ߢ
0
0
0
1
൩
߂ܺ
߂ܻ
߂ܼ
൩
ܿݏߢ
െݏ݅݊݇
0
ݏ݅݊݇
ܿݏߢ
0
0
0
1
൩
1
െ߂ߢ
߂߮
߂ߢ
1
െ߂߱
െ߂߮
߂߱
1
൩ቈ
ݔ
0
ݖ
(2)
where (2) where where – ߂ܺ, ߂ܻ, ߂ܼ are the components of the lever arm vector ܲሬԦீ, – ߂ܺ, ߂ܻ, ߂ܼ are the components of the lever arm vector ܲሬԦீ, – z is the vertical coordinate of the object point with respect to the laser unit coordinate – x is the lateral coordinate of the object point with respect to the laser unit coordinate system,
which is the lateral distance (with the appropriate sign) between the LiDAR point in question and
the projection of the flight trajectory onto the ground. – x is the lateral coordinate of the object point with respect to the laser unit coordinate system,
which is the lateral distance (with the appropriate sign) between the LiDAR point in question and
the projection of the flight trajectory onto the ground. The LiDAR point positioning mathematical model presented in Equation (1) can be simplified to
the form in Equation (2) after considering the following assumptions: linear scanner, vertical system,
and small boresight angles. For a linear scanner, the mirror is rotated in one direction only, leading to
zero values for ߙ and ܵఈ. To simplify the utilized symbols, the scale factor ܵఉ will be represented here
forth by ܵ. One should note that in Equation (2), we consider the convention of right-forward-up
(X-Y-Z) coordinate systems for the laser unit and IMU body frame with the Y-axis aligned along the
flight direction. The deterministic LiDAR equations for point positioning (Equations (1) and (2)) can
be represented in a symbolic form by Equation (3). This equation indicates that the true coordinates of 9074 Sensors 2011, 11 Once these biases have been estimated,
the adjusted coordinates of the LiDAR points can be derived according to Equation (13): ܺԦீಲሺܾ/݊) െܺԦீಳሺܾ/݊) = ܺԦீಲ ሺܶݎݑ݁) ߜܺԦீ݁െܺԦீಳሺܶݎݑ݁) െߜܺԦீെ݁
(9)
ܺԦீಲሺܾ/݊) െܺԦீಳሺܾ/݊) = ߜܺԦீെߜܺԦீ݁െ݁= ߜܺԦீെߜܺԦீ݁
(10)
ܺԦீಲሺܾ/݊) െܺԦீಳሺܾ/݊) =
ሺܿݏߢെܿݏߢ)ߜ߂ܺെሺݏ݅݊ߢെݏ݅݊ߢ)ߜ߂ܻ
ሺݏ݅݊ߢെݏ݅݊ߢ)ߜ߂ܺሺܿݏߢെܿݏߢ)ߜ߂ܻ
0
൩ (9) (10)
ሺݏ݅݊ߢݖെݏ݅݊ߢݖ)ߜ߂߱ሺܿݏߢݖെܿݏߢݖ)ߜ߂߮െሺݏ݅݊ߢݔെݏ݅݊ߢݔ)ߜ߂ߢ
െሺܿݏߢݖെܿݏߢݖ)ߜ߂߱ሺݏ݅݊ߢݖെݏ݅݊ߢݖ)ߜ߂߮ሺܿݏߢݔെܿݏߢݔ)ߜ߂ߢ
െሺݔെݔ)ߜ߂߮
(11)
ሺݏ݅݊ߢݖെݏ݅݊ߢݖ)ߜ߂߱ሺܿݏߢݖെܿݏߢݖ)ߜ߂߮െሺݏ݅݊ߢݔെݏ݅݊ߢݔ)ߜ߂ߢ
െሺܿݏߢݖെܿݏߢݖ)ߜ߂߱ሺݏ݅݊ߢݖെݏ݅݊ߢݖ)ߜ߂߮ሺܿݏߢݔെܿݏߢݔ)ߜ߂ߢ
െሺݔെݔ)ߜ߂߮
(11) (11) ሺ
)
ሺ
)
߮
ሺ
)
െሺݔെݔ)ߜ߂߮
െሾܿݏ݇ ݏ݅݊ሺܵߚ) െܿݏߢ ݏ݅݊ሺܵߚ)ሿߜ߂ݎሺܿݏߢݖߚെܿݏߢݖߚ)ߜܵ
െሾݏ݅݊݇ ݏ݅݊ሺܵߚ) െݏ݅݊ߢ ݏ݅݊ሺܵߚ)ሿߜ߂ݎሺݏ݅݊ߢݖߚെݏ݅݊ߢݖߚ)ߜܵ
െሾܿݏሺܵߚ) െܿݏሺܵߚ)ሿߜ߂ݎെሺݔߚെݔߚ)ߜܵ
݁
(11)
ܺԦீೝെܺԦீಳሺܾ/݊) =
െܿݏߢߜ߂ܺݏ݅݊ߢߜ߂ܻ
െݏ݅݊ߢߜ߂ܺെܿݏߢߜ߂ܻ
െߜ߂ܼ
൩
െݏ݅݊ߢݖ ߜ߂߱െܿݏߢݖ ߜ߂߮ݏ݅݊ߢݔߜ߂ߢ
ܿݏߢݖ ߜ߂߱െݏ݅݊ߢݖ ߜ߂߮െܿݏߢݔ ߜ߂ߢ
ݔ ߜ߂߮
൩
ܿݏߢ ݏ݅݊ሺܵߚ)ߜ߂ݎെܿݏߢݖߚ ߜܵ
ݏ݅݊ߢݏ݅݊ሺܵߚ) ߜ߂ݎെݏ݅݊ߢݖߚߜܵ
ܿݏሺܵߚ) ߜ߂ݎݔߚߜܵ
ሺ݁௧െ݁)
(12)
ܺ෨
ܻ෨
ܼ෨
൩
௧ௗ
=
ܺ
ܻ
ܼ
൩
௦ௗ
െ
ܿݏߢߜ߂መܺെݏ݅݊ߢߜ߂መܻݏ݅݊ߢݖߜ߂መ߱ܿݏߢ ݖ ߜ߂መ߮
ݏ݅݊ߢ ߜ߂መܺܿݏߢߜ߂መܻെܿݏߢݖߜ߂መ߱ݏ݅݊ߢ ݖ ߜ߂መ߮
ߜ߂መܼെݔ ߜ߂መ߮
െ
െݏ݅݊ߢ ݔ ߜ߂መߢെܿݏߢ ݏ݅݊ሺܵߚ)ߜ߂መݎܿݏߢ ݖ ߚ ߜܵመ
ܿݏߢ ݔ ߜ߂መߢെݏ݅݊ߢݏ݅݊ሺܵߚ)ߜ߂መݎݏ݅݊ߢݖߚߜܵመ
െܿݏሺܵߚ) ߜ߂መݎെݔߚߜܵመ
(13)
െሾܿݏ݇ ݏ݅݊ሺܵߚ) െܿݏߢ ݏ݅݊ሺܵߚ)ሿߜ߂ݎሺܿݏߢݖߚെܿݏߢݖߚ)ߜܵ
െሾݏ݅݊݇ ݏ݅݊ሺܵߚ) െݏ݅݊ߢ ݏ݅݊ሺܵߚ)ሿߜ߂ݎሺݏ݅݊ߢݖߚെݏ݅݊ߢݖߚ)ߜܵ
െሾܿݏሺܵߚ) െܿݏሺܵߚ)ሿߜ߂ݎെሺݔߚെݔߚ)ߜܵ
݁
ܺԦீೝെܺԦீಳሺܾ/݊) =
െܿݏߢߜ߂ܺݏ݅݊ߢߜ߂ܻ
െݏ݅݊ߢߜ߂ܺെܿݏߢߜ߂ܻ
െߜ߂ܼ
൩
െݏ݅݊ߢݖ ߜ߂߱െܿݏߢݖ ߜ߂߮ݏ݅݊ߢݔߜ߂ߢ
ܿݏߢݖ ߜ߂߱െݏ݅݊ߢݖ ߜ߂߮െܿݏߢݔ ߜ߂ߢ
ݔ ߜ߂߮
൩
(12)
െሾܿݏ݇ ݏ݅݊ሺܵߚ) െܿݏߢ ݏ݅݊ሺܵߚ)ሿߜ߂ݎሺܿݏߢݖߚെܿݏߢݖߚ)ߜܵ
െሾݏ݅݊݇ ݏ݅݊ሺܵߚ) െݏ݅݊ߢ ݏ݅݊ሺܵߚ)ሿߜ߂ݎሺݏ݅݊ߢݖߚെݏ݅݊ߢݖߚ)ߜܵ
െሾܿݏሺܵߚ) െܿݏሺܵߚ)ሿߜ߂ݎെሺݔߚെݔߚ)ߜܵ
݁
െሾܿݏ݇ ݏ݅݊ሺܵߚ) െܿݏߢ ݏ݅݊ሺܵߚ)ሿߜ߂ݎሺܿݏߢݖߚെܿݏߢݖߚ)ߜܵ
െሾݏ݅݊݇ ݏ݅݊ሺܵߚ) െݏ݅݊ߢ ݏ݅݊ሺܵߚ)ሿߜ߂ݎሺݏ݅݊ߢݖߚെݏ݅݊ߢݖߚ)ߜܵ
െሾܿݏሺܵߚ) െܿݏሺܵߚ)ሿߜ߂ݎെሺݔߚെݔߚ)ߜܵ
݁ ܺԦீೝെܺԦீಳሺܾ/݊) =
െܿݏߢߜ߂ܺݏ݅݊ߢߜ߂ܻ
െݏ݅݊ߢߜ߂ܺെܿݏߢߜ߂ܻ
െߜ߂ܼ
൩
െݏ݅݊ߢݖ ߜ߂߱െܿݏߢݖ ߜ߂߮ݏ݅݊ߢݔߜ߂ߢ
ܿݏߢݖ ߜ߂߱െݏ݅݊ߢݖ ߜ߂߮െܿݏߢݔ ߜ߂ߢ
ݔ ߜ߂߮
൩
(12)
െݏ݅݊ߢݖ ߜ߂߱െܿݏߢݖ ߜ߂߮ݏ݅݊ߢݔߜ߂ߢ
ܿݏߢݖ ߜ߂߱െݏ݅݊ߢݖ ߜ߂߮െܿݏߢݔ ߜ߂ߢ
ݔ ߜ߂߮
൩
(12) ܿݏߢݖ ߜ߂߱െݏ݅݊ߢݖ ߜ߂߮െܿݏߢݔ ߜ߂ߢ
ݔ ߜ߂߮
൩
(12) (12)
ܿݏߢ ݏ݅݊ሺܵߚ)ߜ߂ݎെܿݏߢݖߚ ߜܵ
ݏ݅݊ߢݏ݅݊ሺܵߚ) ߜ߂ݎെݏ݅݊ߢݖߚߜܵ
ܿݏሺܵߚ) ߜ߂ݎݔߚߜܵ
ሺ݁௧െ݁)
ܺ෨
ܻ෨
ܼ෨
൩
௧ௗ
=
ܺ
ܻ
ܼ
൩
௦ௗ
െ
ܿݏߢߜ߂መܺെݏ݅݊ߢߜ߂መܻݏ݅݊ߢݖߜ߂መ߱ܿݏߢ ݖ ߜ߂መ߮
ݏ݅݊ߢ ߜ߂መܺܿݏߢߜ߂መܻെܿݏߢݖߜ߂መ߱ݏ݅݊ߢ ݖ ߜ߂መ߮
ߜ߂መܼെݔ ߜ߂መ߮
െ
െݏ݅݊ߢ ݔ ߜ߂መߢെܿݏߢ ݏ݅݊ሺܵߚ)ߜ߂መݎܿݏߢ ݖ ߚ ߜܵመ
ܿݏߢ ݔ ߜ߂መߢെݏ݅݊ߢݏ݅݊ሺܵߚ)ߜ߂መݎݏ݅݊ߢݖߚߜܵመ
െܿݏሺܵߚ) ߜ߂መݎെݔߚߜܵመ
(13) (13) The procedure for estimating the quantities ሺݔ, ݖ, ߢ and ߚ) in Equations (11) and (12) using the
available data (time-tagged point cloud and trajectory positions) can be summarized as follows: The procedure for estimating the quantities ሺݔ, ݖ, ߢ and ߚ) in Equations (11) and (12) using the
available data (time-tagged point cloud and trajectory positions) can be summarized as follows: (I) For a LiDAR point mapped at time ݐ, we identify trajectory positions within a certain time
interval ሺݐെΔݐ, ݐΔݐ); (I) For a LiDAR point mapped at time ݐ, we identify trajectory positions within a certain time
interval ሺݐെΔݐ, ݐΔݐ); (II) Then, a straight line is fitted through the selected trajectory positions to come up with a local
estimate of the trajectory. Sensors 2011, 11 Therefore, Equation (9) would reduce to the form in Equation (10), where ݁ is the combined
vector of random errors. Using the established form of the impact of the biases in the system
parameters on the ground coordinates (ߜܺԦீ in Equation (8)), the discrepancy between the bias/noise
contaminated coordinates of conjugate points in overlapping strips would take the form in
Equation (11). In a similar fashion, if we are dealing with conjugate points in a LiDAR strip and
control data, the difference between the coordinates of the control point and the bias/noise
contaminated coordinates of the LiDAR point in the strip denoted by the subscript B can be expressed
according to the form in Equation (12). Equations (11) and (12) are the final linear observation
equations when dealing with overlapping strips and control data. Using conjugate points in
overlapping LiDAR strips and control data, these equations allow for the recovery of biases in the 9075 Sensors 2011, 11 system parameters (ߜ߂ܺ, ߜ߂ܻ, ߜ߂ܼ, ߜ߂߱, ߜ߂߮, ߜ߂ߢ, ߜ߂ݎ, ߜܵ). arks Regarding the Necessary Flight and Control Configuration for LiDAR Calibration Analyzing the mathematical expressions for the discrepancies between bias/noise contaminated
coordinates of conjugate points in overlapping strips—Equation (11)—as well as the discrepancies
when
dealing
with
control
data—Equation
(12),
one
can
make
the
following
observations/recommendations regarding the preferred flight and ground control configuration for
reliable estimation of biases in the system parameters: 1. Using only overlapping strips, one cannot estimate the vertical bias in the lever arm ߜ߂ܼ—this
term is absent in Equation (11). Such inability is caused by the fact that a vertical bias in the
lever arm produces the same effect regardless of the flying direction, flying height, or scan
angle—refer to Equation (8). In other words, a bias in the vertical component of the lever arm
would not lead to discrepancies among overlapping strips regardless of the flight configuration. 2. The impact of the range bias ߜ߂ݎ is flight direction independent for parallel flight lines (i.e., the
impact is identical for flight lines flown in opposite directions). As can be seen in Equation (8),
the range bias affects the coordinates of the points in the vertical direction (major component)
as well as the planimetric component across the flight direction (minor component)—
sinሺܵߚ) ا cosሺܵߚ) in the range of the utilized scan angles (e.g., ߚ א ሾെ25°, 25°ሿ). Therefore, the vertical discrepancy is expected to be more useful than the planimetric
discrepancy for the estimation of the range bias. However, the vertical discrepancy – which is
caused by the range bias—among overlapping strips will be almost zero since cosሺܵߚ)≈ 1 for
the above mentioned scan angle range. Therefore, the estimation of the range bias would be
quite difficult to estimate by evaluating/observing the discrepancies among overlapping strips. 3. The impacts of biases in the boresight heading angle ߜ߂ߢ and the mirror angle scale ߜܵ are
flight direction independent for parallel flight lines (i.e., the impact is identical for flight lines
flown in opposite directions). 4. Having opposite flight lines with 100% overlap is optimal for the recovery of the planimetric
biases in the lever arm (ߜΔܺ, ߜΔܻ) as well as the biases in the boresight pitch and roll angles
(ߜΔ߱, ߜΔ߮). In such a case, the impact of these biases on the strip-pairs will be doubled while
the discrepancies arising from other biases will be eliminated. 5. The impact of biases in the lever arm on the introduced discrepancies is independent of the
flying height. Sensors 2011, 11 After defining the local trajectory, the necessary quantities can be
estimated as follows: ݔ, which is the ݔ-coordinate of the LiDAR point with respect to the laser unit coordinate
system, i.e., the lateral distance with the appropriate sign between the LiDAR point in
question and the projection of the flight trajectory onto the ground, can be determined by
computing the normal distance between the LiDAR point and the interpolated trajectory
that have been projected onto the ground; 9076 Sensors 2011, 11 Sensors 2011, 11 ݖ, which is the vertical coordinate of the LiDAR point with respect to the laser unit
coordinate system, can be determine by subtracting the elevation of the laser beam firing
point at time t, given by the interpolated flight trajectory, from the LiDAR point elevation; ߢ, which is the trajectory heading, can be computed once we have the local estimate of the
trajectory and its direction (defined by the neighboring trajectory positions); and ߢ, which is the trajectory heading, can be computed once we have the local estimate of the
trajectory and its direction (defined by the neighboring trajectory positions); and β, which is the scan angle, can be computed by simple trigonometric operation using the
estimated lateral distance ݔ and the trajectory height above ground ݖ. β, which is the scan angle, can be computed by simple trigonometric operation using the
estimated lateral distance ݔ and the trajectory height above ground ݖ. 2.2. Remarks Regarding the Necessary Flight and Control Configuration for LiDAR Calibration arks Regarding the Necessary Flight and Control Configuration for LiDAR Calibration Moreover, biases in the boresight pitch and
roll angles (ߜΔ߱ and ߜΔ߮, respectively) would not lead to any planimetric discrepancies
among conjugate points in overlapping strips. The bias in the boresight heading angle ߜΔߢ
would cause a constant planimetric discrepancy among overlapping strips (along the flight
direction). The bias in the boresight roll angle ߜΔ߮ would cause a constant vertical discrepancy
among overlapping strips. An increase in the lateral distance between the parallel strips would
lead to larger discrepancies, which in turn will have a positive impact on the reliability of the
boresight heading and roll bias estimation (for parallel flight lines in the same direction,
ݔെݔ = lateral distance between the strips). 7. To estimate biases in the vertical component of the lever arm ߜΔܼ and range measurement ߜΔݎ,
control information is necessary. For these biases, vertical control would be sufficient since it
will allow us to observe the discrepancy between the LiDAR and the control surfaces in the
vertical direction—refer to Equation (12). Having said that, one should note that there will be
high correlation between the impacts of these biases. As can be observed in the third line in
Equation (12), the impact of the range bias (ܿݏሺܵߚ) ߜ߂ݎ) is almost constant in the range of the
utilized scan angles (i.e., ܿݏሺܵߚ) ൎ 1 for ߚ א ሾെ25°, 25°ሿ) and therefore, it will be
correlated with the impact of the bias in the vertical component of the lever arm (െߜΔܼ). To
avoid such a problem, one can rely on in-situ measurements of the vertical lever arm and only
solving for the range bias during the calibration process. 8. For reliable estimation of the bias in the mirror angle scale ߜܵ, well distributed data in the
across flight direction should be used (this distribution will exhibit the nonlinear discrepancy
pattern associated with this bias as can be seen in Equations (11) and (12)). Based on the above discussion, one can conclude that the optimal/minimal flight and control
configuration for the estimation of the system parameters consists of three overlapping pairs and one
vertical control point. More specifically, two strip pairs, which are captured from two flying heights in
opposite directions with 100% overlap, and two parallel flight lines, which are flown in the same
direction with the least overlap possible, are needed. arks Regarding the Necessary Flight and Control Configuration for LiDAR Calibration As for the parameters to be estimated, due to high
correlation, it is not recommended to simultaneously solve for the vertical bias in the lever arm ߜ߂ܼ
and the range error ߜΔݎ. arks Regarding the Necessary Flight and Control Configuration for LiDAR Calibration However, the impact of the biases in the boresight angles increases with an
increase in the flying height. Moreover, for a given flight height and relatively flat terrain
(i.e., ݖ ܽ݊݀ ݖ are almost constant in the overlap area), there is a strong correlation between
the biases in boresight pitch ߜΔ߱ and the planimetric component of the lever arm in the flight Sensors 2011, 11 9077 direction ߜΔܻ. To avoid such a correlation, it is recommended to have opposite flight lines with
100% overlap ratio captured at different flying heights. Larger height difference between the
flight pairs would be recommended as long as the point density in the higher flight lines is not
too low to jeopardize the ability of identifying conjugate primitives. direction ߜΔܻ. To avoid such a correlation, it is recommended to have opposite flight lines with
100% overlap ratio captured at different flying heights. Larger height difference between the
flight pairs would be recommended as long as the point density in the higher flight lines is not
too low to jeopardize the ability of identifying conjugate primitives. 6. Having two parallel flight lines with less than 100% overlap is necessary for the estimation of
the bias in the boresight heading angle ߜΔߢ. For such a case, biases in the lever arm would not
lead to any discrepancies among overlapping strips. Moreover, biases in the boresight pitch and
roll angles (ߜΔ߱ and ߜΔ߮, respectively) would not lead to any planimetric discrepancies
among conjugate points in overlapping strips. The bias in the boresight heading angle ߜΔߢ
would cause a constant planimetric discrepancy among overlapping strips (along the flight
direction). The bias in the boresight roll angle ߜΔ߮ would cause a constant vertical discrepancy
among overlapping strips. An increase in the lateral distance between the parallel strips would
lead to larger discrepancies, which in turn will have a positive impact on the reliability of the
boresight heading and roll bias estimation (for parallel flight lines in the same direction,
ݔെݔ = lateral distance between the strips). 6. Having two parallel flight lines with less than 100% overlap is necessary for the estimation of
the bias in the boresight heading angle ߜΔߢ. For such a case, biases in the lever arm would not
lead to any discrepancies among overlapping strips. 2.3. Least Squares Adjustment Model and Primitives The mathematical model that has been developed so far is based on the availability of conjugate
points in overlapping strips, Equation (11), or a LiDAR strip and a control surface, Equation (12). Assuming that such conjugate points exist, observations representing the discrepancy between these
points follow the traditional Gauss Markov Stochastic model in Equation (14). The Least Squares Sensors 2011, 11 9078 Adjustment (LSA) procedure aims at estimating the unknown parameters, which minimize the sum of
squares of weighted residuals in Equation (15), which would lead to the solution in Equations (16–19) [25]: : ݕ= ܣݔ݁ ݁~ሺ0, ߑ)
ݓ݄݁ݎ݁ߑ= ߪ
ଶܲିଵ
(14) (14) where – ݕ is the nx1 vector of observations (discrepancies between conjugate points), – ݔ is the mx1 vector of unknowns (biases in the system parameters), – ݔ is the mx1 vector of unknowns (biases in the system parameters), – ݔ is the mx1 vector of unknowns (biases in the system parameters), – ܣ is the nxm design matrix, and – ݁ is the nx1 vector of random noise, which is normally distributed with a zero mean and variance-
covariance matrix Σ – ߪ
ଶ is the a-priori variance factor – ߪ
ଶ is the a-priori variance factor – ܲ is the weight matrix of the noise vector – ܲ is the weight matrix of the noise vector ்݁ܲ݁= ݉݅݊|௫
(LSA Target Function)
(15)
ݔො= ሺܣ்ܲܣ)ିଵܣ்ܲݕ= ܰିଵܣ்ܲݕ
(Solution Vector)
(16)
݁̃ = ݕെܣݔො
(Predicted Residuals)
(17)
ߑሼݔොሽ= ߪො
ଶሺܣ்ܲܣ)ିଵ= ߪො
ଶܰିଵ
(Variance-Covariance Matrix)
(18)
ߪො
ଶ= ሺ்݁̃ܲ݁̃)/ሺ݊െ݉)
(A-posteriori Variance Factor)
(19) ்݁ܲ݁= ݉݅݊|௫
ݔො= ሺܣ்ܲܣ)ିଵܣ்ܲݕ= ܰିଵܣ்ܲݕ
݁̃ = ݕെܣݔො
ߑሼݔොሽ= ߪො
ଶሺܣ்ܲܣ)ିଵ= ߪො
ଶܰିଵ
ߪො
ଶ= ሺ்݁̃ܲ݁̃)/ሺ݊െ݉) ்݁ܲ݁= ݉݅݊|௫
(LSA Target Function)
(15)
ݔො= ሺܣ்ܲܣ)ିଵܣ்ܲݕ= ܰିଵܣ்ܲݕ
(Solution Vector)
(16)
݁̃ = ݕെܣݔො
(Predicted Residuals)
(17)
ߑሼݔොሽ= ߪො
ଶሺܣ்ܲܣ)ିଵ= ߪො
ଶܰିଵ
(Variance-Covariance Matrix)
(18)
ߪො
ଶ= ሺ்݁̃ܲ݁̃)/ሺ݊െ݉)
(A-posteriori Variance Factor)
(19) (15) (16) (19) For LiDAR data, there is no point-to-point correspondence between overlapping strips or between a
given strip and a control surface. Therefore, the abovementioned LSA solution cannot be used to come
up with an estimate of the biases in the system parameters. Therefore, the following subsections will
deal with the necessary modification to the stochastic model in Equation (14) to allow for the
estimation of biases in the system parameters without having conjugate points in overlapping strips. First, we will discuss the conjugate primitives that could be identified in overlapping LiDAR strips
(point-patch pairs). 2.3. Least Squares Adjustment Model and Primitives Then, we will discuss the modification of the stochastic model that would allow for
the utilization of these conjugate primitives for the estimation of biases in the system parameters. 2.3.1. Proposed Primitives In this research, one of the strips, denoted by S1, is represented by the original points while the
second strip, denoted by S2, is represented by triangular patches, which can be derived from a TIN
generation procedure. When control surface is used, it will be represented by the original control points
(due to its sparse nature) and the LiDAR strips will be represented by triangular patches. It is important
to note that the utilized discrete control points do not need be identifiable in the LiDAR surfaces. Having TIN patches with varying orientation (slope and aspect) overlapping the control points will
contribute towards better estimation of all biases. The correspondence between points in S1 and patches
in S2 is established using the closest patch procedure as shown in [26-27]. In this procedure, a TIN
patch is deemed conjugate to a given point if it is the closest patch to this point. In addition, the point-
patch separation (normal distance n) should not exceed a pre-defined threshold. Finally, the point
projection onto the patch should be located inside the TIN patch (Figure 2a). 9079 Sensors 2011, 11 Figure 2. (a) Point-patch correspondence procedure and (b) the additional unknown vector
D following the calibration procedure. Figure 2. (a) Point-patch correspondence procedure and (b) the additional unknown vector
D following the calibration procedure. Figure 2. (a) Point-patch correspondence procedure and (b) the additional unknown vector
D following the calibration procedure. (a)
(b)
For a given point-patch pair, we will assume that one of the vertices of the TIN patch in S2 is
conjugate to the corresponding point in S1. 2.3.1. Proposed Primitives Note that any vertex of the TIN patch can be selected, as
n
D
Calibration Procedure
Points in S1
Points in S2
Point-Patch Correspondence
Pseudo-conjugate points
Biased
a
'P
threshold
n <
abc
'P
Δ
∈
Corrected
P
Biased
c
Biased
b
Biased
P
Corrected
c
Corrected
b
Corrected
a (a)
n
Point-Patch Correspondence
Biased
a
'P
threshold
n <
abc
'P
Δ
∈
Biased
c
Biased
b
Biased
P (a)
(b)
n
D
Calibration Procedure
Points in S1
Points in S2
Point-Patch Correspondence
Pseudo-conjugate points
Biased
a
'P
threshold
n <
abc
'P
Δ
∈
Corrected
P
Biased
c
Biased
b
Biased
P
Corrected
c
Corrected
b
Corrected
a (a)
(b)
For a given point-patch pair, we will assume that one of the vertices of the TIN patch in S2 is
conjugate to the corresponding point in S1. Note that any vertex of the TIN patch can be selected, as
will be clarified in the next section. The TIN vertex in S2 and the point in S1 for a point-patch pair are
denoted as pseudo-conjugate points (Figure 2b). Starting from Equation (9), the mathematical model
describing the discrepancy between pseudo-conjugate points will take the form in Equation (20). The
stochastic model describing the discrepancy in Equation (20) can be represented by the Gauss Markov
Stochastic model in Equation (21). The difference between this model and the one in Equation (14) is
the additional unknown vector D resulting from using non-conjugate points along a point-patch pair
(Figure 2b). It should be noted that the additional unknown vector D is in the plane of the TIN patch
under consideration (i.e., the component of this vector along the normal to the TIN patch is zero):
n
D
Calibration Procedure
Points in S1
Points in S2
Point-Patch Correspondence
Pseudo-conjugate points
Biased
a
'P
threshold
n <
abc
'P
Δ
∈
Corrected
P
Biased
c
Biased
b
Biased
P
Corrected
c
Corrected
b
Corrected
a Biased
P threshold
n <
abc
'P
Δ
∈ threshold
n < Calibration Procedure (b)
D
Pseudo-conjugate points
Corrected
P
Corrected
c
Corrected
b
Corrected
a Corrected
b Biased
a Point-Patch Correspondence (b) (a) For a given point-patch pair, we will assume that one of the vertices of the TIN patch in S2 is
conjugate to the corresponding point in S1. 2.3.1. Proposed Primitives Note that any vertex of the TIN patch can be selected, as
will be clarified in the next section. The TIN vertex in S2 and the point in S1 for a point-patch pair are
denoted as pseudo-conjugate points (Figure 2b). Starting from Equation (9), the mathematical model
describing the discrepancy between pseudo-conjugate points will take the form in Equation (20). The
stochastic model describing the discrepancy in Equation (20) can be represented by the Gauss Markov
Stochastic model in Equation (21). The difference between this model and the one in Equation (14) is
the additional unknown vector D resulting from using non-conjugate points along a point-patch pair
(Figure 2b). It should be noted that the additional unknown vector D is in the plane of the TIN patch
under consideration (i.e., the component of this vector along the normal to the TIN patch is zero): For a given point-patch pair, we will assume that one of the vertices of the TIN patch in S2 is
conjugate to the corresponding point in S1. Note that any vertex of the TIN patch can be selected, as
will be clarified in the next section. The TIN vertex in S2 and the point in S1 for a point-patch pair are
denoted as pseudo-conjugate points (Figure 2b). Starting from Equation (9), the mathematical model
describing the discrepancy between pseudo-conjugate points will take the form in Equation (20). The
stochastic model describing the discrepancy in Equation (20) can be represented by the Gauss Markov
Stochastic model in Equation (21). The difference between this model and the one in Equation (14) is
the additional unknown vector D resulting from using non-conjugate points along a point-patch pair
(Figure 2b). It should be noted that the additional unknown vector D is in the plane of the TIN patch
under consideration (i.e., the component of this vector along the normal to the TIN patch is zero): ܺԦீಲሺܾ/݊) െܺԦீಳሺܾ/݊) = ܺԦீಲሺܶݎݑ݁) ߜܺԦீെܺԦீಳሺܶݎݑ݁) െߜܺԦீ݁
= ߜܺԦீെߜܺԦீܦ݁ (20) where: – ܦ= ܺԦீಲሺܶݎݑ݁) െܺԦீಳሺܶݎݑ݁)
ݕ= ܣݔܦ݁ ݁~ሺ0, ߑ)
ݓ݄݁ݎ݁ߑ= ߪ
ଶܲିଵ
(21) – ܦ= ܺԦீಲሺܶݎݑ݁) െܺԦீಳሺܶݎݑ݁) ) െܺԦீಳሺܶݎݑ݁) ݕ= ܣݔܦ݁ ݁~ሺ0, ߑ)
ݓ݄݁ݎ݁ߑ= ߪ
ଶܲିଵ
(21) (21) ݕ= ܣݔܦ݁ ݁~ሺ0, ߑ)
ݓ݄݁ݎ݁ߑ= ߪ
ଶܲିଵ Sensors 2011, 11 Sensors 2011, 11 9080 Using the modified weight matrix, the LSA target function can be redefined as per Equation (23). Since the additional unknown vector D belongs to the null space of the modified weight matrix
(i.e., ܲ`ܦ= 0), then the LSA target function in Equation (23) reduces to the form in Equation (24). Thus, the solution ݔො to the LSA target function is defined by Equation (25), refer to Appendix A for
more details. Using the law of error propagation, the variance-covariance matrix of the solution
vector Σሼݔොሽ is shown in Equation (26), refer to Appendix A for the detailed derivation: ்݁ܲ`݁= ሺݕെܣ ݔെܦ)்ܲ`ሺݕെܣ ݔെܦ) = ݉݅݊|௫,
LSA Target Function
(23)
்݁ܲ`݁= ሺݕെܣݔ)்ܲ`ሺݕെܣݔ) = ݉݅݊|௫
LSA Target Function
(24)
ݔො= ሺܣ்ܲ`ܣ)ିଵܣ்ܲ`ݕ= ܰିଵܣ்ܲ`ݕ
Estimated Unknowns
(25)
ߑሼݔොሽ= ߪ
ଶܰିଵ
Variance-Covariance Matrix
(26) ்݁ܲ`݁= ሺݕെܣ ݔെܦ)்ܲ`ሺݕെܣ ݔെܦ) = ݉݅݊|௫,
LSA Target Function
(23)
்݁ܲ`݁= ሺݕെܣݔ)்ܲ`ሺݕെܣݔ) = ݉݅݊|௫
LSA Target Function
(24)
ݔො= ሺܣ்ܲ`ܣ)ିଵܣ்ܲ`ݕ= ܰିଵܣ்ܲ`ݕ
Estimated Unknowns
(25)
ߑሼݔොሽ= ߪ
ଶܰିଵ
Variance-Covariance Matrix
(26) (23) (24) (25) ߑሼݔොሽ= ߪ
ଶܰିଵ (26) The last step is to estimate the a-posteriori variance factor ߪො
ଶ by deriving the expected value of the
sum of squares of weighted predicted residuals. Starting from Equation (27), one can derive an
estimate for the a-posteriori variance factor according to Equation (28), where q is the rank of the
modified weight matrix ܲ` – refer to Appendix A for the detailed derivation: ܧ൫்݁̃ܲ`݁̃൯= ܧሼሺݕെܣݔොെܦ)்ܲ`ሺݕെܣݔොെܦ)ሽ= ܧ൛ሺݕെܣݔො)்ܲ`ሺݕെܣݔො)ൟ= ሺݍെ݉)ߪ
ଶ
(27)
ߪො
ଶ= ሺݕെܣݔො)்ܲ`ሺݕെܣݔො)/ሺݍെ݉)
(28) (27) (28) In summary, from an implementation point of view, the LSA solution to the stochastic model in
Equation (29) can be derived using Equations (25), (26) and (28). This solution is similar to that of the
traditional Gauss Markov model (Equations (16), (18) and (19)) with the exception that the redundancy
is evaluated as the difference between the rank of the modified weight matrix and the number of
unknowns. 2.3.2. Modified Least Squares Adjustment 2.3.2. Modified Least Squares Adjustment The main objective for the development of the modified LSA is to deal with the model in
Equation (21) while eliminating the unknown vector D from the parameters to be estimated. To
explain the modification process, we will start by changing the stochastic properties of the random
noise vector as represented by Equation (22). As it can be seen in this equation, the unknown vector D
belongs to the null space of the modified weight matrix ܲ`. Such a condition signifies that the modified
weight matrix is not positive-definite (i.e., the inverse matrix ܲ`ିଵ does not exist). Therefore, the
modified variance-covariance matrix will be represented as follows: Σ`ሼ݁ሽ= ߪ
ଶ ܲ`ା, where the plus
sign indicates the Moore-Penrose pseudoinverse). Starting from the modified variance-covariance/weight
matrix and the LSA principles, the solution will be derived below: ߑ`ሼ݁ሽ= ߪ
ଶ ܲ`ା
where
ܲ`ܦ= 0 ߑ`ሼ݁ሽ= ߪ
ଶ ܲ`ା
where
ܲ`ܦ= 0
(22) (22) Sensors 2011, 11 Thus, the modification in the weights of the noise vector allows for the elimination of the
additional unknown vector D while having almost no impact on the traditional LSA (i.e., the solution
is obtained using the traditional solution for the Gauss Markov Model in the absence of the additional
unknown vector): ݕ= ܣݔܦ݁ ݁~൫0, ߑ`൯
ݓ݄݁ݎ݁ߑ` = ߪ
ଶܲ`ାܽ݊݀ܲ`ܦ= 0
( (29) So far, we established that by modifying the weight matrix to satisfy the condition in Equation (22),
one can derive an estimate of the biases in the system parameters while dealing with non-conjugate
points along corresponding point-patch pairs. The question now is how to derive the modified weight
matrix ܲ`. This can be established according the following procedure. First, one starts by defining a
new coordinate system (ܷܸܹ), where the ܷܸ axes are aligned along the TIN patch involved in the
current point-patch pair (i.e., W is parallel to the TIN patch normal). The relationship between the ܻܼܺ
and ܷܸܹ coordinates—assuming that the two systems share the same origin—can be expressed by
Equation (30). The rotation matrix ܯ can be defined using the direction of the normal to the TIN patch
as well as the direction of the ܷ and ܸ axes along the TIN plane (one should note that the choice of the
ܷ or ܸ directions is quite arbitrary):
ܷ
ܸ
ܹ
൩= ܯ
ܺ
ܻ
ܼ
൩
ܷ
ܸ
ܹ
൩= ܯ
ܺ
ܻ
ܼ
൩
(30) (30) Sensors 2011, 11 9081 The weight matrix of the transformed coordinates in the ܷܸܹ system can be derived from the law
of error propagation according to Equation (31). This weight matrix is then modified according to
Equation (32): ܲௐ= ܯܲܯ்=
ܲ
ܲ
ܲௐ
ܲ
ܲ
ܲௐ
ܲௐ
ܲௐ
ܲௐ
൩
(31)
ܲௐ
`
=
0
0
0
0
0
0
0
0
ܲௐ
൩
(32) (31) ܲௐ
`
=
0
0
0
0
0
0
0
0
ܲௐ
൩
(32) (32) Finally, the modified weight matrix in the ܻܼܺ coordinate system is defined by Equation (33). Using the modified weight matrix in Equation (33), one can show that ܲ
`
ܦ= 0 —Refer to
Equations (34) while noting that the ܦ vector is aligned along the TIN plane (refer to Figure 2b). Sensors 2011, 11 It
should be noted that the weight modification will be carried out for all the TIN patches of the
established correspondences; whether for point-patch pairs in overlapping strips or point-patch pairs in
LiDAR and control surfaces: ܲ
`
= ܯ்ܲௐ
`
ܯ
(33)
ܲ
`
ܦ= ܯ் ܲௐ
`
ܯ
݀ܺ
ܻ݀
ܼ݀
൩= ܯ்ܲௐ
`
ܷ݀
ܸ݀
0
൩= ܯ்
0
0
0
0
0
0
0
0
ܲௐ
൩
ܷ݀
ܸ݀
0
൩= 0
(34) (33) (34) In summary, the proposed Quasi-rigorous calibration procedure proceeds as follows: 1. The correspondence between points in S1 and patches in S2 is established using the closest
patch procedure as explained in [26] and [27]. 1. The correspondence between points in S1 and patches in S2 is established using the closest
patch procedure as explained in [26] and [27]. 2. For each conjugate point-patch pair from overlapping LiDAR strips, (e.g., patch vertex
ܺԦீಳሺܾ/݊) in S2 and point ܺԦீಲሺܾ/݊) in S1), one can write the observation equations similar to
those in Equation (11). 2. For each conjugate point-patch pair from overlapping LiDAR strips, (e.g., patch vertex
ܺԦீಳሺܾ/݊) in S2 and point ܺԦீಲሺܾ/݊) in S1), one can write the observation equations similar to
those in Equation (11). 3. When a control surface is used, it is represented by the original points (due to its sparse
nature) and the LiDAR strips are represented by triangular patches. Then, for each
conjugate point-patch pair, one can write the observation equations similar to those in
Equation (12). It is important to mention that the observation equations can be written for
all LiDAR strips which overlap with the control surface. 4. For each conjugate point-patch pair, the weight of the observed discrepancy vector
ሺܺԦீಲሺܾ/݊) െܺԦீಳሺܾ/݊)) should be modified using Equations (31–33). 5. The established correspondences and the corresponding observations with their modified
weight matrices are used to derive an estimate of the system biases. Then, the estimated
system biases are used to reconstruct an adjusted point cloud using Equation (13). 6. Following the reconstruction of the LiDAR point cloud, the correspondence between
point-patch pairs might change. Therefore, a new set of correspondences has to be
established. The new correspondences are then utilized to derive a better estimate of the
system biases. 7. Such a procedure is repeated until the corrections to the estimated calibration parameters
are almost zero. 7. 2.4. Impact of the Geometric Calibration on the Relative and Absolute Accuracy The impact of the geometric calibration on the relative accuracy of the LiDAR point cloud can be
assessed by checking the degree of compatibility between conjugate surface elements in overlapping
strips before and after reconstructing the point cloud using the estimated biases. In this work, the
compatibility will be evaluated qualitatively and quantitatively. The qualitative evaluation will be
performed by visual inspection of profiles generated using the original and adjusted point cloud to
check any improvements in the quality of fit between overlapping strips. The quantitative assessment,
on the other hand, will be performed by computing the necessary 3D transformation parameters for the
co-alignment of overlapping strips before and after the calibration procedure. For the computation of
the 3D transformation parameters, the proposed Iterative Closest Patch (ICPatch) procedure in [26]
and [27] will be employed. The impact of the geometric calibration on the absolute accuracy can be evaluated by quantifying
the degree of compatibility between LiDAR and control surfaces before and after the calibration
process. From an implementation point of view, such a procedure would not always be feasible due to
the control surface requirement. In this work, linear features extracted from the LiDAR data before and
after the calibration process are used for the geo-referencing of an image block covering the same area. The absolute accuracy of the derived ground coordinates from the geo-referenced image block is
evaluated using a check point analysis. The adopted methodologies for linear features extraction and
their utilization for the photogrammetric geo-referencing are detailed in [27] and [28], respectively. Sensors 2011, 11 One should note that each pair of pseudo-conjugate points provides three observations of the form
in Equation (11) or Equation (12). However, these three observations increase the redundancy by only
one (the rank of the modified weight matrix for these three equations is one—refer to Equation (32)). In other words, the three observations would increase the redundancy by three if and only if the 3D
discrepancy between the pseudo-conjugate points is considered. After the weight modification process,
the 2D discrepancy between the pseudo-conjugate points along the TIN plane (the unknown vector ܦ)
is ignored during the minimization process. Therefore, only the discrepancy between the
pseudo-conjugate points along the TIN normal is only minimized during the modified LSA (thus, the
three observations only increase the redundancy by one). Since the modified LSA only minimizes the
normal distance between a point in S1 and the corresponding TIN in S2, the topography in the overlap
area should have different slope and aspect values to ensure reliable estimates of the bias parameters. Sensors 2011, 11 Such a procedure is repeated until the corrections to the estimated calibration parameters
are almost zero. 9082 3. Radiometric Correction and Land Cover Classification The physical properties of the laser energy are considered with respect to the sensor configuration
and environmental parameters using the radar equation, which is proposed to model the power of the
received signal [29]. The radar equation can be presented as follows: ܲ=
ܲ௧ܦ
ଶ
4ߨݎସߚ௧
ଶߟ௦௬௦ߟ௧ߪ
(35) ܲ=
ܲ௧ܦ
ଶ
4ߨݎସߚ௧
ଶߟ௦௬௦ߟ௧ߪ (35) In this equation, the received signal power Pr depends on the transmitted signal power Pt, receiver
aperture diameter Dr, range from the sensor to the target r, laser beam width βt, system-specific factor Sensors 2011, 11 9083 ηsys, atmospheric transmission factor ηatm, and target (backscattering) cross-section σ, which depends
on the target characteristics and is defined as follows: ߪ= 4ߨ
Ωߩ௦ܣ௦
(36) (36) In the above equation, Ω is the scattering solid angle, As is the target area, and ρs is the target
spectral reflectance. The radiometric correction aims at converting the intensity value I into the spectral reflectance ρs by
considering the parameters described in the radar equation. It is assumed that the intensity I represents
the peak value of Pr and the LiDAR data providers linearly transform Pr into 8 bit values to represent
the intensity data, which is used in the radar equation [17]. The receiver aperture diameter Dr, laser
beam width βt, and system-specific factor ηsys are considered to be constant during the flight mission. The range r of each laser pulse is the distance between the instantaneous location of the sensor and the
target. This range is critical for the radiometric correction since the attenuation of the laser energy is
directly proportional to the distance of travel [19]. The scattering solid angle is derived as the cosine of
the angle between the surface normal and the direction of the laser pulse [30] based on the assumption
of a Lambertian reflectance. The transmitted laser energy Pt, though is usually unknown, can be
assumed as constant or can be related to the pulse repetition frequency (PRF) [20]. The atmospheric
attenuation ηatm depends on the temperature, pressure, and humidity during the flight survey. These
parameters can be obtained from the nearest weather observatory station. Details on the radiometric
correction process can be found in [31]. Land cover classification is conducted to evaluate the impact of the geometric calibration and the
radiometric correction of the LiDAR data on the final data products. 3. Radiometric Correction and Land Cover Classification Two datasets are prepared from
the original and modified LiDAR data after geometric calibration and radiometric correction. Each set
of data includes the interpolated digital surface model and the interpolated intensity. Four land cover
classes (tree, grass, soil and built-up area) are identified in both datasets. Using the same training sites,
Maximum likelihood classification is conducted on both datasets and finally the classification results
are evaluated by conducting an accuracy assessment using check points, which can be generated from
an orthorectified aerial photo. The overall accuracy and kappa coefficient are used to evaluate the
classification results. Sensors 2011, 11 data consists of a 3D point cloud with multiple returns in LAS format together with the trajectory data. The average point density of the LiDAR data acquired using the flying height H1 (1150 m) is
1.5 points/m2 while for the flying height H2 (540 m) the average point density is 3.7 points/m2. In the
surveyed area, thirty-seven control points were established by a GPS survey. These control points were
used for the geometric calibration and check point analysis to evaluate the absolute accuracy of the
adjusted point cloud. Figure 3. Flight and control configuration of the LiDAR dataset. 5. Results and Discussion
5.1. Geometric Calibration 4. Study Area and Dataset A real LiDAR dataset was acquired to test the feasibility of the proposed methods. The study area
covers the British Columbia Institute of Technology (BCIT) located at Burnaby, British Columbia,
Canada (122°59’W, 49°15’N). The area contains buildings and parking lots connected by sidewalks
and paved road segments. Individual shrubs and open spaces covered by grass can also be found in the
surveyed area. The LiDAR mission was conducted on July 17, 2009 from 14:37 to 15:15 local time. The day of the mission was a sunny day with a temperature of 29.8 °C. The visibility and the pressure
were 48.3 km and 101.81 kPa, respectively, as delivered by the National Climate Data and Information
Archive from Environment Canada. The LiDAR sensor used was a Leica ALS50 operating at a 1.064
μm wavelength with 0.33 mrad beam divergence. The captured LiDAR data consists of six strips that
cover a 1 km by 2 km area. The configuration of the flight lines is shown in Figure 3. The acquired 9084 5.1. Geometric Calibration 5.1. Geometric Calibration In addition to testing the feasibility of the proposed geometric calibration procedure, we would like
to investigate whether the calibration results are significantly different when using more overlapping
strip-pairs than the minimum recommended configuration as discussed in Section 2.2. Table 1 shows
the different investigated scenarios. Experiment “I” utilizes all the available overlapping strip-pairs
(4 overlapping strip-pairs) without any control information. Experiment “II”, on the other hand,
utilizes all the available overlapping strip-pairs and 37 vertical ground control points. Experiment “III”
utilizes the 4 overlapping strip-pairs with only 1 vertical control point. Finally, experiment “IV”
utilizes the minimum recommended overlapping strip-pairs configuration (3 overlapping strip-pairs)
and 37 vertical ground control points. 9085 Sensors 2011, 11 1. Experiments description (used overlapping strip-pairs and number of control points). Table 1. Experiments description (used overlapping strip-pairs and number of control p
Experiment
Overlapping Strip-Pairs Number of Control Points
I
1&2, 3&4, 4&5, 5&6
0
II
1&2, 3&4, 4&5, 5&6
37
III
1&2, 3&4, 4&5, 5&6
1
IV
1&2, 4&5, 5&6
37 Experiment
Overlapping Strip-Pairs Number of Control Points As already mentioned, for the estimation of the biases in the vertical component of the lever arm
ሺߜΔܼ) and range measurement ሺߜΔݎ), control information is necessary. However, they cannot be
estimated simultaneously due to the high correlation among them. Therefore, in the calibration
process, we only solve for the range bias while relying on the field/in-situ measurements for the
vertical component of the lever arm. For reliable estimation of the bias in the mirror angle scale ሺߜS),
well distributed data in the overlapping area in the across flight direction was manually selected for the
calibration process. Table 2 reports the estimated biases in the system parameters for the different
experiments. One can note in Table 2 that a significant bias in the boresight roll angle ሺߜΔ߮) is
detected in all experiments (refer to the highlighted cells in Table 2). One can also observe a
non-negligible bias in the boresight pitch angle ሺߜΔ߱). By comparing experiments “I” and “II”, we
can observe compatible results for the estimated mounting parameters. This is expected, since the
addition of vertical control information will mainly contribute to the estimation of the range bias. When comparing experiments “II” and “III”, we can note that with the reduction in the number of
utilized GCP, the estimated range bias is not significantly changed. 5.2. Impact of the Geometric Calibration on the Relative and Absolute Accuracy To evaluate the impact of the geometric calibration on the relative accuracy, the compatibility of
overlapping strips before and after the calibration procedure (using the different experiment scenarios)
is assessed. The compatibility of the point cloud is evaluated qualitatively and quantitatively. The
qualitative evaluation is performed by visual inspection of profiles generated using the original and
adjusted point cloud to check any improvements in the quality of fit between overlapping strips. The
improvement in the strips compatibility is illustrated in Figure 4, which shows a profile covered by
strips 1 (in blue), 2 (in red), 3 (in green), and 4 (in magenta) along the X direction before and after the
calibration procedure using the different scenarios. The quantitative assessment, on the other hand, is
performed by computing the necessary 3D transformation parameters for the co-alignment of
overlapping strips before and after the calibration procedure. The computed transformation parameters
are reported in Table 3. As it can be seen in this table, large discrepancies can be observed before the
calibration procedure, especially in the across flight direction between strips flown in opposite
directions (XT direction for strips 1&2, and YT direction for strips 3&4 and strips 4&5—refer to the
circled cells in Table 3). This is expected since a larger bias was estimated in the boresight roll angle,
which mainly affects the across-flight direction, i.e., constant shift across the flight direction and a
rotation around the flight direction [27]. One can also observe significant improvement after the
calibration procedure for such strips in the across and along flight directions (refer to the highlighted
cells in Table 3) due to the accurate estimation of the biases in the boresight roll and pitch angles. On
the other hand, minor improvement can be observed for strips 5&6. This is due to the fact that for
strips flown in the same direction the boresight roll bias only causes a constant vertical shift between
conjugate surfaces elements with a much smaller magnitude while the boresight pitch bias would not
lead to any discrepancies. The qualitative and quantitative evaluations demonstrate compatible results from the different
investigated scenarios. One should finally note that the range bias, which was removed in the
experiments using control information, does not lead to significant discrepancies among conjugate
surface elements in overlapping strips. Sensors 2011, 11 Sensors 2011, 11 Sensors 2011, 11 9086 5.2. Impact of the Geometric Calibration on the Relative and Absolute Accuracy 5.1. Geometric Calibration Hence, one can conclude that a
single vertical GCP would be sufficient for the estimation of the range bias as long as it is observed in
several strips (in this dataset, the control point was visible in 5 strips). Finally, experiment IV shows
that the recommended minimum flight configuration is capable of producing reliable estimates for the
biases in question. In experiment IV, the estimated parameters, which are showing the largest variation
when compared to the estimated parameters in experiments I, II, and III, are the boresight pitch and
heading biases. This can be attributed to a correlation of 0.63 between the boresight heading and pitch
biases and a correlation of 0.72 between the boresight heading bias and the planimetric component of
the lever arm in the along-flight direction. In experiments I, II, and III, these correlations are close to
zero. This increased correlation can be explained by the fact that the overlapping strip-pair 5&6, which
is the overlapping strip-pair whose configuration contributes most towards the estimation of the
boresight heading bias (i.e., parallel strips flown in the same direction), didn’t have enough features in
the overlapping region with topography/buildings exhibiting varying slope and aspect. Therefore the
addition of one more overlapping strip-pair improved the results. Table 2. Estimated biases in the system parameters for the different experiments. Table 2. Estimated biases in the system parameters for the different experiments. Experiment
ࢾઢࢄ
(m)
ࢾઢࢅ
(m)
ࢾઢ࣓
(")
ࢾઢ࣐
(")
ࢾઢࣄ
(")
ࢾઢ࣋
(m)
ࢾ܁
I
0.01
0.00
−29.5
−88.7
3.0
-
0.0002656
II
0.00
0.00
−29.0
−91.0
3.6
0.118
0.00010377
III
0.01
0.00
−29.5
−89.6
3.5
0.142
0.00009348
IV
0.02
0.05
−38.8
−90.7
−32.1
0.115
0.00003607 5.2. Impact of the Geometric Calibration on the Relative and Absolute Accuracy Therefore, we cannot evaluate the introduced improvement
when adding the range bias in the calibration process by checking the compatibility among
overlapping strip-pairs. Such an analysis is done next in the absolute accuracy verification. The qualitative and quantitative evaluations demonstrate compatible results from the different
investigated scenarios. One should finally note that the range bias, which was removed in the
experiments using control information, does not lead to significant discrepancies among conjugate
surface elements in overlapping strips. Therefore, we cannot evaluate the introduced improvement
when adding the range bias in the calibration process by checking the compatibility among
overlapping strip-pairs. Such an analysis is done next in the absolute accuracy verification. For all investigated scenarios, significant improvement in the planimetric accuracy can be observed
(refer to the highlighted cells). This can be explained by the fact that the main detected bias in the
studied dataset is in the boresight roll and pitch angles, which mostly affect the horizontal accuracy. One can also note that when reducing the number of utilized overlapping strip-pairs (experiment IV),
the results are not negatively affected. For the experiments using control information (experiments II,
III, and IV), where we solved for the range bias, we can observe that the bias value in the vertical
direction has been significantly reduced (refer to the circled values in Table 4). Similar to what has
been established in the relative accuracy evaluation, increasing the number of ground control points
did not improve the absolute accuracy. 9087 Sensors 2011, 11 Sensors 2011, 11 Figure 4. Profiles along the X direction over a building covered by strips “1” (in blue), “2”
(in red), “3” (in green), and “4” (in magenta); (a) Before the calibration procedure;
(b) After the calibration procedure using the configuration in “I”; (c) After the calibration
procedure using the configuration in “II”; (d) After the calibration procedure using the
configuration in “III”; and (e) After the calibration procedure using the configuration
in “IV”. Figure 4. Profiles along the X direction over a building covered by strips “1” (in blue), “2”
(in red), “3” (in green), and “4” (in magenta); (a) Before the calibration procedure;
(b) After the calibration procedure using the configuration in “I”; (c) After the calibration
procedure using the configuration in “II”; (d) After the calibration procedure using the
configuration in “III”; and (e) After the calibration procedure using the configuration
in “IV”. (a)
(b)
(c)
(d)
(e)
1m
1m
1m
1m
1m (a) (c) 088 088 e and after applying the calibration parameters estimated using the different scenarios
After Calibration
II
III
IV
1&2
XT (m)
YT (m)
ZT(m)
XT (m)
YT (m)
ZT(m)
XT (m)
YT (m)
ZT(m
0.0976
0.0266
−0.0495
0.1025
0.0297
−0.0489
0.0845
0.0211
−0.048
ω(deg)
φ(deg)
κ(deg)
ω(deg)
φ(deg)
κ(deg)
ω(deg)
φ(deg)
κ(deg
0.001
−0.0032
−0.0041
0.001
−0.004
−0.0041
0.0011
−0.004
−0.003
3&4
XT (m)
YT (m)
ZT(m)
XT (m)
YT (m)
ZT(m)
XT (m)
YT (m)
ZT(m
−0.0098
0.0467
−0.0042
−0.01
0.0378
−0.0044
0.0046
0.0589
−0.003
ω(deg)
φ(deg)
κ(deg)
ω(deg)
φ(deg)
κ(deg)
ω(deg)
φ(deg)
κ(deg
−0.0003
−0.0008
0.0022
−0.0014
−0.0007
0.0026
0.001
−0.0008
0.002
4&5
XT (m)
YT (m)
ZT(m)
XT (m)
YT (m)
ZT(m)
XT (m)
YT (m)
ZT(m
−0.0107
0.0128
−0.024
−0.0106
0.0024
−0.0244
−0.1032
0.0212
−0.026
ω(deg)
φ(deg)
κ(deg)
ω(deg)
φ(deg)
κ(deg)
ω(deg)
φ(deg)
κ(deg
−0.0053
0.0009
0.0026
−0.0049
0.0009
0.0026
−0.0027
0.0008
0.002
5&6
XT (m)
YT (m)
ZT(m)
XT (m)
YT (m)
ZT(m)
XT (m)
YT (m)
ZT(m
0.0375
0.0606
−0.0281
0.0379
0.0536
−0.0271
−0.002
0.0702
−0.024
ω(deg)
φ(deg)
κ(deg)
ω(deg)
φ(deg)
κ(deg)
ω(deg)
φ(deg)
κ(deg
−0.0008
−0.0009
−0.0087
−0.0012
−0.0009
−0.0088
0.0011
−0.0009
−0.008 9089 Sensors 2011, 11 Sensors 2011, 11 To further assess the impact of the radiometric correction, the intensity values before and after
radiometric correction are compared for different land cover classes that have been identified with the
help of an overlapping orthophoto of the study area. This comparison is conducted to check whether
the proposed procedure would have any impact on the homogeneity and separability of the
investigated classes. Table 5 shows the mean and standard deviation of the intensity values for four
land cover classes, which are used to investigate if there is any overlap of intensity values amongst
different land cover features. In the original intensity data, there is an overlap amongst all the land
cover classes. After radiometric correction, the mean values of the built-up areas, grassland, and soil
are reduced by 17% to 24%. The standard deviation values are also reduced for these three classes by
20% to 47%. There is also significant change in the intensity and standard deviation values for the
grassland and tree classes. For the tree class, the standard deviation after radiometric correction is
significantly increased (from 9.3 to 44.2) and the mean intensity value changed from 21.4 to 82.7. Despite this significant increase in the standard deviation of the intensity value after radiometric
correction, the intensity range of the tree class does not have any overlap with the other three land
cover classes. These results indicate that the homogeneity within most of the specified land cover
classes is enhanced and the separability amongst all the different land cover classes is improved. Table 5. Mean and standard deviation of the intensity data for different land cover classes
before and after radiometric correction. 5. Mean and standard deviation of the intensity data for different land cover classes
and after radiometric correction. Before Correction
After Correction
Built-Up Area
13.9 ± 4.1
10.6 ± 3.3
Grassland
40.6 ± 9.5
32.7 ± 5.7
Soil
24.7 ± 5.1
20.5 ± 2.7
Tree
21.4 ± 9.3
82.7 ± 44.2
5.4. Impact of the Geometric Calibration and Radiometric Correction on Land Cover Classification 5.4. Impact of the Geometric Calibration and Radiometric Correction on Land Cover Classification Using more than 1,000 check points that have been identified with the help of an orthophoto over
the study area, we compared the four-land cover classification results (Tree, Built-up Area, Grass, and
Soil) with the manual classification of the check points. Sensors 2011, 11 Sensors 2011, 11 Table 4. RMSE analysis of the photogrammetric check points using extracted control
linear features from the LiDAR data before and after the calibration procedure. Before Calibration
After Calibration
I
II
III
IV
Mean ΔX (m)
−0.03
0.00
0.01
0.03
−0.01
Mean ΔY (m)
−0.18
0.04
0.03
0.04
−0.01
Mean ΔZ (m)
0.15
0.13
0.02
−0.01
0.02
σX (m)
0.11
0.06
0.06
0.06
0.06
σY (m)
0.15
0.06
0.07
0.06
0.07
σZ (m)
0.17
0.17
0.18
0.17
0.17
RMSEX (m)
0.11
0.05
0.06
0.06
0.06
RMSEY (m)
0.23
0.07
0.07
0.07
0.07
RMSEZ (m)
0.23
0.22
0.17
0.17
0.17
RMSETOTAL (m)
0.34
0.23
0.20
0.19
0.19
5.3. Radiometric Correction 5.3. Radiometric Correction To assess the impact of radiometric correction, the variance-to-mean ratio of the intensity values for
the whole dataset are plotted against the surface slope before and after the radiometric correction
(Figure 5). The variance-to-mean ratio represents the inverse of the signal-to-noise ratio where small
amount of variance-to-mean ratio implies less noise within the data. Therefore, a reduction in the
variance-to-mean ratio indicates a reduction in the noise within the intensity values. The purpose of
plotting this ratio against the slope is to evaluate the variation of the intensity values with respect to the
different topographic conditions. As can be seen in Figure 5, the variance-to-mean ratio is reduced
after the radiometric correction by approximately 20% to 30% (therefore, improved signal content is
achieved by the radiometric correction). In general, the reduction is larger when dealing with slopes of
30° or less. For slopes greater than 30°, less pronounced reduction can be observed. This can be
explained by the fact that the majority of features with slope greater than 30° belong to tree clusters
where high variance of the intensity value is expected due to the irregular tree canopy. Figure 5. Variance-to-mean ratio of the intensity data (before and after radiometric
correction) for different slopes. 6
8
10
12
14
16
18
20
3
9
15
21
27
33
39
45
51
Variance-to-mean ratio
Slope (Deg.)
Original Intensity
Radiometrically Corrected Intensity 6
8
10
12
14
16
18
20
3
9
15
21
27
33
39
45
51
Variance-to-mean ratio
Slope (Deg.)
Original Intensity
Radiometrically Corrected Intensity 9090 Sensors 2011, 11 Tables 6 and 7 show the confusion matrix,
overall accuracy, and kappa coefficient of the classification results for the original and the
geometrically calibrated/radiometrically corrected LiDAR data. Table 6. Confusion matrix of the classification results using original LiDAR dataset
Tree Built-up
Grass
Soil
Total
KC
Tree
130
40
20
18
208
0.464
Built-up
119
386
12
16
533
0.486
Grass
34
26
59
43
162
0.294
Soil
20
16
10
61
107
0.502
Total
303
468
101
138
1010
Overall Accuracy = 63.0%
Average Kappa Coefficient (KC) = 0.442 Table 6. Confusion matrix of the classification results using original LiDAR dataset. 9091 Sensors 2011, 11 Table 7. Confusion matrix of the classification result using geometrically calibrated and
radiometrically corrected LiDAR dataset. Tree Built-up
Grass
Soil
Total
KC
Tree
153
23
12
4
192
0.725
Built-up
62
399
23
23
507
0.593
Grass
37
35
94
26
192
0.402
Soil
11
24
19
65
119
0.486
Total
263
481
148
118
1010
Overall Accuracy = 70.4%
Average Kappa Coefficient (KC) = 0.558 The overall classification accuracy using the intensity from the original LiDAR data is about 63.0%. After geometric calibration and radiometric correction, the overall accuracy increased to 70.5%. The
kappa coefficient has also increased from 0.442 to 0.558. Considering each individual land cover class,
the kappa coefficient of the tree and built-up features are found to be always lower than 0.5 using the
original LiDAR dataset. After geometric calibration and radiometric correction, the kappa coefficient
of the tree class increases from 0.464 to 0.725 and the kappa coefficient of the built-up class increases
from 0.486 to 0.593. This improvement in the classification results is due to the high separability of the
intensity values between different class features after the radiometric correction. A slight improvement
of the kappa coefficient of the grass class can also be detected after the geometric calibration and
radiometric correction. It can be also observed that the proposed process helps in reducing the
confusion between grass and tree classes in LiDAR data classification. Figure 6. Comparison of classification results of original and the geometrically calibrated
and radiometerically corrected LiDAR dataset. Sensors 2011, 11 (c) Original Intensity
(d) GC & RC Intensity
(a) Original Intensity
(b) GC & RC Intensity (a) Original Intensity (b) GC & RC Intensity (c) Original Intensity (d) GC & RC Intensity Sensors 2011, 11 9092 Figure 6 shows the orthophoto used in the accuracy assessment process and the classification results
of the intensity data before and after the geometric calibration and radiometric correction of the
LiDAR data. Figure 6 shows the orthophoto used in the accuracy assessment process and the classification results
of the intensity data before and after the geometric calibration and radiometric correction of the
LiDAR data. One can note a significant impact of the geometric calibration and radiometric correction in the tree
class areas. In Figure 6a, the tree cluster along the main road is misclassified as built-up area using the
original intensity data. However, the intensity values of tree clusters have significantly increased after
radiometric correction. This leads to correct classification result as shown in Figure 6b since the
difference of intensity values between built-up area and tree has increased after radiometric correction. In figure 6c, the roof top in the study area is entirely misclassified as tree in the result from the original
intensity data. The results are improved after using the geometrically calibrated and radiometrically
corrected intensity data (Figure 6d). Generally, the results show that the geometric calibration and
radiometric correction of the airborne LiDAR data has a significant positive impact on the
classification process, which would ultimately lead to better feature extraction and object recognition. Sensors 2011, 11 Sensors 2011, 11 used for radiometric correction of the intensity data. The correction considers the system parameters,
topographic effect, and atmospheric attenuation. After the radiometric correction, the homogeneity of
the land cover classes is improved and the variance-to-mean ratio of the intensity data is reduced. It
also enhances the separability amongst the different land cover classes. Finally, land cover
classification is performed to investigate the impacts of the geometric calibration and radiometric
correction on the classification process. A 7.5% accuracy improvement has been achieved in the
classification results after conducting the proposed procedures. Future work will focus on more testing using real datasets from operational systems. Also, the
quasi-rigorous geometric calibration will be extended to include the attitude information in the
calibration process. Furthermore, an automated procedure for the identification of useful areas within
the data for reliable and faster estimation of the parameters will be implemented in the geometric
calibration process. In addition, radiometric correction and land cover classification will be
investigated by using full-waveform LiDAR data as it provides additional information (such as the
transmitted laser pulse, the echo width, the cross section of the echo, etc.) comparing to the traditional
multi-return LiDAR data. It is expected that the full-waveform LiDAR data will improve the point
cloud density and the dimensionality of feature space leading to better classification and segmentation. Acknowledgments This research work is supported by a Discovery Grant from the Natural Sciences and Engineering
Research Council of Canada (NSERC) and the GEOIDE Canadian Network of Excellence, Strategic
Investment Initiative (SII) project SII P-IV # 72. The authors would like to thank McElhanney
Consulting Services Ltd, BC, Canada for providing the real LiDAR and image datasets. Also, the
authors are indebted to Dan Tresa, McElhanney Consulting Services Ltd, for the valuable feedback. 6. Conclusions and Recommendations for Future Work In this research, methodologies for the geometric calibration and radiometric correction of the
LiDAR system and collected data have been presented. The introduced geometric calibration
procedure is denoted as the Quasi-rigorous due the fact that few reasonable assumptions are made for
its development. This method only assumes that we are dealing with an almost vertical LiDAR system,
which is quite realistic for flight missions with a steady platform. To conduct such a calibration, we
require time-tagged point cloud and trajectory position data. In contrast to the position and orientation
information requirement for each pulse in the rigorous calibration, the Quasi-rigorous procedure only
requires a sample of the trajectory positions at a much lower rate. Access to this type of data is not a
concern. Since this calibration procedure derives approximations of some of the system raw
measurements, the proposed procedure can provide as a by-product the necessary information for the
radiometric correction of the LiDAR intensity data when system raw measurements are not available
(i.e., improved scan mirror angles and ranges). It is important to mention that the introduced method is
based on appropriate primitives (point-patch pairs) and do not require pre-processing of the LiDAR
data. Moreover, the developed procedure provided a detailed analysis of the optimum flight and
control configuration for reliable estimation of residual biases in the system parameters. The impact of
the calibration process on the relative and absolute accuracy has been verified. The impact on the
relative accuracy was evaluated by assessing the degree of compatibility between conjugate surface
elements in overlapping strips before and after the geometric system calibration. The qualitative and
quantitative assessments have demonstrated a significant improvement in the quality of fit between
overlapping strips. The impact on the absolute accuracy was assessed by using the LiDAR data for
photogrammetric georeferencing before and after performing the proposed geometric calibration
procedure. The outcome of the photogrammetric reconstruction was evaluated through check point
analysis. Significant improvement in the horizontal and vertical accuracy was demonstrated after
removing the effect of estimated biases in the system parameters. Radiometric correction of the
intensity data is also conducted on the geometrically calibrated LiDAR data to reduce the effects of
atmospheric attenuation and scanning configuration. A physical model based on the radar equation is 9093 Sensors 2011, 11 Sensors 2011, 11 Sensors 2011, 11 9094 8. Maas, H.G. Method for measuring height and planimetry discrepancies in airborne laserscanner
data. Photogramm. Eng. Remote Sens.2002, 68, 933-940. 8. Maas, H.G. Method for measuring height and planimetry discrepancies in airborne laserscanner
data. Photogramm. Eng. Remote Sens.2002, 68, 933-940. 9. Filin, S.; Vosselman, G. Adjustment of airborne laser altimetry strips. Int. Arch. Photogramm. Remote Sens. 2004, 35, 285-289. 9. Filin, S.; Vosselman, G. Adjustment of airborne laser altimetry strips. Int. Arch. Photogramm. Remote Sens. 2004, 35, 285-289. 10. Filin, S. Calibration of Spaceborne and Airborne Laser Altimeters Using Natural Surfaces. Ph.D. Dissertation, The Ohio-State University, Columbus, OH, USA, 2001. 10. Filin, S. Calibration of Spaceborne and Airborne Laser Altimeters Using Natural Surfaces. Ph.D. Dissertation, The Ohio-State University, Columbus, OH, USA, 2001. 11. Skaloud, J.; Lichti, D. Rigorous approach to bore-sight self-calibration in airborne laser scanning. ISPRS J. Photogramm. Remote Sens. 2006, 61, 47-59. 12. Friess, P. Toward a rigorous methodology for airborne laser mapping. In Proceedings of
EuroCOW, Castelldefels, Spain, January 2006; [CD-ROM]. 13. Burman, H. Calibration and Orientation of Airborne Image and Laser Scanner Data Using GPS
and INS. Ph.D. Thesis, Royal Institute of Technology, Stockholm, Sweden, 2000. 14. Toth, C.K. Calibrating airborne lidar systems. In Proceedings of ISPRS Commission II
Symposium, Xi’an, China, August 2002; pp. 475-480. 15. Morin, K.W. Calibration of Airborne Laser Scanners. M.Sc. Thesis, University of Calgary,
Alberta, Canada, 2002. 16. Coren, F.; Sterzai, P. Radiometric correction in laser scanning. Int. J. Remote Sens. 2006, 27,
3097-3104. 17. Höfle, B.; Pfeifer, N. Correction of laser scanning intensity data: Data and model-driven
approaches. ISPRS J. Photogramm. Remote Sens. 2007, 62, 415-433. 18. Kaasalainen, S.; Hyyppä, H.; Kukko, A.; Litkey, P.; Ahokas, E.; Hyyppä, J.; Lehner, H.; Jaakkola,
A.; Suomalainen, J.; Akujärvi, A.; et al. Radiometric calibration of LIDAR Intensity with
commercially available reference targets. IEEE Trans. Geosci. Remote Sens. 2009, 47, 588-598. 19. Kaasalainen, S.; Krooks, A.; Kukko, A.; Kaartinen, H. Radiometric calibration of terrestrial laser
scanners with external reference targets. Remote Sens. 2009, 1, 144-158. 20. Vain, A.; Kaasalainen, S.; Pyysalo, U.; Krooks, A.; Litkey, P. Use of naturally available reference
targets to calibrate airborne laser scanning intensity data. Sensors 2009, 9, 2780-2796. 21. Kaasalainen, M.; Kaasalainen, S. Aperture size effects on backscatter intensity measurements in
Earth and space remote sensing. J. Opt. Soc. Am. 2008, 25, 1142-1146. 22. Vain, A.; Yu, X.; Kaasalainen, S.; Hyyppä, J. References 1. Shimada, M. Radiometric and geometric calibration of JERS-1 SAR. Adv. Space Res. 1996, 17,
79-88. 2. Small, D.; Holecz, F.; Meier, E.; Nüesch, D.; Barmettler, A. Geometric and radiometric calibration
of RADARSAT Images. In Proceedings of Geomatics in the Era of RADARSAT, Ottawa, Canada,
24-30 May 1997; [CD-ROM]. 2. Small, D.; Holecz, F.; Meier, E.; Nüesch, D.; Barmettler, A. Geometric and radiometric calibration
of RADARSAT Images. In Proceedings of Geomatics in the Era of RADARSAT, Ottawa, Canada,
24-30 May 1997; [CD-ROM]. 3. Riegler, G.; Stolz, R.; Mauser, W. Geometric and radiometric corrections of ERS SAR data for
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image products. Remote Sens. Environ. 2007, 106, 337-349. 5. Loew, A.; Mauser, W. Generation of geometrically and radiometrically terrain corrected SAR
image products. Remote Sens. Environ. 2007, 106, 337-349. 6. Kilian, J.; Haala, N.; Englich, M. Capture and evaluation of airborne laser scanner data. Int. Arch. Photogramm. Remote Sens. 1996, 31, 383-388. 6. Kilian, J.; Haala, N.; Englich, M. Capture and evaluation of airborne laser scanner data. Int. Arch. Photogramm. Remote Sens. 1996, 31, 383-388. 7. Crombaghs, M.; De Min, E.; Bruegelmann, R. On the adjustment of overlapping strips of laser
altimeter height data. Int. Arch. Photogramm. Remote Sens. 2000, 33, 230-237. 7. Crombaghs, M.; De Min, E.; Bruegelmann, R. On the adjustment of overlapping strips of laser
altimeter height data. Int. Arch. Photogramm. Remote Sens. 2000, 33, 230-237. Appendix A Modified Least Squares Sensors 2011, 11 9095 Sensors 2011, 11 28. Shin, S.; Habib, A.; Ghanma, M.; Kim, C.; Kim, E. Algorithms for Multi-Sensor and Multi-
Primitive Photogrammetric Triangulation. ETRI Journal 2007, 29, 411-420. 29. Baltsavias, E.P. Airborne laser scanning: Basic relations and formulas. ISPRS J. Photogramm. Remote Sens. 1999, 54, 199-214. 30. Shaker, A.; Yan, W.Y.; El-Ashmawy, N. The effects of laser reflection angle on radiometric
correction of airborne LiDAR intensity data. In Proceedings of ISPRS Workshop Laser Scanning
2011, Calgary, Canada, August 2011; [CD-ROM]. 31. Yan, W.Y.; Shaker, A.; Habib, A.; Kersting, A.P. Improving classification accuracy of airborne
LiDAR intensity data by geometric calibration and radiometric correction. ISPRS J. Photogramm. Remote Sens., submitted. 32. Koch, K.R. Parameter Estimation and Hypothesis Testing In Linear Models, 1st ed.;
Springer-Verlag New York: Inc. New York, NY, USA, 1988; p. 378. 32. Koch, K.R. Parameter Estimation and Hypothesis Testing In Linear Models, 1st ed.;
Springer-Verlag New York: Inc. New York, NY, USA, 1988; p. 378. 1. Model ݕ= ܣݔܦ݁ ݁~൫0, ߑ`൯
ݓ݄݁ݎ݁ߑ` = ߪ
ଶܲ`ାܽ݊݀ܲ`ܦ= 0 (A.1) ݕ= ܣݔܦ݁ ݁~൫0, ߑ൯
ݓ݄݁ݎ݁ߑ= ߪ
ଶܲାܽ݊݀ܲܦ= 0
(A.1)
2. LSA Target Function ߶ሺݔ, ܦ) 2. LSA Target Function ߶ሺݔ, ܦ) ߶ሺݔ, ܦ) = ்݁ܲ`݁= ሺݕെܣݔെܦ)்ܲ`ሺݕെܣݔെܦ) = ݉݅݊|௫,
(A.2) (A.2) Since ܲ`ܦ= 0: Since ܲ`ܦ= 0: Since ܲ`ܦ= 0: Since ܲܦ
0:
߶ሺݔ, ܦ) = ߶ሺݔ) = ்݁ܲ`݁= ሺݕെܣݔ)்ܲ`ሺݕെܣݔ) = ݉݅݊|௫
(A.3)
Expanding Equation (A.3) we get:
߶ሺݔ)
ሺݕ
ܣݔ)்ܲ`ሺݕ
ܣݔ)
ݕ்ܲ`ݕ
ݕ்ܲ`ܣݔ
ݔ்ܣ்ܲ`ݕݔ்ܣ்ܲ`ܣݔ
݉݅݊|
(A 4) ߶ሺݔ, ܦ) = ߶ሺݔ) = ்݁ܲ`݁= ሺݕെܣݔ)்ܲ`ሺݕെܣݔ) = ݉݅݊|௫
(A.3) ߶ሺݔ, ܦ) = ߶ሺݔ) = ்݁ܲ`݁= ሺݕെܣݔ)்ܲ`ሺݕെܣݔ) = ݉݅݊|௫ (A.3) Expanding Equation (A.3) we get: ߶ሺݔ) = ሺݕെܣݔ)்ܲ`ሺݕെܣݔ) = ݕ்ܲ`ݕെݕ்ܲ`ܣݔെݔ்ܣ்ܲ`ݕݔ்ܣ்ܲ`ܣݔ= ݉݅݊|௫
(A.4)
E
ti
(A 3)
b
i
lifi d t െܣݔ)்ܲ`ሺݕെܣݔ) = ݕ்ܲ`ݕെݕ்ܲ`ܣݔെݔ்ܣ்ܲ`ݕݔ்ܣ்ܲ`ܣݔ= ݉݅݊|௫
(A.4) ߶ሺݔ) = ሺݕെܣݔ)்ܲ`ሺݕെܣݔ) = ݕ்ܲ`ݕെݕ்ܲ`ܣݔെݔ்ܣ்ܲ`ݕݔ்ܣ்ܲ`ܣݔ= ݉݅݊|௫
(A.4) Equation (A.3) can be simplified to: Equation (A.3) can be simplified to: Equation (A.3) can be simplified to: Equation (A.3) can be simplified to: ߶ሺݔ) = ݕ்ܲ`ݕݔ்ܣ்ܲ`ܣݔെ2ݔ்ܣ்ܲ`ݕ= ݉݅݊|௫
(A.5) (A.5) 3. Solution Vector ݔො Sensors 2011, 11 Correcting airborne laser scanning intensity data for
automatic gain control effect. IEEE Geosci. Remote Sens. Lett. 2010, 7, 511-514. 23. Schenk, T. Modeling and Analyzing Systematic Errors in Airborne Laser Scanners; Technical
Report in Photogrammetry No. 19; Ohio State University: Columbus, OH, USA, 2001; pp. 1-42. 24. El-Sheimy, N.; Valeo, C.; Habib, A. Digital Terrain Modeling: Acquisition, Manipulation and
Applications, 1st ed.; Artech House Remote Sensing Library: Boston, MA, USA, 2005; p. 256. 25. Mikhail, E.M.; Ackerman, F. Observations and Least Squares. University Press of America: New
York, NY, USA; 1976. 26. Habib, A.; Bang K.; Kersting, A.P.; Lee, D.C. Error budget of lidar systems and quality control of
the derived data. Photogramm. Eng. Remote Sens.2009, 75, 1093-1108. 27. Habib, A.; Kersting, A.P.; Bang, K.; Lee, D.C. Alternative methodologies for the internal quality
control of parallel LiDAR strips. IEEE Trans. Geosci. Remote Sens. 2010, 48, 221-236. Modified Least Squares 1. Model 1. Model 5. A-posteriori variance factor ߪො
ଶ The a-posteriori variance factor ߪො
ଶ is obtained by deriving the expected value of the sum squares of the
weighted predicted residuals: The a-posteriori variance factor ߪො
ଶ is obtained by deriving the expected value of the sum squares of the
weighted predicted residuals: ܧ൫்݁̃ܲ`݁̃൯= ܧሼሺݕെܣݔොെܦ)்ܲ`ሺݕെܣݔොെܦ)ሽ
(A.10) (A.10) Since ܲ`ܦ= 0, Equation (A.10) gets the form: Since ܲ`ܦ= 0, Equation (A.10) gets the form: ܧ൫்݁̃ܲ`݁̃൯= ܧ൛ሺݕെܣݔො)்ܲ`ሺݕെܣݔො)ൟ
(A.11) (A.11) Expanding Equation (A.11) while using the derived solution for ݔො in Equation (A.7) we get:
ܧ൫்݁̃ܲ`݁̃൯= ܧ൛ݕ்ܲ`ݕെݕ்ܲ`ܣܰିଵܣ்ܲ`ݕൟ
(A.12) ܧ൫்݁̃ܲ`݁̃൯= ܧ൛ݕ்ܲ`ݕെݕ்ܲ`ܣܰିଵܣ்ܲ`ݕൟ
(A.12) (A.12) Given that the trace of a scalar equals to the scalar, i.e., tr(S) = S and that the trace operation is
commutative, i.e., tr(AB) = tr(BA) [32], Equation (A.12) can be manipulated as follows: Given that the trace of a scalar equals to the scalar, i.e., tr(S) = S and that the trace operation is
commutative, i.e., tr(AB) = tr(BA) [32], Equation (A.12) can be manipulated as follows: ܧ൫்݁̃ܲ`݁̃൯= ܧ൛ݐݎ൫ܲ`ݕݕ்൯െݐݎሺܲ`ܣܰିଵܣ்ܲ`ݕݕ்)ൟ
(A.13) ܧ൫்݁̃ܲ`݁̃൯= ܧ൛ݐݎ൫ܲ`ݕݕ்൯െݐݎሺܲ`ܣܰିଵܣ்ܲ`ݕݕ்)ൟ
(A.13) (A.13) Based on the properties that tr(A) + tr(B) = tr(A+B) and that ܧሼݐݎሺܣ)ሽ= ݐݎܧሺܣ) [32], Equation
(A.13) can be rewritten as follows: Based on the properties that tr(A) + tr(B) = tr(A+B) and that ܧሼݐݎሺܣ)ሽ= ݐݎܧሺܣ) [32], Equation
(A.13) can be rewritten as follows: ܧ൫்݁̃ܲ`݁̃൯= ݐݎܲ`ൣܧሺݕݕ்) െܣܰିଵܣ்ܲ`ܧሺݕݕ்)൧= ݐݎܲ`൫ܫെܣܰିଵܣ்ܲ`൯ܧሺݕݕ்)
(A.14)
where I୬ is an ݊ݔ݊ identity matrix. (A.14) where I୬ is an ݊ݔ݊ identity matrix. The term ܧሺݕݕ்) can be derived from the variance-covariance matrix of the observations vector ߑሼݕሽ
as follows: The term ܧሺݕݕ்) can be derived from the variance-covariance matrix of the observations vector ߑሼݕሽ
as follows: ߑሼݕሽ= ߪ
ଶܲ`ା= ܧሼሺݕെܣݔെܦ)ሺݕെܣݔെܦ)்ሽ
(A.15) (A.15) Expanding Equation (A.15) we get: Expanding Equation (A.15) we get: ܧሺݕݕ்) = ߪ
ଶܲ`ାሺܣݔܦ)ሺܣݔܦ)்= ߪ
ଶܲ`ାܣݔݔ்ܣ்ܣݔܦ்ܦݔ்ܣ்ܦܦ்
(A.16)
S b i
i
E
i
(A 16) i E
i
(A 14) i ld ݕݕ்) = ߪ
ଶܲ`ାሺܣݔܦ)ሺܣݔܦ)்= ߪ
ଶܲ`ାܣݔݔ்ܣ்ܣݔܦ்ܦݔ்ܣ்ܦܦ் Substituting Equation (A.16) in Equation (A.14) yields: Substituting Equation (A.16) in Equation (A.14) yields:
ܧ൫்݁̃ܲ`݁̃൯= ݐݎܲ`൫ܫെܣܰିଵܣ்ܲ`൯ൣߪ
ଶܲ`ାܣݔݔ்ܣ்ܣݔܦ்ܦݔ்ܣ்ܦܦ்൧
(A.17)
Given that P`ܦ= 0, Equation (A.17) can be simplified to:
ܧ൫்݁̃ܲ`݁̃൯= ߪ
ଶݐݎܲ`൫ܫെܣܰିଵܣ்ܲ`൯ܲ`ା=ߪ
ଶݐݎܲ`ܲ`ାെߪ
ଶݐݎܰିଵܣ்ܲ`ܲ`ାܲ`ܣ
(A.18) ܲ`݁̃൯= ݐݎܲ`൫ܫെܣܰିଵܣ்ܲ`൯ൣߪ
ଶܲ`ାܣݔݔ்ܣ்ܣݔܦ்ܦݔ்ܣ்ܦܦ்൧
(A. 3. Solution Vector ݔො The solution vector ݔො that minimizes ߶ሺݔ) can be obtained by differentiating ߶ሺݔ) w.r.t. ݔ and
equating it to zero: The solution vector ݔො that minimizes ߶ሺݔ) can be obtained by differentiating ߶ሺݔ) w.r.t. ݔ and
equating it to zero: ߲߶
߲ݔ= 2ܣ்ܲ`ܣݔെ2ܣ்ܲ`ݕ= 0
(A.6)
ݔො= ൫ܣ்ܲ`ܣ൯
ିଵܣ்ܲ`ݕ= ܰିଵܣ்ܲ`ݕ where ܰ= ܣ்ܲ`ܣ
(A.7) (A.6) ݔො= ൫ܣ்ܲ`ܣ൯
ିଵܣ்ܲ`ݕ= ܰିଵܣ்ܲ`ݕ where ܰ= ܣ்ܲ`ܣ
(A.7) (A.7) variance matrix of the solution vector ߑሼݔොሽ 4. Variance-covariance matrix of the solution vector ߑሼݔොሽ 4. Variance-covariance matrix of the solution vector ߑሼݔොሽ Using the law of error propagation, the variance-covariance matrix of the solution vector Σሼݔොሽ can be
obtained as follows: ߑሼݔොሽ=ߪ
ଶܰିଵܣ்ܲ`ܲ`ାܲ`ܣܰିଵ
(A.8) ߑሼݔොሽ=ߪ
ଶܰିଵܣ்ܲ`ܲ`ାܲ`ܣܰିଵ (A.8) Sensors 2011, 11 9096 Since for a pseudo inverse, ܲ`ܲ`ାܲ` = ܲ` [32]
ߑሼݔොሽ= ߪ
ଶܰିଵܰܰିଵ= ߪ
ଶܰିଵ
(A.9) Since for a pseudo inverse, ܲ`ܲ`ାܲ` = ܲ` [32]
ߑሼݔොሽ= ߪ
ଶܰିଵܰܰିଵ= ߪ
ଶܰିଵ
(A.9) ߑሼݔොሽ= ߪ
ଶܰିଵܰܰିଵ= ߪ
ଶܰିଵ
(A.9) (A.9) variance factor ߪො
ଶ 5. A-posteriori variance factor ߪො
ଶ 5. A-posteriori variance factor ߪො
ଶ 5. A-posteriori variance factor ߪො
ଶ 5. A-posteriori variance factor ߪො
ଶ 5. A-posteriori variance factor ߪො
ଶ 5. A-posteriori variance factor ߪො
ଶ ܧ൫்݁̃ܲ`݁̃൯= ݐݎܲ`൫ܫെܣܰିଵܣ்ܲ`൯ൣߪ
ଶܲ`ାܣݔݔ்ܣ்ܣݔܦ்ܦݔ்ܣ்ܦܦ்൧
(A.17)
Gi
th t P`ܦ
0 E
ti
(A 17)
b
i
lifi d t ܧ൫்݁̃ܲ`݁̃൯= ݐݎܲ`൫ܫെܣܰିଵܣ்ܲ`൯ൣߪ
ଶܲ`ାܣݔݔ்ܣ்ܣݔܦ்ܦݔ்ܣ்ܦܦ்൧
(A.17)
Given that P`ܦ= 0, Equation (A.17) can be simplified to: `݁̃൯= ߪ
ଶݐݎܲ`൫ܫെܣܰିଵܣ்ܲ`൯ܲ`ା=ߪ
ଶݐݎܲ`ܲ`ାെߪ
ଶݐݎܰିଵܣ்ܲ`ܲ`ାܲ`ܣ
(A.18) (A.18) Based on the property that ݐݎሺܣܤ) = ݎܽ݊݇ሺܣܤ) (given that ܣܤ is idempotent) and ݎܽ݊݇ሺܣܤ)
min ሺݎܽ݊݇ܣ, ݎܽ݊݇ܤ) [32], the following can be stated: ܲ`ା) = ݎܽ݊݇ሺܲ`ܲ`ା) = ݉݅݊൫ݎܽ݊݇ܲ`, ݎܽ݊݇ܲ`ା൯= ݎܽ݊݇ܲ` = ݍ
(A.19) ݐݎሺܲ`ܲ`ା) = ݎܽ݊݇ሺܲ`ܲ`ା) = ݉݅݊൫ݎܽ݊݇ܲ`, ݎܽ݊݇ܲ`ା൯= ݎܽ݊݇ܲ` = ݍ
(A.19)
Given that ݐݎሺܲ`ܲ`ା) = ݍ (as shown in Equation (A.19)) and that ܲ`ܲ`ାܲ` = ܲ`, Equation (A.18) can
be simplified to:
ܧ൫்݁̃ܲ`݁̃൯= ߪ
ଶݍെߪ
ଶݐݎܰିଵܰ=ߪ
ଶݍെߪ
ଶݐݎܫ= ߪ
ଶݍെߪ
ଶ݉
(A.20) (A.19) ܧ൫்݁̃ܲ`݁̃൯= ߪ
ଶݍെߪ
ଶݐݎܰିଵܰ=ߪ
ଶݍെߪ
ଶݐݎܫ= ߪ
ଶݍെߪ
ଶ݉
(A.20) ܧ൫்݁̃ܲ`݁̃൯= ߪ
ଶݍെߪ
ଶݐݎܰିଵܰ=ߪ
ଶݍെߪ
ଶݐݎܫ= ߪ
ଶݍെߪ
ଶ݉ ܧ൫்݁̃ܲ`݁̃൯= ߪ
ଶݍെߪ
ଶݐݎܰିଵܰ=ߪ
ଶݍെߪ
ଶݐݎܫ= ߪ
ଶݍെߪ
ଶ݉
(A.20) (A.20) where m is the number of unknown parameters. where m is the number of unknown parameters. rs 2011, 11 9097 Sensors 2011, 11 Finally, we can get the expression for the a-posteriori variance factor ߪො
ଶ as follows: ߪො
ଶ= ܧ൫்݁̃ܲ`݁̃൯
ሺݍെ݉)
(A.21) (A.21) © 2011 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/3.0/). © 2011 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/3.0/).
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https://openalex.org/W3168701222
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http://eprints.gla.ac.uk/241359/1/241359.pdf
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English
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Role of Common Genetic Variants for Drug-Resistance to Specific Anti-Seizure Medications
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Frontiers in pharmacology
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Role of Common Genetic Variants for
Drug-Resistance to Specific
Anti-Seizure Medications Stefan Wolking 1,2†, Ciarán Campbell 3†, Caragh Stapleton 3, Mark McCormack 3,
Norman Delanty 3,4,5, Chantal Depondt 6, Michael R. Johnson 7, Bobby P. C. Koeleman 8,
Roland Krause 9, Wolfram S. Kunz 10, Anthony G. Marson 11,12,13, Josemir W. Sander 14,15,16,
Graeme J. Sills 17, Pasquale Striano 18,19, Federico Zara 18,19, Sanjay M. Sisodiya 14,15,
Gianpiero L. Cavalleri 3,4,7, Holger Lerche 1* and EpiPGX Consortium Stefan Wolking 1,2†, Ciarán Campbell 3†, Caragh Stapleton 3, Mark McCormack 3,
Norman Delanty 3,4,5, Chantal Depondt 6, Michael R. Johnson 7, Bobby P. C. Koeleman 8,
Roland Krause 9, Wolfram S. Kunz 10, Anthony G. Marson 11,12,13, Josemir W. Sander 14,15,16,
Graeme J. Sills 17, Pasquale Striano 18,19, Federico Zara 18,19, Sanjay M. Sisodiya 14,15,
Gianpiero L. Cavalleri 3,4,7, Holger Lerche 1* and EpiPGX Consortium 1Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany,
2Department of Epileptology and Neurology, University of Aachen, Aachen, Germany, 3Department of Molecular and Cellular
Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland, 4FutureNeuro Research Centre, Science Foundation Ireland,
Dublin, Ireland, 5Division of Neurology, Beaumont Hospital, Dublin, Ireland, 6Department of Neurology, H ˆopital Erasme, Université
Libre de Bruxelles, Brussels, Belgium, 7Division of Brain Sciences, Imperial College Faculty of Medicine, London, United Kingdom,
8Department of Genetics, University Medical Center Utrecht, Utrecht, Netherlands, 9Luxembourg Centre for Systems
Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg, 10Institute of Experimental Epileptology and Cognition
Research and Department of Epileptology, University of Bonn, Bonn, Germany, 11Department of Molecular and Clinical
Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom, 12The Walton Centre NHS
Foundation Trust, Liverpool, United Kingdom, 13Liverpool Health Partners, Liverpool, United Kingdom, 14Department of Clinical
and Experimental Epilepsy, UCL Queen Square Institute of Neurology, London, United Kingdom, 15Chalfont Centre for Epilepsy,
Chalfont-St-Peter, United Kingdom, 16Stichting Epilepsie Instellingen Nederland (SEIN), Heemstede, Netherlands, 17School of
Life Sciences, University of Glasgow, Glasgow, United Kingdom, 18IRCCS "G. Gaslini" Institute, Genova, Italy, 19Department of
Neurosciences, University of Genoa, Genova, Italy Edited by:
Heng Xu,
Sichuan University, China
Reviewed by:
Adrian Isaac Campos Gonzalez,
The University of Queensland,
Australia
Lifeng Tian,
University of Pennsylvania,
United States
*Correspondence:
Holger Lerche
Holger.lerche@uni-tuebingen.de
†These authors contributed equally
to this work Edited by:
Heng Xu,
Sichuan University, China
Reviewed by:
Adrian Isaac Campos Gonzalez,
The University of Queensland,
Australia
Lifeng Tian,
University of Pennsylvania,
United States
*Correspondence: Edited by:
Heng Xu,
Sichuan University, China Reviewed by:
Adrian Isaac Campos Gonzalez,
The University of Queensland,
Australia
Lifeng Tian,
University of Pennsylvania,
United States *Correspondence:
Holger Lerche
Holger.lerche@uni-tuebingen.de Objective: Resistance to anti-seizure medications (ASMs) presents a significant hurdle in the
treatmentofpeople withepilepsy. Genetic markersforresistancetoindividual ASMs could support
clinicians to make better-informed choices for their patients. In this study, we aimed to elucidate
whether the response to individual ASMs was associated with common genetic variation. Specialty section:
This article was submitted to
Pharmacogenetics and
Pharmacogenomics,
a section of the journal
Frontiers in Pharmacology
Received: 30 March 2021
Accepted: 10 May 2021
Published: 09 June 2021 Specialty section:
This article was submitted to
Pharmacogenetics and
Pharmacogenomics,
a section of the journal
Frontiers in Pharmacology Specialty section:
This article was submitted to
Pharmacogenetics and
Pharmacogenomics,
a section of the journal
Frontiers in Pharmacology
Received: 30 March 2021
Accepted: 10 May 2021
Published: 09 June 2021 Methods: A cohort of 3,649 individuals of European descent with epilepsy was deeply
phenotyped and underwent single nucleotide polymorphism (SNP)-genotyping. We conducted
genome-wide association analyses (GWASs) on responders to specific ASMs or groups of
functionally related ASMs, using non-responders as controls. We performed a polygenic risk
score (PRS) analyses based on risk variants for epilepsy and neuropsychiatric disorders and
ASM resistance itself to delineate the polygenic burden of ASM-specific drug resistance. Received: 30 March 2021
Accepted: 10 May 2021
Published: 09 June 2021 Results: We identified several potential regions of interest but did not detect genome-wide
significant loci for ASM-specific response. We did not find polygenic risk for epilepsy,
neuropsychiatric disorders, and drug-resistance associated with drug response to specific
ASMs or mechanistically related groups of ASMs. ORIGINAL RESEARCH
published: 09 June 2021
doi: 10.3389/fphar.2021.688386 Keywords: drug-resistant epilepsies, polygenic risk score (PRS), GWAS, anti-seizure medication (ASM), single
nucelotide polymorphisms INTRODUCTION European-wide
epilepsy
research
partnership
under
the
European Commission Seventh Framework Protocol (FP7). This
case-control
study
was
based
on
the
retrospective
evaluation of individual data. Relevant data were extracted
from case charts by trained personnel and collected in a
standard electronic case report form (eCRF) used at all
consortium
sites. Our
cohorts
consisted
exclusively
of
individuals
of
non-Finnish
European
ancestry
with
an
established diagnosis of either focal or genetic generalized
epilepsy according to current ILAE diagnostic criteria (Scheffer
et al., 2017). We tested whether common genetic variants were
significantly associated with drug response to one ASM or groups
of mechanistically related ASMs (sodium channel-active and
calcium channel-active ASMs). We also tested whether the
response profile was associated with an increased burden of
polygenic variants for risk of epilepsy syndromes, other
neuropsychiatric disorders, or whether a burden of risk
variants for drug response itself could predict the outcome. About one-third of people with epilepsy have seizures that are
refractory to anti-seizure medications (ASMs). The International
League against Epilepsy (ILAE) defines drug resistance as
ongoing seizures despite treatment with at least two well-
tolerated and appropriate ASMs (Kwan et al., 2010). With
each additional drug trial, the odds to achieve seizure freedom
decrease (Brodie et al., 2012). The introduction of new ASMs with
alternate mechanisms of action has not significantly changed this
situation (Chen et al., 2018). For certain epilepsy syndromes,
some ASMs have proven to be more beneficial than others:
valproic
acid
(VPA)
and
ethosuximide
are
superior
to
lamotrigine (LTG) in childhood absence epilepsy (Glauser
et al., 2013), VPA is superior to topiramate (TPM) and LTG
in genetic generalized epilepsy (GGE) (Marson et al., 2007a;
Silvennoinen et al., 2019), and carbamazepine (CBZ) and LTG
are superior to TPM and gabapentin (GBP) in focal epilepsy
(Marson
et
al.,
2007b). Yet,
studies
with
head-to-head
comparisons are sparse (Beyenburg et al., 2010; Androsova
et al., 2017). Thus, in clinical practice, ASMs are prescribed
based on age, gender, co-morbidities, electroclinical syndrome,
seizure type, potential drug interactions, or adverse drug
reactions. ASMs were selected based on their usage in the EpiPGX
cohort. ASM-specific analysis was performed for levetiracetam
(LEV), lamotrigine (LTG), valproic acid (VPA) for focal
epilepsies and all epilepsies. For focal epilepsies only, we
performed
additional
ASM-specific
GWAS
for
phenytoin
(PHT), oxcarbazepine (OXC), and carbamazepine (CBZ). Citation: Wolking S, Campbell C, Stapleton C,
McCormack M, Delanty N, Depondt C,
Johnson MR, Koeleman BPC,
Krause R, Kunz WS, Marson AG,
Sander JW, Sills GJ, Striano P, Zara F,
Sisodiya SM, Cavalleri GL, Lerche H
and EpiPGX Consortium (2021) Role of
Common Genetic Variants for Drug-
Resistance to Specific Anti-
Seizure Medications. Front. Pharmacol. 12:688386. doi: 10.3389/fphar.2021.688386 Significance: This study could not ascertain the predictive value of common genetic
variants for ASM responder status. The identified suggestive loci will need replication in
future studies of a larger scale. June 2021 | Volume 12 | Article 688386 1 Frontiers in Pharmacology | www.frontiersin.org Common Variants in Drug-Resistant Epilepsy Wolking et al. Cohorts and Phenotype Definition The individuals in this study were selected from more than 12.000
individuals that were documented in the EpiPGX eCRF. Thereof,
3,649 individuals fulfilled the inclusion criteria, 2,762 with focal
epilepsy, and 887 with generalized genetic epilepsy. The latter
group has been part of a previous study (Wolking et al., 2020a). A
more detailed cohort description is provided in Table 1. Individuals
were
classified
as
responders
or
non-
responders. The response was defined as seizure freedom
under ongoing treatment for at least one year and before
initiation of any other treatment; non-response as recurring
seizures at ≥50% of pretreatment seizure frequency given
adequate dosage of the trial drug. Individuals with recurrent
non-compliance for ASM intake were excluded. The response
or non-response groups’ assignment was based on the
evaluation of one or more epilepsy specialists at the source
center. To harmonize phenotyping procedures a phenotyping
manual was created at the start of the EpiPGX project. At the
beginning and on a yearly basis throughout the recruitment
phase phenotyping workshops were held. To assess cross-
center consistency of data interpretation, a cross-center
phenotyping validation test was performed at the outset of
the EpiPGX project, using anonymized medical records. An
overall inter-rater agreement of 74.2% was reached. Stark
disagreement, e.g ASM response vs. non-response, occurred
in 5.1% of recorded ASM trials. We assessed common variants’ role and common variant
burden for drug response to common ASMs using genome-
wide association studies (GWAS) and polygenic risk score
(PRS) analyses in a cohort of 3,649 individuals. Ethics Statement All study participants provided written, informed consent for
genetic analyses. Local institutional review boards reviewed and
approved study protocols at each contributing site. INTRODUCTION ASM
groups comprised T-type calcium channel-active ASMs (valproic
acid, ethosuximide, and zonisamide [ZNS]) for focal and all
epilepsies; and sodium channel-active ASMs (LTG, lacosamide
[LCM], ZNS, PHT, CBZ, OXC, and eslicarbazepine [ESL]) for
focal epilepsies only. The breakdown of the sample size per
analysis is depicted in Table 1. Pharmacogenomics, i.e. the influence of genetic variants on
drug response or adverse effects, bear the potential to support the
choice of the most suitable ASM (Löscher et al., 2009). Other
medical fields have seen the integration of pharmacogenomics in
clinical routine (Daly, 2017). For epilepsies, reproducible
pharmacogenomic findings are limited to cutaneous adverse
reactions caused by aromatic ASMs (Chung et al., 2004;
McCormack et al., 2011; McCormack et al., 2018). The utility
of these findings in individuals’ care remains a matter of debate
(Chen et al., 2014). The endeavor to identify common genetic
variants associated with drug response is still elusive, also due to
small sample sizes (Heavin et al., 2019; Wolking et al., 2020a). There is some evidence that enrichment of ultra-rare variants in
genes associated with pharmacodynamics and pharmacokinetics
can modify ASM response, but further replication of these results
is needed (Wolking et al., 2020a). Study Design This
cohort
was
derived
from
the
EpiPGX
Consortium
established in 2012 to identify genetic biomarkers of epilepsy
treatment response and adverse drug reactions. EpiPGX is a June 2021 | Volume 12 | Article 688386 Frontiers in Pharmacology | www.frontiersin.org 2 Common Variants in Drug-Resistant Epilepsy Wolking et al. TABLE 1 | Sample numbers, estimated power, and clinical details for GWAS cohorts. ASM
Status
n
Study power
Female (%)
GGE (%)
AOO (mean, SD)
Ethnicity %
South Europe
Central Europe
British Isles
LEV
R
343
1.55
58.9
24.8
24.7 (±19.0)
10.2
32.7
57.1
N
895
56.6
24.2
18.2 (±14.8)
9.1
43.4
47.6
Na-C-ASMs
R
910
1.37
50.7
0
30.9 (±19.7)
14.6
17.5
67.9
N
1,286
54.2
0
21.5 (±16.5)
6.5
31.5
62.0
LTG
R
471
1.49
58.0
29.1
26.3 (±19.0)
7.5
36.5
56.0
N
929
61.9
26.8
19.1 (±15.2)
6.8
40.5
52.7
CBZ
R
424
1.57
47.6
0
30.4 (±19.6)
21.7
12.0
66.3
N
591
55.8
0
20.7 (±16.6)
13.7
27.2
59.1
OXC
R
98
2.08
55.1
0
29.6 (±19.6)
21.4
15.3
63.3
N
296
50.7
0
18.1 (±14.1)
10.1
48.6
41.2
PHT
R
71
2.30
47.9
0
28.2 (±20.3)
14.1
18.3
67.6
N
218
54.1
0
18.0 (±14.6)
15.6
20.2
64.2
Ca-C-ASMs
R
690
1.45
59.3
69.0
16.6 (±14.2)
14.6
54.1
31.3
N
848
51.7
20.6
18.9 (±15.3)
9.4
39.6
50.9
VPA
R
612
1.49
56.9
67.0
17.6 (±14.5)
15.4
53.9
30.7
N
690
51.3
23.5
20.0 (±16.0)
8.4
44.5
47.1
All samples
—
3,649
55.0
24.3
22.8 (±17.7)
8.9
34.8
56.4
Depiction of sample size per ASM and responder status, study power, gender distribution, mean age at seizure onset, and distribution of ethnicity. Study power shows relative risk for 80%
study power, given an allele frequency of ≥20%, an α level of 5 × 10–8 and a prevalence of drug-resistance of 30%. AOO age of onset of first seizure, ASM anti-seizure medication, Ca-
C-ASMs T-type calcium channel-active anti-seizure medications, CBZ carbamazepine, GGE genetic generalized epilepsy, LEV levetiracetam, LTG lamotrigine, n number, N
non-responders, Na-C-ASMs sodium channel-active anti-seizure medications, OXC oxcarbazepine, PHT phenytoin, R responders, SD standard deviation, VPA valproic acid. Ca-C-ASMs comprised VPA, zonisamide, and ethosuximide; Na-C-ASMs comprised LTG, lacosamide, zonisdamide, PHT, CBZ, OXC, and eslicarbazepine. Genome-wide Association Analysis y
GWAS power was calculated using PGA (Menashe et al.,
2008). Association analysis was performed using SNPTEST
in a frequentist model with the top 10 main components, sex,
and
epilepsy
subtype
(where
appropriate)
included
as
covariates. The
statistical
threshold
for
genome-wide
significance was set at p < 5 × 10–8. Post-association QC
removed SNPs with INFO scores lower than 0.95, missingness
rates >0.10, Hardy-Weinberg deviations p < 5 × 10–6, and
minor allele frequencies <5%. Study Design Depiction of sample size per ASM and responder status, study power, gender distribution, mean age at seizure onset, and distribution of ethnicity. Study power shows relative risk for 80%
study power, given an allele frequency of ≥20%, an α level of 5 × 10–8 and a prevalence of drug-resistance of 30%. AOO age of onset of first seizure, ASM anti-seizure medication, Ca-
C-ASMs T-type calcium channel-active anti-seizure medications, CBZ carbamazepine, GGE genetic generalized epilepsy, LEV levetiracetam, LTG lamotrigine, n number, N
non-responders, Na-C-ASMs sodium channel-active anti-seizure medications, OXC oxcarbazepine, PHT phenytoin, R responders, SD standard deviation, VPA valproic acid. Ca-C-ASMs comprised VPA, zonisamide, and ethosuximide; Na-C-ASMs comprised LTG, lacosamide, zonisdamide, PHT, CBZ, OXC, and eslicarbazepine. drug-resistance of 30%. The study power for the other analyses is
shown in Table 1. Imputation and Genotyping Quality Controls
GWASs were conducted separately for each ASM-response
cohort using imputed genotypes. Genotyping of a subset of
samples was performed at deCODE Genetics on Illumina
OmniExpress-12
v1.1
and
-24
v1.1
single
nucleotide
polymorphism (SNP) arrays. The remainder of samples
were genotyped locally on various Illumina beadchip SNP
arrays. Detailed genotyping, imputation and quality control
methods have been described previously (McCormack et al.,
2018). Population structure was controlled via principal
component
analysis
as
reported
previously
(Wolking
et
al.,
2020a)
(Supplementary
Figure
S1). European
ancestry
was
established
by
a
principal
component
analysis
comparison
to
1,000
genomes
data
(Supplementary Figure S2). Polygenic Risk Score Analysis for Epilepsy
and Neuropsychiatric Disorders and Neuropsychiatric Disorders
GWAS summary statistics for epilepsy (focal, GGE, and all
epilepsies) were downloaded from the ILAE study (Cross-
Disorder Group of the Psychiatric Genomics Consortium,
2013) using the EpiGAD server. These statistics were
remade with the overlapping samples between the larger
ILAE cohort and our EpiPGX samples removed. GWAS
results for a broad psychiatric disorder study (covering
autism,
attention
deficit
hyperactivity
disorder,
bipolar
disorder,
major
depression
and
schizophrenia)
were
downloaded from the psychiatric genomics consortium
(International
League
Against
Epilepsy
Consortium
on
Complex Epilepsies, 2018). PRS for each phenotype were
calculated for all samples our study cohorts using PRSice
(Euesden et al., 2015), using all SNPs from the base GWAS
with p-values ≤0.5. The threshold of ≤0.5 was selected because
for most complex traits the most predictive p-thresholds will
typically be between 0.3 and 0.5, including epilepsy (Leu et al.,
2019). The PRS were then regressed onto responder status
using R 3.6, with the top six principle components, sex, and
epilepsy subtype (where appropriate) included as covariates. SNP-Heritability Testing We calculated SNP-based heritability [SNP-h2] as a measure of
the proportion of variance in ASM response status, which could
be attributed to common genetic variants for the largest cohort of
samples treated with sodium-blocking ASMs. The result was not
significant, SNP-h2 was estimated to be 0.3108, with a standard
error of 0.2868 (Lower CI: −0.252, Upper CI: 0.873). Using
GCTA-GREML to calculate h2, the result was not significant
[h2 0.000002, standard error 0.178,925, p 0.5]. Cohort Description After per individual quality check, 3,649 individuals were
included in the GWAS analyses. The breakdown of the GWAS
cohorts is shown in Table 1. The proportion of individuals with
GGE was 25%. For the GWAS for VPA response and in
consequence
for
T-type
calcium
channel-active
ASMs
(including VPA, ESX, and ZNS), GGE was overrepresented in
the responder group. The mean age of onset tended to be higher
for responders than non-responders except for VPA and T-type
calcium channel-active ASMs. Polygenic Risk Score Analysis for Drug
Response We estimated that our most extensive analysis for sodium
channel-active ASMs had 80% power to detect a genetic
predictor of the relative risk of 1.37 with an allele frequency of
≥20%, based on an α level of 5 × 10–8 and given a prevalence of To test whether responsiveness to individuals ASMs or groups of
ASMs had a distinct polygenic component, we split our cohorts June 2021 | Volume 12 | Article 688386 Frontiers in Pharmacology | www.frontiersin.org 3 Common Variants in Drug-Resistant Epilepsy Wolking et al. TABLE 2 | Top genome-wide association study results (p < 5 × 10–6) for ASM
responder status
SNP
Location (hg19)
p-value
Gene
Focal Epilepsies
Levetiracetam
rs10191428
2:62,725,407
2.37 × 10–6
TMEM17
rs6455984
6:1,65,419,809
2.98 × 10–6
—
rs10786411
10:100091761
4.01 × 10–6
—
Sodium channel-Active ASMs
rs2600151
3:4148058
2.83 × 10–6
SUMF1
rs60350499
17:71111631
6.89 × 10–8
—
Lamotrigine
rs7811069
7:32,003,223
1.75 × 10–6
PDE1C
rs1859577
7:68254624
4.80 × 10–7
—
rs2028234
8:4747736
6.90 × 10–7
CSMD1
Carbamazepine
rs4078065
2:238110123
3.88 × 10–6
—
rs13150739
4:128045535
8.95 × 10–7
—
rs4243569
14:51536146
4.49 × 10–6
TRIM9
Oxcarbazepine
rs6552076
4:68014557
4.71 × 10–6
—
rs1816237
5:33040812
1.00 × 10–6
—
rs2944715
8:69346689
3.10 × 10–6
C8orf34
rs34744859
18:65165115
4.44 × 10–6
—
Phenytoin
rs12038219
1:167503917
6.07 × 10–8
—
rs28740860
3:3277529
8.72 × 10–7
—
rs188002
6:140473067
4.60 × 10–7
—
rs16945236
15:91664327
8.36 × 10–7
—
Calcium channel-Active ASMs
rs11125398
2:52227824
2.77 × 10–6
—
rs73104283
2:231,130,300
3.64 × 10–6
SP140
rs7092992
10:20,922,643
3.56 × 10–6
—
Valproic acid
rs2700204
3:112,841,569
4.52 × 10–6
—
rs1952670
9:128,654,392
9.11 × 10–7
PBX3
rs7092992
10:20,922,643
4.07 × 10–6
—
All Epilepsies
Levetiracetam
rs10191428
2:62,725,407
2.30 × 10–6
TMEM17
rs9390556
6:148,643,960
4.80 × 10–6
—
Lamotrigine
rs12468936
2:106,116,654
2.60 × 10–6
—
rs7811069
7:32,003,223
8.44 × 10–7
PDE1C
rs7859863
9:104,337,744
4.52 × 10–6
GRIN3A
rs28776624
14:41,898,817
3.90 × 10–6
—
Calcium channel Active ASMs
rs73104283
2:231,130,300
1.14 × 10–6
SP140
Valproic acid
rs3936663
4:7,185,699
3.83 × 10–6
-- TABLE 2 | Top genome-wide association study results (p < 5 × 10–6) for ASM
responder status Assessing the Polygenic Risk of Epilepsy
and Neuropsychiatric Disorders for Drug
Response First, we tested whether the responder status to individual
ASMs and the groups of sodium channel active-, and T-type
calcium channel-active ASMs correlated with the genetic load
for epilepsy (focal, generalized, and combined), Figure 1C. Second, we tested whether the responder status correlated
with the genetic load for five neuropsychiatric disorders was
associated with responder status. In both cases, we found no
significant association of polygenic risk scores with any ASM
drug-responder status. into
a
discovery
and
replication
cohort,
depending
on
recruitment site (Test: 636 cases, 890 controls; discovery: 229
cases, 323 controls). A GWAS was run in the test cohort
(following the protocol from above) and used a PRS analysis
base in the discovery cohort (same methods as above). Genome-wide Association Studies for Drug
Response p
We performed GWAS for drug response for specific ASMs and
groups of ASMs (as shown in Table 1) for focal epilepsy and all
epilepsies. Results for GGE alone have been published previously
(Wolking et al., 2020b). We found no evidence for a relevant
GWAS p-value inflation (lambda-range between 0.99 and 1.06). We did not find any genome-wide markers that exceeded the
significance threshold (5 × 10–8). We identified 30 loci suggestive
for an association with ASM response (<5 × 10–6) as shown in
Table 2. To exemplify the findings, QQ- and Manhattan plots for
the largest GWAS of sodium channel-active ASMs are shown in
Figures 1A,B; the results of the other GWAS are depicted in
Supplementary Figures S3 to S12. SNP-Heritability Testing Third, we assumed that the responder status itself harbored a
polygenic component, which is largely distinct from the polygenic
component for epilepsy risk. We split the ASM cohorts in half to
calculate a GWAS for the first half. This discovery cohort was
used to calculate PRS for individual ASM responder status in the Linkage disequilibrium score-regression (Bulik-Sullivan et al.,
2015) was used to calculate SNP-based heritability in the
cohort
of
sodium-channel
actives
ASM
treated
study
participants. We also used GCTA-GREML to estimate the
heritability (Yang et al., 2011). June 2021 | Volume 12 | Article 688386 Frontiers in Pharmacology | www.frontiersin.org 4 Common Variants in Drug-Resistant Epilepsy Wolking et al. FIGURE 1 | Results for sodium channel-active ASMs. A and B show results for GWAS, C and D for PRS analysis. A: QQ plot shows no relevant p-value inflation;
lambda-value 1.005. B: Manhattan plot of GWAS; locations of SNPs with p-value ≤5 × 10–6 are marked. C: Results for PRS-analysis PRS for epilepsy risk based on
the ILAE 2018 metadata. D: Results for PRS-analysis for PRS for risk of ASM-specific drug response. FIGURE 1 | Results for sodium channel-active ASMs. A and B show results for GWAS, C and D for PRS analysis. A: QQ plot shows no relevant p-value inflation;
lambda-value 1.005. B: Manhattan plot of GWAS; locations of SNPs with p-value ≤5 × 10–6 are marked. C: Results for PRS-analysis PRS for epilepsy risk based on
the ILAE 2018 metadata. D: Results for PRS-analysis for PRS for risk of ASM-specific drug response. FIGURE 1 | Results for sodium channel-active ASMs. A and B show results for GWAS, C and D for PRS analysis. A: QQ plot shows no relevant p-value inflation;
lambda-value 1.005. B: Manhattan plot of GWAS; locations of SNPs with p-value ≤5 × 10–6 are marked. C: Results for PRS-analysis PRS for epilepsy risk based on
the ILAE 2018 metadata. D: Results for PRS-analysis for PRS for risk of ASM-specific drug response. second half. We also did not find a significant association for drug
response PRS with responder status (Figure 1D). not
show
that
polygenic
risk
scores
were
significantly
associated with ASM response within the limits of study size. SNP-Heritability Testing Other methods of PRS calculation also exist, such as LDpred
(Vilhjálmsson et al., 2015), which may prove more successful at
finding polygenic signals associated with drug response to ASMs
and could be further explored in future studies. DISCUSSION We tested whether common genetic variation could predict drug
response to various commonly used ASMs. We identified several
loci of potential interest for ASM response but found no
significant
genome-wide
association. Our
analysis
was
underpowered to detect small effect size variants, but the
results suggest that there are no large-effect size variants
associated with drug response. We further tested whether the
polygenic
burden
for
epilepsy
risk,
risk
for
various
neuropsychiatric disorders, or drug-resistance itself had a
predictive value for the drug response phenotype. We could This study was limited to the sample size of the sub-analyses. This study does not prove that drug response is without genetic
influence. The results could imply that drug response is a far more
complex trait with multiple influencing parameters beyond
genomic factors alone. While PRS for epilepsy is a reliable
predictor for the risk of epilepsy and epilepsy sub-phenotypes
itself (Leu et al., 2019; Moreau et al., 2020), this approach was not
beneficial to predict drug response within this study’s limitations. The results align with our previous studies that found no
common genetic variants in association to VPA, LTG, and LEV June 2021 | Volume 12 | Article 688386 Frontiers in Pharmacology | www.frontiersin.org 5 Wolking et al. Common Variants in Drug-Resistant Epilepsy AUTHOR CONTRIBUTIONS SW, CC, GLC, and HL conceived the study concept and design. SW, CC, HL, SS, JS, PS wrote the manuscript. SW, CC, CS, GLC,
and MM performed the analyses. SW, ND, CD, MJ, BK, RK, WK,
AM, JS, GS, PS, FZ, SS, GC, and HL acquired the data and
oversaw phenotyping. All authors interpreted the data. HL, SS,
and GC supervised the study. ETHICS STATEMENT The studies involving human participants were reviewed and
approved by Ethics Comittee, University Hospital Tübingen and
other ethics comittees of recruitment sites. Written informed
consent to participate in this study was provided by the
participants’ legal guardian/next of kin. FUNDING The EpiPGX Consortium was funded by FP7 grant 279062
“EpiPGX” from the European Commission. SW received
funding from the German Research Foundation (DFG) (WO
2385/1-1). The EpiPGX Consortium was funded by FP7 grant 279062
“EpiPGX” from the European Commission. SW received
funding from the German Research Foundation (DFG) (WO
2385/1-1). DATA AVAILABILITY STATEMENT response in genetic generalized epilepsy (Wolking et al., 2020b) or
for the response to lacosamide in focal epilepsy (Heavin et al.,
2019). One previous study suggests that rare genetic variants in
genes related to drug targets and pharmacokinetics might be
involved (Wolking et al., 2020b). Given that many individuals
with epilepsy exhibit a broad pharmacoresistance, regardless of
the drugs’ mechanism of action, other factors are probably at play
(Löscher et al., 2020). Epigenetic mechanisms such as altered
DNA
methylation
(Kobow
et
al.,
2013),
seizure-induced
alterations of neural networks (Fang et al., 2011), or intrinsic
factors mediating disease severity (Rogawski, 2013) should be
further explored. The original contributions presented in the study are included
in the article/Supplementary Material. The raw SNP datasets
presented in this article are not readily available due to ethical and
privacy restrictions. Further inquiries should be directed to the
corresponding author. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fphar.2021.688386/
full#supplementary-material Chen, Z., Liew, D., and Kwan, P. (2014). Effects of a HLA-B*15:02 Screening Policy
on Antiepileptic Drug Use and Severe Skin Reactions. Neurology 83 (22),
2077–2084. doi:10.1212/wnl.0000000000001034 CONCLUSION No genome-wide significant variants could be identified in
association with drug response to various widely used ASMs. We identified several suggestive risk loci. Future hypothesis-
driven association studies should attempt to reproduce our findings. EPIPGX-CONSORTIUM Andreja Avbersek, Costin Leu, Kristin Heggeli, Rita Demurtas,
Joseph
Willis,
Douglas
Speed,
Narek
Sargsyan,
Krishna
Chinthapalli, Mojgansadat Borghei, Antonietta Coppola, Antonio
Gambardella, Stefan Wolking, Felicitas Becker, Sarah Rau, Christian
Hengsbach, Yvonne G. Weber, Bianca Berghuis, Wolfram S. Kunz,
Mark McCormack, Norman Delanty, Ellen Campbell, Lárus J. Gudmundsson,
Andres
Ingason,
Kári
Stefánsson,
Reinhard
Schneider, Rudi Balling, Pauls Auce, Ben Francis, Andrea
Jorgensen, Andrew Morris, Sarah Langley, Prashant Srivastava,
Martin Brodie, Marian Todaro, Slave Petrovski, Jane Hutton,
Fritz Zimprich, Martin Krenn, Hiltrud Muhle, Karl Martin Klein,
Rikke S Møller, Marina Nikanorova, Sarah Weckhuysen, Zvonka
Rener-Primec, Gianpiero L. Cavalleri, John Craig, Chantal Depondt,
Michael R. Johnson, Bobby P. C. Koeleman, Roland Krause, Holger
Lerche, Anthony G. Marson, Terence J. O’Brien, Slave Petrovski,
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5585821. Wolking, S., Schulz, H., Nies, A. T., McCormack, M., Schaeffeler, E., Auce, P., et al. (2020a). Pharmacoresponse in Genetic Generalized Epilepsy: a Genome-wide
Association Study. Available:at: https://www.futuremedicine.com/doi/10.2217/
pgs-2019-0179. Löscher, W., Klotz, U., Zimprich, F., and Schmidt, D. (2009). The Clinical Impact
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Common Antiseizure Medications. Available:at: https://onlinelibrary.wiley. com/doi/abs/10.1111/epi.16467. Löscher, W., Potschka, H., Sisodiya, S. M., and Vezzani, A. (2020). Drug Resistance
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Genome-wide Complex Trait Analysis. Am. J. Hum. Genet. 88 (1), 76–82. doi:10.1016/j.ajhg.2010.11.011 Marson, A. G., Al-Kharusi, A. M., Alwaidh, M., Appleton, R., Baker, G. A.,
Chadwick, D. W., et al. (2007a). The SANAD Study of Effectiveness of
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60460-7 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Copyright © 2021 Wolking, Campbell, Stapleton, McCormack, Delanty, Depondt,
Johnson, Koeleman, Krause, Kunz, Marson, Sander, Sills, Striano, Zara, Sisodiya,
Cavalleri, Lerche and EpiPGX Consortium. 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) and the copyright owner(s) 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. Marson, A. G., Al-Kharusi, A. M., Alwaidh, M., Appleton, R., Baker, G. A.,
Chadwick, D. W., et al. (2007b). The SANAD Study of Effectiveness of
Valproate, Lamotrigine, or Topiramate for Generalised and Unclassifiable
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(9566), 1016–1026. doi:10.1016/s0140-6736(07)60461-9 McCormack, M., Alfirevic, A., Bourgeois, S., Farrell, J. J., Kasperaviˇciut˙e, D.,
Carrington, M., et al. (2011). HLA-A*3101 and Carbamazepine-Induced June 2021 | Volume 12 | Article 688386 Frontiers in Pharmacology | www.frontiersin.org 7
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Being Friends with or Rejected by Classmates: Aggression Toward Same- and Cross-Ethnic Peers
|
Journal of youth and adolescence
| 2,019
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cc-by
| 11,671
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Abstract Whereas previous research suggests that adolescents’ aggressive behavior in itself does not highlight ethnic boundaries, it
remains unclear whether classmates’ responses to same- and cross-ethnic aggression strengthen ethnic boundaries. This
study examined how adolescents’ aggression toward same- and cross-ethnic peers relates to the positive (friendship) and
negative (rejection) relationship nominations they receive from same- and cross-ethnic classmates. Cross-sectional peer
nomination data on 917 Dutch and 125 Turkish adolescents in 56 secondary schools were analyzed (mean age = 14.9 year;
51.4% boys). Adolescents received more friendship nominations from same-ethnic than from cross-ethnic classmates, but
were not more rejected by cross-ethnic than same-ethnic classmates. Multilevel Poisson and negative binomial regression
models showed that, irrespective of aggressor’s ethnic background, adolescents’ aggressive behavior was related to rejection
by classmates from the ethnic group that was the target of aggression and to being befriended by classmates from the ethnic
group that was not the target of aggression. Specifically, both Dutch and Turkish adolescents who were aggressive toward
Dutch peers were rejected by Dutch classmates and befriended by Turkish classmates and vice versa. These findings suggest
that classmates’ positive and negative responses to adolescents are related to adolescents’ aggressive behavior based on the
ethnic background of the victim, not on the ethnic background of the aggressor. This suggests that integration between ethnic
groups in schools relates to aggression in general, not only cross-ethnic aggression. Keywords
Aggression
●Ethnicity
●Friendship
●Rejection
●Adolescence Introduction behavior might indirectly affect ethnic boundaries in
classrooms. In this study, it is questioned how adolescents’
aggressive behavior toward same- and cross-ethnic peers
relates to the positive (friendship) and negative (rejection)
relationship nominations they receive from their classmates. Do these responses to adolescents relate to their aggressive
behavior in ways that reflect or emphasize ethnic boundaries
in the classroom? In the context of ethnically diverse classrooms, interethnic
aggression may be perceived as behavior that reinforces
ethnic boundaries. Previous research has shown that
although ethnic groups might slightly differ in the extent to
which they behave aggressively, same- and cross-ethnic
aggression was equally common (Tolsma et al. 2013;
Vitoroulis and Vaillancourt 2018). Whereas this suggests
that adolescents’ aggressive behavior in itself may not
highlight ethnic boundaries, it remains unclear how class-
mates respond to adolescents’ same- and cross-ethnic
aggression
and,
consequently,
whether
aggressive Being Friends with or Rejected by Classmates: Aggression Toward
Same- and Cross-Ethnic Peers Marianne Hooijsma
1
●Gijs Huitsing
1
●Jan Kornelis Dijkstra
1
●Andreas Flache
1
●René Veenstra
1 Received: 16 September 2019 / Accepted: 13 November 2019 / Published online: 6 December 2019
© The Author(s) 2019 Journal of Youth and Adolescence (2020) 49:678–692
https://doi.org/10.1007/s10964-019-01173-1 Journal of Youth and Adolescence (2020) 49:678–692
https://doi.org/10.1007/s10964-019-01173-1 EMPIRICAL RESEARCH 1
Department of Sociology and Interuniversity Center for Social
Science Theory and Methodology (ICS), University of Groningen,
Groningen, the Netherlands * Marianne Hooijsma
m.hooijsma@rug.nl The Role of Ethnicity in Friendship and Rejection In the context of ethnic heterogeneity, social identity theory
proposes that through identification with the in-group (i.e.,
same-ethnic peers), individuals have the tendency to eval-
uate the in-group positively (Tajfel and Turner 1979). Aiming to achieve a positive social identity, individuals
categorize their environment into groups and, specifically,
compare their own group to other groups (i.e., cross-ethnic
peers). In this process of differentiating the in-group from
out-groups, individuals develop in-group favoritism, refer-
ring to individuals’ preference to affiliate with others whom * Marianne Hooijsma
m.hooijsma@rug.nl Journal of Youth and Adolescence (2020) 49:678–692 679 they perceive to belong to their in-group (Tajfel and Turner
1979). Consequently, individuals are more likely to feel
connected to in-group members than to out-group members. Positive peer relationships, such as friendships, are more
likely between same-ethnic peers (Rivera et al. 2010). Similarity in, for example, ethnicity, enhances both agree-
ment and understanding and makes the other’s behavior
predictable (Hamm 2000; Ibarra 1992). Same-ethnic peers
have a similar cultural background which relates to more
similar norms and values compared to cross-ethnic peers. This similarity facilitates the initiation and maintenance of
positive peer relationships. As ethnicity is an important
characteristic in categorizing groups in adolescence (Boda
and Néray 2015; Leszczensky and Pink 2015), adolescents
are expected to favor their same-ethnic peers over their
cross-ethnic peers. Among early adolescents it has been found that victims’
same-sex peers rejected the victims’ bullies (Veenstra et al. 2010). As peers who do not belong to the victim’s in-group
are less likely to be threatened by the behavior of the
aggressor, aggressors were not rejected by their victims’
cross-sex peers (Veenstra et al. 2010). Similarly, it may be
expected that aggression toward members of a specific
ethnic group in the classroom is only related to rejection by
classmates from that specific ethnic group, and not to
rejection by classmates from other groups. Although it can be expected that adolescents would
generally tend to evaluate aggression toward same-ethnic
peers negatively, it can also be argued that this relation is
stronger if the aggressor is same-ethnic than if the aggressor
is cross-ethnic. In the context of differentiating and com-
paring of the in-group to out-groups, the most common
pattern would be that aggression by cross-ethnic peers may
be perceived as norm-conforming behavior. Aggression to
same-ethnic peers, however, is likely to be perceived as
norm-deviating behavior. Aggression Toward Peers of Different Ethnic Groups Aggressive behavior toward peers is a powerful means to
gain status in the classroom (Sijtsema et al. 2009). More-
over, when used strategically, aggressive behavior poses a
minimum risk for loss of connectedness by significant
peers, for instance by selecting victims, both same- and
cross-ethnic, who are rejected by the aggressor’s significant
others (Veenstra et al. 2010). Victims of aggression are,
however, at risk of losing both status and connectedness as
they are likely to be avoided and less accepted by class-
mates (Van der Ploeg et al. 2015). Classmates may not want
to affiliate with victims as this puts them at risk of being
victimized as well (Sentse et al. 2013). Given the assump-
tion that individuals act as a response to the awareness of
factors that foster or threaten their goal pursuit by liking the
former and disliking the latter (Lindenberg 2001), aggres-
sors are likely to be rejected by their victims because they
threaten their victim’s goal pursuit. The Role of Ethnicity in Friendship and Rejection Specifically, aggression by an in-
group member toward one’s in-group can be seen as a form
of betrayal of the in-group. Adolescents who feel betrayed
by their significant peers distrust and avoid interactions with
these peers. In an experimental setting, it was indeed found
that individuals negatively evaluate in-group members who
are disloyal to their group (Travaglino et al. 2014). Whereas several studies have found evidence for ethnic
boundaries and segregation in adolescents’ positive peer
relationships (Boda and Néray 2015; Leszczensky and Pink
2015; Stark and Flache 2012), less is known about the role
of ethnicity in adolescents’ negative peer relationships. In
line with social identity theory (Tajfel and Turner 1979),
group categorization and comparison of the in-group to out-
groups may lead to more negative evaluations of out-group
members. Categorizing groups along ethnic boundaries
signals differences between groups, for example in terms of
cultural norms and practices (Strohmeier et al. 2008). Consequently, negative peer relationships can be expected
to be more likely between adolescents of different ethnic
groups. Whereas cross-ethnic aggression is expected to be related
to rejection by the cross-ethnic group as the target of
aggression, it can be argued that cross-ethnic aggression is
also related to positive evaluations by same-ethnic peers. Next to social needs, such as achieving status and being
connected,
adolescents
have
individual
psychological
needs, such as developing a positive social identity. In order
to achieve a positive social identity, individuals tend to
differentiate the in-group from and compare the in-group to
out-groups in ways to devaluate out-groups (Tajfel and
Turner 1979). Aggressive behavior toward out-group peers
is an example of a way to devaluate the out-group. Aggressive behavior toward out-groups may highlight dif-
ferences between groups and consequently benefit in-group
members’ development of a positive social identity. Given
that individuals tend to like characteristics that foster their
goal pursuit (Lindenberg 2001), such as achieving a posi-
tive social identity, it can be expected that cross-ethnic
aggression would be related to positive evaluations of same-
ethnic classmates of the aggressor. Procedure Data was collected in the Netherlands for the first wave
(2010/2011) of the Children of Immigrants Longitudinal
Survey in Four European Countries (CILS4EU; Kalter et al. 2016). The CILS4EU project focuses on the intergenera-
tional integration of children of immigrants. Early adoles-
cent immigrants and their majority peers around the age of
14 to 15 (3rd grade of secondary school) were the target
population. A three-stage sampling method was used. Second, the current study examined how adolescents’
same- and cross-ethnic aggressive behavior relates to the
positive and negative relationship nominations they receive
from their same- and cross-ethnic classmates. As adolescents’
aggressive behavior threatens the goal pursuit of their victims
and their victims’ in-group classmates, but not that of the
victims’ out-group classmates, it is hypothesized (H3) that
aggressive adolescents would be rejected by classmates from
the ethnic group to whom the victim belongs, but not by
classmates from other ethnic groups in the classroom. Aggression toward same-ethnic classmates may be perceived
as norm-deviating behavior and betrayal. Consequently, it is
hypothesized (H4) that adolescents’ aggression toward same-
ethnic peers would relate more to rejection by same-ethnic
classmates than aggression toward cross-ethnic peers. Finally,
aggression toward cross-ethnic peers may be a way to
devaluate the out-group, benefiting the development of same-
ethnic peers’ positive social identity. Therefore, it is hypo-
thesized (H5) that adolescents who are aggressive toward
cross-ethnic peers would receive more friendship nomina-
tions from same-ethnic classmates than cross-ethnic adoles-
cents who are aggressive toward this cross-ethnic group. First, secondary schools were selected based on a prob-
ability proportional to size as well as the proportion of stu-
dents with an immigrant background (referring to students
who were themselves, or had at least one parent who was,
born in a non-Western country). Schools were excluded
from the school sample when the total number of students
was less than 2% of the total target population and the target
classroom size was smaller than one quarter of the average
classroom size, or when it were special schools for students
with cognitive, emotional, or physical disabilities. 34.9% of
the 109 originally targeted schools participated. To increase
the number of participating schools, a replacement strategy
was implemented in which replacement schools were
selected which matched the non-participating schools based
on proportion of students with an immigrant background and
school type. After this replacement strategy was imple-
mented, 100 schools participated. Current Study As previous research has shown that cross-ethnic aggression
was not more likely than same-ethnic aggression (Tolsma
et al. 2013), aggressive behavior in itself may not highlight Moreover, as victims’ in-group members may feel
threatened by the aggressor as well, aggressive behavior is
also likely to relate to rejection by victims’ in-group peers. 680 Journal of Youth and Adolescence (2020) 49:678–692 The extent to which adolescents are aggressive toward,
as well as befriended or rejected by, Dutch or Turkish
classmates depends partially on the opportunity structure,
i.e., whether adolescents have Dutch or Turkish classmates. Similarly, the extent to which adolescents are befriended or
rejected by classmates may differ between boys and girls. Previous research has, for example, shown that boys are
more aggressive toward and more rejected by classmates
(Veenstra et al. 2010). In examining adolescents’ aggres-
sive behavior and the relationship nominations they receive
from classmates, this study therefore controls for the
number of Dutch and Turkish classmates as well as
adolescents’ sex. ethnic boundaries. It is unclear, however, whether class-
mates’ responses to adolescents’ same- and cross-ethnic
aggression emphasize ethnic boundaries in the classroom. In
the current study, it is first examined how the positive and
negative relationship nominations adolescents receive from
same- and cross-ethnic classmates differ by their ethnic
background. In the process of differentiating the in-group
from out-groups to achieve a positive social identity, indi-
viduals tend to develop a preference for affiliating with in-
group members over out-group members. Moreover, simi-
larity between peers enhances agreement, understanding,
and predictability in relationships. Consequently, it is
hypothesized (H1) that adolescents would receive relatively
more friendship nominations from same-ethnic classmates
than cross-ethnic classmates. Furthermore, comparing the in-
group to out-groups may lead to more negative evaluations
of out-group members. Therefore, it is hypothesized (H2)
that adolescents would be rejected by relatively more cross-
ethnic classmates than same-ethnic classmates. Rejection Participants were also asked to nominate classmates they did
not want to sit close to (“Who would you not want to sit
by?”), which is taken as a proxy for rejection. Participants
could nominate up to five classmates. To measure how often
students were rejected by their Dutch and Turkish class-
mates, the number and proportion of rejection nominations
students received from each of these groups were calculated. Procedure Second, in most schools,
two classrooms were selected. In schools with more than
60% immigrant students, as many classrooms as possible
were selected to increase the number of participating
immigrants. Of the selected classrooms, 94.5% (222 class-
rooms) participated in the study. Third, only students not
being able to respond to the questionnaire in the language of
the host country were excluded from the sample. Exclusions
within schools were negligible. In total, 91.1% (4363 stu-
dents) of the selected students participated in the study. This study examined classmates’ response to adoles-
cents’ aggressive behavior toward same- and cross-ethnic
peers in secondary school classrooms in the Netherlands. Specifically, this study examined these relationships among
Dutch and Turkish adolescents. As part of labor migration,
the first Turkish immigrants came to the Netherlands in the
1960s. Although the Dutch society’s ethnic composition is
changing, with immigrants from more diverse ethnic
backgrounds coming to the Netherlands, immigrants with a
Turkish background remain to form the largest ethnic
minority group in the Netherlands and in Dutch secondary
schools (Statistics Netherlands 2018). Journal of Youth and Adolescence (2020) 49:678–692 681 (maternal grand)mother (for details on the definition of
adolescents’ ethnic origin: Dollmann et al. 2014). The CILS4EU survey was administered using paper-and-
pencil questionnaires. Extensively trained students assis-
tants were selected to administer the survey. In order to
ensure privacy, questionnaires were identified with a unique
ID number, which was linked to the specific student on a
separate class list. Immigrant background Immigrant background was assessed using information on
students’, their parents’, and their grandparents’ country of
birth and country of origin as provided by the student as
well as their parents. Students who themselves, their par-
ents, and grandparents were born in the Netherlands, were
classified as ‘Dutch’. Students who themselves, their par-
ents or grandparents were born outside of the Netherlands
were classified as having an immigrant background. A
bottom-up approach was used to define immigrant adoles-
cents’ ethnic origin in which information at the grandparent
level was used first, followed by information about the
parents and the adolescent. For the grandparent and parent
levels, two decision rules were applied to define adoles-
cents’ ethnic origin. First, the majority rule indicated that if
the majority of (grand)parents was born in a certain country,
this information was used to define adolescent’s ethnic
origin. Second, the priority rule indicated that if there was
no majority, priority was given to the country of birth of the Aggression Participants were asked to nominate an unlimited number of
classmates they perceived as being mean to them (“Who is
sometimes mean to you?”). To measure students’ aggres-
sion toward their Dutch and Turkish classmates, the number
and proportion of nominations students received from each
of these groups were calculated. Friendship Participants responded to the question: “Who are your best
friends?”. They received a list of names and random iden-
tification numbers of all of their classmates (also non-
participating classmates) and were asked to nominate up to
five classmates as their best friends. To measure the extent
to which students were befriended by their Dutch and
Turkish classmates, the number of nominations for best
friends they received from each of the groups was calcu-
lated. Furthermore, the proportion of nominations for best
friends students received from each of the groups compared
to the total number of nominations they could have received
from these groups was calculated. Sample This study investigated the relation between aggression
toward and being befriended and rejected by classmates
specifically for the two largest ethnic groups in the Dutch
CILS4EU data, namely non-immigrant Dutch adolescents
and adolescents with an immigrant background from Turkish
origin. Of these adolescents, those who had at least one
Dutch and one Turkish classmate were selected. This resulted
in a sample of 1042 adolescents (917 Dutch and 125 Turkish
adolescents) in 85 classes in 56 schools. Adolescents in the
sample were on average 14.9 years old (SD = 6.8 months)
and there were about as many boys (51.4%) as girls. On
average, Dutch adolescents’ socio-economic background was
higher than that of Turkish adolescents (based on the inter-
national socio-economic index of occupational status, ISEI,
ranging from 0 to 100; Ganzeboom et al. 1992; average ISEI
for Dutch adolescents = 51.8, average ISEI for Turkish
adolescents = 35.6). Only one participant in the sample had a
missing value on one of the study variables. For this Turkish
adolescent, information on sex was missing. As the exclusion
of this participant will not likely influence the results, it was
decided to exclude the participant from the analyses. Control variables Self-reported sex was included to control for possible sex
differences in being befriended or rejected by classmates
(girls were coded as 0 and boys were coded as 1). In
addition, to control for the availability of same- and cross-
ethnic classmates, the number of Dutch and Turkish
classmates adolescents have were included in the analyses. Main analyses Poisson regression models in Stata version 15.1 (StataCorp
2017) were used to examine the relation between aggres-
sion, friendship, and rejection because of the non-negative
count characteristic of the dependent variables (Cameron
and Trivedi 2013). Given that adolescents are more likely to
be befriended or rejected by Dutch or Turkish classmates in
classrooms with, respectively, more Dutch or Turkish stu-
dents, the number of Dutch or Turkish classmates was used
as an exposure variable, indicating the number of nomina-
tions an adolescent could have received. That is, the number
of Dutch or Turkish classmates was used as on offset to
account for opportunity differences (see Long and Freese
2006). Furthermore, robust standard errors for the parameter
estimates were examined to control for mild violation of
underlying assumptions (Cameron and Trivedi 2009). The regression analyses were conducted separately for
being befriended by and rejected by either Dutch or Turkish
classmates as the dependent variables. Each regression
analysis consisted of three models. In Model 1, we added a
dummy
indicating
adolescents’
ethnic
background
to
examine whether being befriended by (hypothesis 1) or
rejected by (hypothesis 2) classmates differed between
cross- and same-ethnic peers. In Model 2, we added
aggression toward Dutch and Turkish peers as independent
variables to examine the association between aggression
toward each of these groups and being befriended or
rejected by (hypothesis 3) classmates from each of these
groups. In Model 3, we added interactions between ado-
lescents’ ethnic background and their aggression toward
Dutch and Turkish peers to examine whether the relation
between aggression toward each ethnic group and being
rejected (hypothesis 4) or befriended by (hypothesis 5)
classmates differed between cross- and same-ethnic peers. All models controlled for adolescents’ sex and availability
of Dutch or Turkish classmates. All continuous independent
variables were grand-mean centered. Before conducting the regression models, it was checked
whether the dependent variables’ variance exceeded the
mean, i.e., whether there was an issue of overdispersion. This
was checked by comparing the full regression model using a
regular Poisson distribution to a model using a negative
binomial distribution, which accounts for overdispersion. The difference between the model was tested using a like-
lihood ratio test of the overdispersion parameter alpha. Analytical Strategy To account for the availability of Dutch and Turkish
classmates, proportions of nominations are discussed in the
descriptive statistics. In the confirmatory analyses, the
absolute number of nominations is used while accounting
for the number of Dutch and Turkish classmates. Journal of Youth and Adolescence (2020) 49:678–692 682 0.001). To account for the two-level structure, multilevel
Poisson and negative binomial regression models were used. Main analyses For
being befriended by Dutch or Turkish classmates, over-
dispersion was not found to be an issue (Χ2 = 0.94, p = 0.17;
negative binomial models could not be estimated for being
befriended by Turkish classmates due to convergence pro-
blems). For being rejected by Dutch or Turkish classmates,
however, overdispersion was found (Χ2 = 363.37, p < 0.001;
Χ2 = 8.75, p = 0.002). Therefore, Poisson regression models
were used to examine the extent to which adolescents were
befriended by Dutch and Turkish classmates, whereas for
their rejection by Dutch and Turkish classmates negative
binomial regression models were used. Disclosure statement Students were nested in classrooms which were nested in
schools. Given the low number of selected classrooms in
each school, school-level variation was comparable to
classroom-level variation. The intraclass correlation at the
classroom-level was high for being befriended (0.34 and
0.27) and rejected by classmates (0.13 and 0.27) and the
design effect was larger than 2 indicating the importance of
accounting for the two-level structure in our data (Muthen
and Satorra 1995). Moreover, likelihood ratio tests comparing
the full regression model using a single-level model to a two-
level model showed that the two-level Poisson regression
model was more appropriate to examine the extent to which
adolescents received friendship nominations from Dutch and
Turkish classmates (Χ2 = 28.66, p < 0.001; Χ2 = 4.67, p =
0.02) and the two-level negative binomial models were more
appropriate to examine adolescents’ rejection by Dutch and
Turkish classmates (Χ2 = 15.15, p < 0.001; Χ2 = 11.58, p < All information regarding the selection of the sample, data
exclusions, manipulations, and measures in the study are
reported. Additional analyses In addition to the analyses among Dutch and Turkish ado-
lescents, analyses were conducted for two other immigrant
groups: Dutch and Moroccan (810 Dutch and 117 Mor-
occan adolescents in 75 classes), and Dutch and Surinamese
(1099 Dutch and 121 Surinamese adolescents in 91 classes)
adolescents. The measures and analytical strategy for these
additional analyses are similar to the analyses for Dutch and
Turkish adolescents. Results of the additional analyses can
be found in Appendix 1. Descriptive Results For friendships, adolescents received higher pro-
portions of nominations from same-ethnic classmates than
from cross-ethnic classmates (Dutch to Dutch: M = 0.17;
Dutch to Turkish: M = 0.12; Turkish to Dutch: M = 0.09;
Turkish to Turkish: M = 0.33). Table 2 presents correlations between aggression toward
and being befriended and rejected by the two ethnic groups. The table shows that adolescents who were aggressive
toward one of the groups were also more likely to be
aggressive toward peers from the other ethnic group (r =
0.16, p < 0.001). Similarly, adolescents who were rejected
by classmates from one group were also likely to be rejected
by classmates from the other group (r = 0.25, p < 0.001). No association was found between being befriended by the
two groups (r = −0.03, p = 0.28). adolescents were befriended by more Dutch classmates (PE
= 0.77, p < 0.001) and befriended by fewer Turkish class-
mates (PE = −1.02, p < 0.001) than Turkish adolescents,
see Model 1 in Table 3 (left panel for being befriended by
Dutch classmates, right panel for being befriended by
Turkish classmates). Descriptively it was found that aggression toward one of
the groups was positively related to rejection by classmates
from this specific group (rDutch = 0.40, p < 0.001; rTurkish =
0.20, p < 0.001) but not to rejection by classmates from the
other group. Also, although with smaller correlations,
aggression toward one group related positively to being
befriended by classmates from the other group: aggression
toward Dutch related positively to being befriended by
Turkish classmates (r = 0.15, p < 0.001) and aggression
toward Turkish related positively to being befriended by
Dutch classmates (r = 0.07, p = 0.04). Similarly, Models 1 in Table 4 were used to test
hypothesis 2 that adolescents would be rejected by rela-
tively
more
cross-ethnic
classmates
than
same-ethnic
classmates. In contrast to the hypothesis, no evidence was
found that Dutch adolescents were rejected by fewer Dutch
classmates than Turkish adolescents (PE = −0.24, p =
0.20). In line with the hypothesis, however, Dutch adoles-
cents were rejected by slightly more Turkish classmates
than Turkish adolescents (PE = 0.67, p = 0.05). Thus,
partial support was found for hypothesis 2. The Role of Ethnicity in Friendship and Rejection In line with hypothesis 1 (adolescents would receive rela-
tively
more friendship nominations
from
same-ethnic
classmates than cross-ethnic classmates), it was found in
the multilevel Poisson regression models that Dutch Descriptive Results Table 1 shows that overall, Dutch and Turkish adolescents did
not differ much in their general aggression toward (MDutch =
0.02,
MTurkish = 0.04),
being
befriended
(MDutch = 0.16,
MTurkish = 0.15), and rejected (MDutch = 0.10, MTurkish = 0.12)
by Dutch and Turkish classmates. Moreover, adolescents
were not more aggressive toward same- or cross-ethnic peers Journal of Youth and Adolescence (2020) 49:678–692 683 Table 1 Descriptive statistics on aggression, friendships, and rejection for Dutch and Turkish adolescents
Aggression toward
Befriended by
Rejected by
M (SD)
Max. number of
nominations
M (SD)
Max. number of
nominations
M (SD)
Max. number of
nominations
Dutch adolescent
0.02 (0.05) 10
0.16 (0.10) 11
0.10 (0.14) 18
Dutch classmates
0.03 (0.07) 5
0.17 (0.13) 10
0.11 (0.15) 16
Turkish classmates
0.02 (0.13) 2
0.12 (0.31) 3
0.10 (0.29) 5
Turkish adolescent
0.04 (0.06) 6
0.15 (0.12) 7
0.12 (0.13) 17
Dutch classmates
0.05 (0.13) 5
0.09 (0.19) 4
0.13 (0.24) 9
Turkish classmates
0.04 (0.16) 2
0.33 (0.37) 4
0.08 (0.20) 2
Means and standard deviations are based on proportion of nominations. Minimum number of nominations is 0 in all cases ive statistics on aggression, friendships, and rejection for Dutch and Turkish adolescents Table 2 Correlations
1. 2. 3. 4. 5. 6. Aggression toward
1. Dutch
2. Turkish
0.16**
Befriended by
3. Dutch
−0.04
0.07*
4. Turkish
0.15**
0.05
−0.03
Rejected by
5. Dutch
0.40**
0.06
−0.16**
0.03
6. Turkish
0.02
0.20**
−0.04
−0.10*
0.25**
Correlations are calculated using proportion of nominations
*p < 0.05; **p < 0.001 Table 2 Correlations
1. 2. 3. 4. 5. 6. Aggression toward
1. Dutch
2. Turkish
0.16**
Befriended by
3. Dutch
−0.04
0.07*
4. Turkish
0.15**
0.05
−0.03
Rejected by
5. Dutch
0.40**
0.06
−0.16**
0.03
6. Turkish
0.02
0.20**
−0.04
−0.10*
0.25**
Correlations are calculated using proportion of nominations
*p < 0.05; **p < 0.001 (Dutch toward Dutch: M = 0.03; Dutch toward Turkish: M =
0.02; Turkish toward Dutch: M = 0.05; Turkish toward
Turkish: M = 0.04), and did not receive more proportions of
nominations for rejection by cross-ethnic than same-ethnic
classmates (Dutch by Dutch: M = 0.11; Dutch by Turkish:
M = 0.10; Turkish by Dutch: M = 0.13; Turkish by Turkish:
M = 0.08). Rejection Hypothesis 3 stated that aggressive adolescents would be
rejected by classmates from the ethnic group to whom the Journal of Youth and Adolescence (2020) 49:678–692 684 Table 3 Results of multiple multilevel Poisson regression models for friendship
Befriended by Dutch
Befriended by Turkish
Model 1
Model 2
Model 3
Model 1
Model 2
Model 3
Parameter
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
Constant
−2.58
0.13
−2.59
0.13
−2.59
0.14
−1.17
0.18
−1.18
0.17
−1.20
0.18
Ethnic background (Dutch = 1)
0.77
0.13
<0.001
0.78
0.14
<0.001
0.78
0.14
<0.001
−1.02
0.19
<0.001
−1.01
0.19
<0.001
−0.98
0.20
<0.001
Aggression toward
Dutch
−0.04
0.03
0.18
−0.10
0.08
0.19
0.24
0.07
0.001
0.30
0.09
0.001
*Ethnic background (Dutch = 1)
0.06
0.08
0.42
−0.07
0.14
0.59
Turkish
0.25
0.11
0.02
0.26
0.26
0.32
0.04
0.20
0.84
0.20
0.21
0.34
*Ethnic background (Dutch = 1)
−0.02
0.28
0.95
−0.63
0.57
0.28
Sex (boy = 1)
0.05
0.06
0.39
0.05
0.06
0.39
0.05
0.06
0.39
−0.09
0.16
0.59
−0.12
0.16
0.46
−0.12
0.16
0.46
Number of Dutch classmates
Exp. Exp. Exp. −0.01
0.02
0.65
−0.01
0.02
0.46
−0.01
0.02
0.43
Number of Turkish classmates
−0.02
0.03
0.41
−0.03
0.03
0.30
−0.03
0.03
0.30
Exp. Exp. Exp. Rejection Variance classroom
0.05
0.03
0.05
0.03
0.05
0.03
0.11
0.06
0.10
0.06
0.09
0.05
Log likelihood
−1698.40
−1696.21
−1696.13
−586.70
−582.80
−581.91
The number of Dutch or Turkish classmates was used as an exposure variable to account for opportunity differences
*It indicates an interaction effect
Table 4 Results of multiple multilevel negative binomial regression models for rejection
Rejected by Dutch
Rejected by Turkish
Model 1
Model 2
Model 3
Model 1
Model 2
Model 3
Parameter
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
Constant
−2.16
0.17
−2.19
0.16
−2.21
0.17
−3.21
0.39
−3.25
0.40
−3.24
0.42
Ethnic background (Dutch = 1)
−0.24
0.18
0.20
−0.21
0.17
0.24
−0.18
0.19
0.32
0.67
0.34
0.05
0.71
0.35
0.04
0.70
0.36
0.05
Aggression toward
Dutch
0.35
0.05
<0.001
0.54
0.23
0.02
−0.05
0.13
0.70
−0.18
0.33
0.59
*Ethnic background (Dutch = 1)
−0.20
0.22
0.36
0.14
0.36
0.70
Turkish
0.11
0.16
0.50
0.14
0.39
0.72
0.55
0.28
0.05
0.39
0.34
0.24
*Ethnic background (Dutch = 1)
−0.05
0.50
0.93
0.27
0.57
0.63
Sex (boy = 1)
0.13
0.09
0.12
0.08
0.09
0.40
0.08
0.09
0.40
0.17
0.19
0.37
0.17
0.20
0.38
0.17
0.20
0.38
Number of Dutch classmates
Exp. Exp. Exp. −0.06
0.02
0.004
−0.06
0.02
.004
−0.06
0.02
0.01
Number of Turkish classmates
−0.04
0.06
0.47
−0.03
0.05
0.60
−0.03
0.05
0.63
Exp. Exp. Exp. Rejection Variance classroom
0.12
0.06
0.11
0.05
0.11
0.05
0.36
0.17
0.35
0.17
0.35
0.17
Log likelihood
−1573.52
−1547.40
−1546.82
−476.19
−474.34
−474.17
The number of Dutch or Turkish classmates was used as an exposure variable to account for opportunity differences Table 3 Results of multiple multilevel Poisson regression models for friendship
Befriended by Dutch
Befriended by Turkish
Model 1
Model 2
Model 3
Model 1
Model 2
Model 3
Parameter
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
Constant
−2.58
0.13
−2.59
0.13
−2.59
0.14
−1.17
0.18
−1.18
0.17
−1.20
0.18
Ethnic background (Dutch = 1)
0.77
0.13
<0.001
0.78
0.14
<0.001
0.78
0.14
<0.001
−1.02
0.19
<0.001
−1.01
0.19
<0.001
−0.98
0.20
<0.001
Aggression toward
Dutch
−0.04
0.03
0.18
−0.10
0.08
0.19
0.24
0.07
0.001
0.30
0.09
0.001
*Ethnic background (Dutch = 1)
0.06
0.08
0.42
−0.07
0.14
0.59
Turkish
0.25
0.11
0.02
0.26
0.26
0.32
0.04
0.20
0.84
0.20
0.21
0.34
*Ethnic background (Dutch = 1)
−0.02
0.28
0.95
−0.63
0.57
0.28
Sex (boy = 1)
0.05
0.06
0.39
0.05
0.06
0.39
0.05
0.06
0.39
−0.09
0.16
0.59
−0.12
0.16
0.46
−0.12
0.16
0.46
Number of Dutch classmates
Exp. Exp. Exp. −0.01
0.02
0.65
−0.01
0.02
0.46
−0.01
0.02
0.43
Number of Turkish classmates
−0.02
0.03
0.41
−0.03
0.03
0.30
−0.03
0.03
0.30
Exp. Exp. Exp. Rejection Variance classroom
0.05
0.03
0.05
0.03
0.05
0.03
0.11
0.06
0.10
0.06
0.09
0.05
Log likelihood
−1698.40
−1696.21
−1696.13
−586.70
−582.80
−581.91 Table 4 Results of multiple multilevel negative binomial regression models for rejection
Rejected by Dutch
Rejected by Turkish
Model 1
Model 2
Model 3
Model 1
Model 2
Model 3
Parameter
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
Constant
−2.16
0.17
−2.19
0.16
−2.21
0.17
−3.21
0.39
−3.25
0.40
−3.24
0.42
Ethnic background (Dutch = 1)
−0.24
0.18
0.20
−0.21
0.17
0.24
−0.18
0.19
0.32
0.67
0.34
0.05
0.71
0.35
0.04
0.70
0.36
0.05
Aggression toward
Dutch
0.35
0.05
<0.001
0.54
0.23
0.02
−0.05
0.13
0.70
−0.18
0.33
0.59
*Ethnic background (Dutch = 1)
−0.20
0.22
0.36
0.14
0.36
0.70
Turkish
0.11
0.16
0.50
0.14
0.39
0.72
0.55
0.28
0.05
0.39
0.34
0.24
*Ethnic background (Dutch = 1)
−0.05
0.50
0.93
0.27
0.57
0.63
Sex (boy = 1)
0.13
0.09
0.12
0.08
0.09
0.40
0.08
0.09
0.40
0.17
0.19
0.37
0.17
0.20
0.38
0.17
0.20
0.38
Number of Dutch classmates
Exp. Exp. Exp. −0.06
0.02
0.004
−0.06
0.02
.004
−0.06
0.02
0.01
Number of Turkish classmates
−0.04
0.06
0.47
−0.03
0.05
0.60
−0.03
0.05
0.63
Exp. Exp. Exp. Variance classroom
0.12
0.06
0.11
0.05
0.11
0.05
0.36
0.17
0.35
0.17
0.35
0.17
Log likelihood
−1573.52
−1547.40
−1546.82
−476.19
−474.34
−474.17
The number of Dutch or Turkish classmates was used as an exposure variable to account for opportunity differences Journal of Youth and Adolescence (2020) 49:678–692 685 victim belongs, but not by classmates from other ethnic
groups in the classroom. Models 2 in Table 4 were used to
test this hypothesis. Adding the variables on aggression
toward Dutch and Turkish peers resulted in a small decrease
of the log likelihood in Model 2 (rejection by Dutch: Χ2(2)
= 5.88, p = 0.05; rejection by Turkish: Χ2(2) = 5.88, p =
0.14), indicating that the additional effects improved the
model only slightly. Friendship Models 2 in Table 3 examined the association between
aggression
toward
Dutch
and
Turkish
and
receiving
friendship nominations from Dutch and Turkish classmates. The table shows that adding aggression to the model did not
improve the model substantively (befriended by Dutch: Χ2
(2) = 2.19, p = 0.33; befriended by Turkish: Χ2(2) = 3.90,
p = 0.14). Results show that aggression toward Turkish
peers was related to being befriended by more Dutch
classmates (PE = 0.25, p = 0.02). Similarly, aggression
toward Dutch peers was related to being befriended by more
Turkish classmates (PE = 0.24, p = 0.001). In Models 3,
interactions between aggression and adolescents’ ethnic
background were added to examine whether these associa-
tions differ by adolescents’ ethnic background. Adding the
interactions to the model did not improve the model
(befriended by Dutch: Χ2(2) = 0.08, p = 0.96; befriended by
Turkish: Χ2(2) = 0.89, p = 0.64). It was not found that the
relation between aggression toward Turkish peers and being Rejection Model 2 shows that aggression toward
Dutch was related to being rejected by more Dutch class-
mates (PE = 0.35, p < 0.001) but not by more Turkish
classmates (PE = −0.05, p = 0.70), and that aggression
toward Turkish was related to being rejected by slightly
more Turkish classmates (PE = 0.55, p = 0.05) but not by
more Dutch classmates (PE = 0.11, p = 0.50). befriended by Dutch classmates differed for Dutch or Turkish
adolescents. That is, Dutch adolescents who were aggressive
toward their Turkish peers were not befriended by more
Dutch classmates than Turkish adolescents who were
aggressive toward Turkish peers (PE = −0.02, p = 0.95). For
Turkish adolescents, it was also not found that they were
befriended by more same-ethnic classmates when they were
aggressive toward their Dutch, cross-ethnic peers (difference
estimated = −0.07, p = 0.59). Therefore, no support was
found for hypothesis 5, stating that adolescents who are
aggressive toward cross-ethnic peers would be befriended by
more same-ethnic classmates than cross-ethnic adolescents
who are aggressive toward this cross-ethnic group. Additional Analyses Results on the interactions between aggression and
adolescents’ ethnic background (Models 3 in Table 4) were
used to examine hypothesis 4, stating that adolescents’
aggression toward same-ethnic peers would relate more to
rejection by classmates from the ethnic group to whom the
victim belongs than aggression toward cross-ethnic peers. Table 4 shows that adding the interactions did not improve
the model (rejection by Dutch: Χ2(2) = 0.78, p = 0.68;
rejection by Turkish: Χ2(2) = 0.37, p = 0.83). Results show
that Dutch adolescents who were aggressive toward Dutch
were not rejected by more Dutch classmates than Turkish
adolescents who were aggressive toward Dutch (PE =
−0.20, p = 0.36). Similarly, Turkish adolescents who were
aggressive toward their same-ethnic Turkish peers were not
found to be rejected by more same-ethnic classmates than
Dutch adolescents who were aggressive toward Turkish
(difference estimated = 0.27, p = 0.63). Thus, no support
was found for hypothesis 4. In Appendix 1 models of the additional analyses for
friendship and rejection among Dutch-Moroccan and
Dutch-Surinamese adolescents can be found. Overall, the
findings on Dutch and Moroccan adolescents were in line
with those for Dutch and Turkish adolescents. The results
for Dutch and Surinamese adolescents were less in line
with the findings for Dutch and Turkish adolescents. Whereas among Dutch and Turkish adolescents, Dutch
adolescents were befriended by more Dutch classmates
than Turkish adolescents, Dutch adolescents were not
befriended by more Dutch classmates than Surinamese
adolescents. That is, receiving friendship nominations
from Dutch classmates was not related to adolescents’
ethnic background among Dutch and Surinamese adoles-
cents. Furthermore, in contrast to hypothesis 4, it was
found that Dutch adolescents who were aggressive toward
Dutch peers were rejected by fewer Dutch classmates (PE
= 0.21, p < 0.001) than Surinamese adolescents who were
aggressive toward Dutch peers (PE = 0.45, p < 0.001). Aggression toward Dutch was thus more strongly related
to rejection by Dutch classmates if the aggressor was
Surinamese, i.e., cross-ethnic. The Role of Ethnicity in Friendship and Rejection threatened by peers who are aggressive toward adolescents’
out-group peers (Veenstra et al. 2010). In line with previous research (Boda and Néray 2015;
Leszczensky and Pink 2015) and this study’s first hypoth-
esis, it was found that adolescents received overall more
friendship nominations from same-ethnic classmates than
cross-ethnic classmates. Partially in line with our second
hypothesis, Dutch adolescents were only slightly more
rejected by Turkish classmates than Turkish adolescents. In
contrast to the hypothesis, Turkish adolescents were not
found to be more rejected by Dutch classmates than Dutch
classmates. These findings partially support arguments
based on social identity theory, stating that in order to
achieve a positive social identity, which is closely linked to
individual’s group membership, individuals favor their in-
group and devaluate out-groups (Tajfel 1982; Tajfel and
Turner 1979). The results of this study showed that
although adolescents were more likely to be befriended by
same-ethnic classmates (in-group favoritism), they were
generally not more likely to be rejected by cross-ethnic
classmates (out-group devaluation). Based on these find-
ings, it could be concluded that overall, ethnic boundaries
are more important for adolescents’ positive peer relation-
ships than negative peer relationships. This is in line with
previous research, showing that although adolescents from
different ethnic backgrounds may vary in the extent to
which they have negative peer relationships, cross-ethnic
negative peer relationships were not more common than
same-ethnic negative peer relationships (Tolsma et al. 2013). The findings from this study also relates to the idea
that same-ethnic peers are more important for adolescents’
goal pursuit than cross-ethnic peers. As youth’s positive
peer relationships are more important for their goal pursuit
than negative peer relationships, they will be less selective
regarding negative peer relationships. Thus, youth may be
less likely to direct their positive peer relationships toward
cross-ethnic peers but are less likely to base their negative
peer relationships on ethnic background. Additionally, it was expected that adolescents’ aggres-
sion toward same-ethnic peers would more strongly relate to
rejection by classmates from the ethnic group to whom the
victim belongs than aggression toward cross-ethnic peers. However, in contrast to the fourth hypothesis, it was not
found that the relation between adolescents’ aggression and
rejection by classmates from the group that is the target of
aggression was stronger if the adolescent had the same
ethnic background as the victimized group. Discussion In the context of ethnically diverse classrooms, interethnic
aggression may be perceived as behavior that reinforces
ethnic boundaries. Whereas previous research suggests that
adolescents' aggressive behavior in itself does not highlight
ethnic boundaries, it remains unclear whether classmates’
responses to same- and cross-ethnic aggression strengthen
ethnic boundaries. This article examined how adolescents’
aggression toward same- and cross-ethnic peers relates to
the positive (friendship) and negative (rejection) relation-
ship nominations they receive from same- and cross-ethnic
classmates. Journal of Youth and Adolescence (2020) 49:678–692 686 The Role of Ethnicity in Friendship and Rejection Instead, it was
found that adolescents’ aggression was related to rejection
by classmates from the group that was the target of
aggression irrespective of adolescents’ ethnic background. Thus, adolescents’ aggression toward classmates’ same-
ethnic peers was related to rejection by classmates in gen-
eral, irrespective of whom the aggressor was. This suggests
that aggression toward same-ethnic peers might not be
perceived as betrayal by same-ethnic classmates. Although it was found that adolescents who were
aggressive toward Dutch were more befriended by Turkish
classmates and vice versa, this was found irrespective of
adolescents’ ethnic background. For example, both Turkish
and Dutch adolescents who were aggressive toward Dutch
peers were more befriended by Turkish classmates. This
meant that no support was found for the fifth hypothesis that
adolescents who were aggressive toward cross-ethnic peers
were more befriended by same-ethnic peers. It was argued
that adolescents may be rewarded by their same-ethnic
classmates for being aggressive toward cross-ethnic peers
because cross-ethnic aggression is a way to devaluate the
out-group and consequently benefit adolescents’ positive
social identity (Tajfel and Turner 1979). With the current
findings, this argumentation might still hold. Irrespective of
the ethnic background of the aggressor, classmates’ social
identity development might benefit from aggression toward
out-groups by devaluating the out-group. Overall, these findings suggest that classmates’ positive
and negative responses to adolescents were related to ado-
lescents’ aggressive behavior based on the ethnic back-
ground of the victim, not on the ethnic background of the
aggressor. Both Dutch and Turkish adolescents who were
aggressive toward Dutch peers were rejected by Dutch
classmates and befriended by Turkish classmates and vice
versa. That is, it was not found that the ethnic composition
of the aggressor-victim dyad, i.e., same- or cross-ethnic,
was related to the relationship nominations adolescents
receive from classmates. Instead, only the ethnic back-
ground of the victim was related to the relationship nomi-
nations
adolescents
receive
from
classmates. Ethnic
boundaries in friendship and rejection were therefore rein-
forced by the relation between adolescents’ aggressive
behavior toward same- and cross-ethnic victims and the Ethnic Group Differences The effects of accounting for classroom norms on the
association between aggression, friendships, and rejection is a
promising avenue for future research. Aggression Toward Peers of Different Ethnic Groups Nevertheless, the
measure used to examine aggressive behavior in this study
(“who is mean to you?”) captures more general forms of
aggressive behavior. Being mean to someone does not only
include strategic behavior to gain status, it could also include
more innocent forms of aggressive behavior, such as teasing
between friends. As, for example, teasing between friends
might not affect the friendship nominations adolescents
receive from classmates, aggressive behavior as measured in
this study might have a smaller impact on friendships and
rejection than pure strategic aggressive behavior. This sug-
gests that the results might have been stronger if a more
accurate measure of strategic aggressive behavior was used. Some of the findings in this study may be explained by
classroom contextual factors such as the ethnic composition
and prevailing classroom norms (Veenstra et al. 2018). In
classrooms with prominent ethnic boundaries, adolescents
may be prone to the risk cross-ethnic aggression poses to their
in-group identity. In such classrooms, classmates are likely to
perceive aggression by cross-ethnic adolescents as an assault
to their in-group, resulting in classmates specifically rejecting
these cross-ethnic aggressors. In these classrooms, classmates
may, however, not punish aggression by same-ethnic ado-
lescents in order to avoid tensions within their ethnic group. In classrooms in which the prevailing norm is that students
with different ethnic backgrounds associate with each other,
however, classmates may be likely to punish adolescents’
aggression in general, not distinguishing between in-group or
out-group aggression. Similarly, in classrooms with promi-
nent ethnic boundaries, cross-ethnic aggression may be more
likely to be related to being rewarded by same-ethnic class-
mates than in classrooms with less prominent ethnic bound-
aries. The effects of accounting for classroom norms on the
association between aggression, friendships, and rejection is a
promising avenue for future research. Ethnic Group Differences Additional analyses showed that the results among Dutch
and Turkish adolescents were relatively robust. Especially
among Dutch and Moroccan adolescents, the findings were
largely comparable to the findings among Dutch and
Turkish adolescents. For Dutch and Surinamese adoles-
cents, however, different results were found, indicating less
in- and out-group processes. These divergent results may be
explained by cultural distance (Beiser et al. 2015; Lundborg
2013; Schiefer et al. 2012). In the Dutch context, Sur-
inamese adolescents are culturally closer to Dutch adoles-
cents than Turkish or Moroccan adolescents: they speak the
same language and share similar religious beliefs. Feelings
of cultural closeness are related to more positive attitudes
(Berry 2003) and may therefore explain why Dutch ado-
lescents were not more befriended by Dutch classmates than
Surinamese adolescents. In contrast, Turkish and Moroccan
adolescents’ culture is more distant from the Dutch culture. In drawing conclusions on the results it should be taken into
account that Dutch and Turkish adolescents’ cultural
backgrounds are relatively distant. For groups that are cul-
turally less distant from each other, such as Dutch and
Surinamese adolescents, in- and out-group distinctions may
be less clear. Consequently, for these groups the relations
between being aggressive toward and being befriended or
rejected by cross-ethnic classmates may be more in line
with in-group processes than out-group processes. g
gg
Some of the findings in this study may be explained by
classroom contextual factors such as the ethnic composition
and prevailing classroom norms (Veenstra et al. 2018). In
classrooms with prominent ethnic boundaries, adolescents
may be prone to the risk cross-ethnic aggression poses to their
in-group identity. In such classrooms, classmates are likely to
perceive aggression by cross-ethnic adolescents as an assault
to their in-group, resulting in classmates specifically rejecting
these cross-ethnic aggressors. In these classrooms, classmates
may, however, not punish aggression by same-ethnic ado-
lescents in order to avoid tensions within their ethnic group. In classrooms in which the prevailing norm is that students
with different ethnic backgrounds associate with each other,
however, classmates may be likely to punish adolescents’
aggression in general, not distinguishing between in-group or
out-group aggression. Similarly, in classrooms with promi-
nent ethnic boundaries, cross-ethnic aggression may be more
likely to be related to being rewarded by same-ethnic class-
mates than in classrooms with less prominent ethnic bound-
aries. Aggression Toward Peers of Different Ethnic Groups In line with the third hypothesis, it was found that adoles-
cents’ aggressive behavior was related to rejection by
classmates from the group that was the target of aggression. This supports the idea that individuals act as a response to
their awareness of characteristics that threaten their goal
pursuit by disliking these characteristics (Lindenberg 2001),
such as aggression to same-ethnic peers. Further, it was
found that adolescents who were aggressive toward one of
the groups were not rejected by classmates from the other
group. That is, aggressors of Dutch peers were not rejected
by their Turkish classmates and vice versa. This is in line
with the assumption that adolescents’ goal pursuit is not Journal of Youth and Adolescence (2020) 49:678–692 687 more complete picture of the relations between being
aggressive toward and being befriended or rejected by
same- and cross-ethnic classmates. nominations they receive from classmates but not by whe-
ther aggression in itself was cross- or same-ethnic. In the
context of interventions aiming to promote integration
between ethnic groups in schools, this implies that inte-
gration
between
ethnic groups
in schools
relates to
aggression in general, not only to cross-ethnic aggression. It was argued that aggressive behavior toward peers is a
powerful means to gain status in the classroom and that,
when used strategically, aggressive behavior poses a mini-
mum risk for loss of connectedness by significant peers
(Sijtsema et al. 2009; Veenstra et al. 2010). Nevertheless, the
measure used to examine aggressive behavior in this study
(“who is mean to you?”) captures more general forms of
aggressive behavior. Being mean to someone does not only
include strategic behavior to gain status, it could also include
more innocent forms of aggressive behavior, such as teasing
between friends. As, for example, teasing between friends
might not affect the friendship nominations adolescents
receive from classmates, aggressive behavior as measured in
this study might have a smaller impact on friendships and
rejection than pure strategic aggressive behavior. This sug-
gests that the results might have been stronger if a more
accurate measure of strategic aggressive behavior was used. It was argued that aggressive behavior toward peers is a
powerful means to gain status in the classroom and that,
when used strategically, aggressive behavior poses a mini-
mum risk for loss of connectedness by significant peers
(Sijtsema et al. 2009; Veenstra et al. 2010). Limitations and Future Directions The present study aimed to gain insights into the role of
adolescents’ ethnic background in peer friendships and
rejection and how this was related to aggression toward
these specific ethnic groups. The sample of this study
restricted the analyses of the data to be cross-sectional
because of considerable attrition between waves, especially
for immigrant adolescents. Therefore, it was not possible to
examine whether adolescents’ aggressive behavior affects
the nominations they receive for friendship and rejection
from classmates, or whether the effect goes in the opposite
direction. For example, it could also be that adolescents
behave aggressively toward classmates from a specific
group because they were rejected by classmates from this
group. Other methods, such as longitudinal social network
analyses (Snijders et al. 2010), might be able to obtain a As adolescents’ peer relationships change by age, the
relation between adolescents’ aggressive behavior toward
same- and cross-ethnic peers and the nominations they
receive for friendship and rejection by classmates might
change as well. For example, previous research found that
boys who bullied girls were more accepted by other boys in
middle childhood, but not in preadolescence (Veenstra et al. 2010). Although such changes in boy-girl interactions are
more typical for adolescence than changes in same- or
cross-ethnic relationships, future research may consider
whether aggression toward same- and cross-ethnic peers is Journal of Youth and Adolescence (2020) 49:678–692 688 were overall not more rejected by cross-ethnic than same-
ethnic classmates. Moreover, adolescents’ aggression was
related to rejection by classmates from the targeted group
only, irrespective of adolescents’ ethnic background. Fur-
thermore, adolescents’ aggressive behavior was related to
being befriended by classmates from the group that was not
the target of the aggression, irrespective of adolescents’
ethnic background. These results suggest that classmates’
positive and negative responses to adolescents are related to
adolescents’ aggression based on the ethnicity of the victim,
not the aggressor. This suggests that integration between
ethnic groups in schools relates to aggression in general, not
only to cross-ethnic aggression. differently related to friendship and rejection in age groups
other than studied here. Furthermore, future research may consider the role of
ethnic identity in adolescents’ response to same- and cross-
ethnic aggressive behavior. Previous research has recog-
nized the importance of ethnic identification for adolescents’
peer relationships (Knifsend et al. 2016; Syed et al. 2018),
showing that stronger ethnic identities hinder opportunities
for cross-ethnic positive relationships. Limitations and Future Directions Little is known,
however, on how adolescents’ ethnic identity development
influences negative peer relationships. Furthermore, in the
case of Turkish adolescents in the Netherlands, immigrant
adolescents’ ethnic identity is likely to be complex, relating
to both their ethnic origin and their identification as Dutch. Investigating how such complex ethnic identities affect
adolescents’ peer relationships and behaviors might be par-
ticularly interesting for future research. Author’s Contributions M.H. conceived of the study, participated in its
design and coordination, performed statistical analyses, and drafted the
manuscript; G.H., J.K.D., A.F. and R.V. participated in the design and
helped to draft and revise the manuscript. All authors read and
approved the final manuscript. Previous studies have argued for differentiating between
various ethnic groups in studying peer relationships and
processes (Bikmen 2011; Vitoroulis and Vaillancourt
2018). By investigating two specific ethnic groups and not
combining multiple immigrant groups, this study aimed to
take into account the possibility that processes of friendship
and rejection differ between ethnic groups. Moreover, by
conducting additional analyses on two other immigrant
groups that differ in their cultural distance to the Dutch
society, nuance was brought to the findings. The findings
highlight the importance of accounting for differences
between various ethnic groups in adolescents’ peer rela-
tionships. Similarly, in interpreting the results of this study
it should be taken into account that this study took place in a
specific context, with a long history of immigration and
integration. In contexts with more recent immigration or
with more prominent tensions between ethnic groups, the
results may differ. Funding This work was supported by the PhD fund of the faculty of
Behavioral and Social Sciences of the University of Groningen. Data Sharing and Declaration The dataset analyzed during the current
study are available from the GESIS Data Archive for the Social Sci-
ences. https://doi.org/10.4232/cils4eu.5656.3.3.0 Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of
interest. Ethical Approval An ethical review of the CILS4EU project has not
been carried out. At the time of data collection (2010–2011) the Dutch
law did not require Internal Review Board permission for this type of
research. Informed Consent Informed consent were obtained from all students
included in the study and their parents. Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Conclusion Open Access This article is distributed under the terms of the Creative
Commons
Attribution
4.0
International
License
(http://crea
tivecommons.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. As previous research found that cross-ethnic aggression was
not more likely than same-ethnic aggression, aggressive
behavior in itself does not seem to emphasize ethnic
boundaries. It was unclear, however, whether classmates’
responses to adolescents’ same- and cross-ethnic aggression
emphasize ethnic boundaries in the classroom. This study
investigated how adolescents’ ethnic background influences
friendships and rejection and how adolescents’ same- and
cross-ethnic aggressive behavior is related to the relation-
ship nominations they receive from classmates. Same-ethnic
classmates were more likely to be friends, but adolescents Appendix 1 Additional analyses on Dutch and Moroccan, and Dutch
and Surinamese adolescents (Table 5–8). Journal of Youth and Adolescence (2020) 49:678–692 689 Table 5 Results of multiple multilevel Poisson and negative binomial regression modelsa for friendship among Dutch and Moroccan adolescents (n = 926)
Befriended by Dutch
Befriended by Moroccan
Model 1
Model 2
Model 3
Model 1
Model 2
Model 3
Parameter
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
Constant
−2.40
0.20
−2.42
0.20
−2.40
0.19
−0.84
0.22
−0.85
0.21
−0.83
0.22
Ethnic background (Dutch = 1)
0.63
0.20
0.002
0.65
0.20
0.002
0.63
0.20
0.002
−1.33
0.22
<0.001
−1.31
0.22
<0.001
−1.33
0.22
<0.001
Aggression toward
Dutch
−0.002
0.04
0.95
0.00
0.14
1.00
0.14
0.09
0.13
0.15
0.23
0.40
*Ethnic background (Dutch = 1)
−0.004
0.15
0.98
−0.04
0.24
0.87
Moroccan
0.17
0.11
0.14
0.02
0.51
0.97
0.02
0.14
0.91
−0.10
0.14
0.47
*Ethnic background (Dutch = 1)
0.18
0.55
0.75
0.34
0.44
0.44
Sex (boy = 1)
0.02
0.06
0.69
0.02
0.06
0.70
0.02
0.06
0.71
−0.04
0.18
0.84
−0.04
0.18
0.80
−0.04
0.17
0.81
Number of Dutch classmates
Exp. Exp. Exp. 0.03
0.02
0.05
0.03
0.02
0.08
0.03
0.02
0.07
Number of Moroccan classmates
0.04
0.06
0.25
0.03
0.03
0.28
0.04
0.03
0.26
Exp. Exp. Exp. Appendix 1 Variance classroom
0.03
0.02
0.03
0.02
0.03
0.02
0.24
0.08
0.23
0.08
0.22
0.08
Log likelihood
−1509.48
−1508.43
−1508.26
−570.67
−569.38
−569.02
The number of Dutch or Moroccan classmates was used as an exposure variable to account for opportunity differences
aBeing befriended by Dutch classmates was examined using multilevel Poisson regression models (test of overdispersion: Χ2 = 0.00, p = 0.50; test of multilevel model: Χ2 = 11.00, p < 0.001),
being befriended by Moroccans was examined using multilevel negative binomial regression models (test of overdispersion: Χ2 = 3.79, p = 0.03; test of multilevel model: Χ2 = 22.90, p < 0.001)
*It indicates an interaction effect
Table 6 Results of multiple (multilevel) negative binomial regression modelsa for rejection among Dutch and Moroccan adolescents (n = 926)
Rejected by Dutch
Rejected by Moroccan
Model 1
Model 2
Model 3
Model 1
Model 2
Model 3
Parameter
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
Constant
−2.11
0.15
−2.18
0.15
−2.21
0.17
−2.61
0.39
−2.80
0.32
−2.84
0.32
Ethnic background (Dutch = 1)
−0.17
0.16
0.27
−0.11
0.16
0.48
−0.08
0.18
0.67
0.04
0.37
0.90
0.08
0.09
0.40
0.27
0.30
0.37
Aggression toward
Dutch
0.25
0.03
<0.001
0.62
0.17
<0.001
0.08
0.09
0.40
0.49
0.29
0.09
*Ethnic background (Dutch = 1)
−0.40
0.17
0.02
−0.45
0.31
0.15
Moroccan
0.22
0.18
0.22
−0.16
0.27
0.55
0.87
0.22
<0.001
0.95
0.18
<0.001
*Ethnic background (Dutch = 1)
0.53
0.34
0.12
−0.10
0.48
0.84
Sex (boy = 1)
0.06
0.09
0.47
0.03
0.09
0.72
0.04
0.09
0.68
0.28
0.19
0.046
0.37
0.19
.045
0.38
0.18
0.04
Number of Dutch classmates
Exp. Exp. Exp. 0.01
0.02
0.76
0.01
0.02
0.74
0.004
0.02
0.85
Number of Moroccan classmates
−0.04
0.04
0.25
−0.04
0.04
0.34
−0.03
0.04
0.45
Exp. Exp. Exp. Appendix 1 Variance classroom
na
na
na
0.26
0.15
0.27
0.13
0.25
0.13
Log likelihood
−1408.29
−1393.34
−1390.79
−452.07
−443.26
−442.26
The number of Dutch or Moroccan classmates was used as an exposure variable to account for opportunity differences
aBeing rejected by Dutch classmates was examined using single-level negative binomial regression models (test of overdispersion: Χ2 = 305.27, p < 0.001; test of multilevel model: Χ2 = 0.01, p =
0.47), being rejected by Moroccans was examined using multi-level negative binomial regression models (test of overdispersion: Χ2 = 2.26, p = 0.07; test of multilevel model: Χ2 = 6.99, p <
0.001) ber of Dutch or Moroccan classmates was used as an exposure variable to account for opportunity differences
jected by Dutch classmates was examined using single-level negative binomial regression models (test of overdispersion: Χ2 = 305.27, p < 0.001; test of multilevel model: Χ2 = 0.01, p =
ng rejected by Moroccans was examined using multi-level negative binomial regression models (test of overdispersion: Χ2 = 2.26, p = 0.07; test of multilevel model: Χ2 = 6.99, p < Journal of Youth and Adolescence (2020) 49:678–692 690 Table 7 Results of multiple (multilevel) Poisson regression modelsa for friendship among Dutch and Surinamese adolescents (n = 1219)
Befriended by Dutch
Befriended by Surinamese
Model 1
Model 2
Model 3
Model 1
Model 2
Model 3
Parameter
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
Constant
−1.84
0.07
−1.84
0.07
−1.84
0.07
−1.64
0.14
−1.65
0.14
−1.65
0.14
Ethnic background (Dutch = 1)
0.002
0.07
0.98
0.003
0.07
0.97
0.01
0.08
0.87
−0.29
0.14
0.04
−0.28
0.14
0.04
−0.29
0.14
0.04
Aggression toward
Dutch
−0.001
0.02
0.97
−0.20
0.13
0.11
0.03
0.06
0.69
0.16
0.16
0.31
*Ethnic background (Dutch = 1)
0.20
0.13
0.11
−0.16
0.17
0.35
Surinamese
−0.12
0.07
0.08
0.32
0.34
0.34
0.19
0.14
0.18
0.13
0.38
0.73
*Ethnic background (Dutch = 1)
−0.48
0.35
0.17
0.08
0.41
0.85
Sex (boy = 1)
0.10
0.05
0.07
0.10
0.05
0.08
0.09
0.05
0.09
0.08
0.10
0.44
0.08
0.10
0.42
0.09
0.10
0.38
Number of Dutch classmates
Exp. Exp. Exp. −0.02
0.01
0.09
−0.02
0.01
0.13
−0.02
0.01
0.13
Number of Surinamese classmates
−0.05
0.03
0.08
−0.05
0.03
0.10
−0.05
0.03
0.10
Exp. Exp. Exp. Appendix 1 Variance classroom
0.01
0.01
0.01
0.01
0.01
0.01
na
na
na
Log likelihood
−2137.03
−2136.08
−2134.09
−746.66
−745.84
−745.51
The number of Dutch or Surinamese classmates was used as an exposure variable to account for opportunity differences
aBeing befriended by Dutch classmates was examined using multilevel Poisson regression models (test of overdispersion: Χ2 = 0.54, p = 0.23; test of multilevel model: Χ2 = 4.71, p = 0.02), being
befriended by Surinamese classmates was examined using single-level Poisson regression models (test of overdispersion: Χ2 = 0.00, p = 0.50; test of multilevel model: Χ2 = 0.00, p = 0.50)
*It indicates an interaction effect
Table 8 Results of multiple Poisson and negative binomial regression modelsa for rejection among Dutch and Surinamese adolescents (n = 1219)
Rejected by Dutch
Rejected by Surinamese
Model 1
Model 2
Model 3
Model 1
Model 2
Model 3
Parameter
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
PE
SE
p
Constant
−2.49
0.14
−2.51
0.13
−2.54
0.14
−2.53
0.26
−2.59
0.27
−2.55
0.26
Ethnic background (Dutch = 1)
0.24
0.14
0.09
0.25
0.14
0.07
0.27
0.15
0.06
0.34
0.26
0.19
0.34
0.26
0.20
0.29
0.25
0.26
Aggression toward
Dutch
0.23
0.04
<0.001
0.45
0.11
<0.001
0.05
0.07
0.45
−0.36
0.48
0.46
*Ethnic background (Dutch = 1)
−0.24
0.12
0.04
0.42
0.49
0.39
Surinamese
0.19
0.15
0.21
−0.06
0.42
0.89
0.63
0.12
<0.001
−0.37
0.79
0.64
*Ethnic background (Dutch = 1)
0.27
0.45
0.54
1.04
0.79
0.19
Sex (boy = 1)
−0.07
0.08
0.40
−0.10
0.09
0.26
−0.09
0.09
0.30
−0.01
0.14
0.95
0.03
0.14
0.85
0.03
0.14
0.84
Number of Dutch classmates
Exp. Exp. Exp. −0.01
0.01
0.69
−0.001
0.02
0.96
−0.002
0.02
0.88
Number of Surinamese classmates
0.02
0.04
0.60
0.01
0.04
0.75
0.01
0.04
0.77
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na
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−1876.39
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−600.32
−590.63
−588.85
The number of Dutch or Surinamese classmates was used as an exposure variable to account for opportunity differences
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0.19), being rejected by Surinamese classmates was examined using single-level Poisson regression models (test of overdispersion: Χ2 = 0.20, p = 0.33; test of multilevel model: Χ2 = 0.45, p =
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*It indicates an interaction effect ber of Dutch or Surinamese classmates was used as an exposure variable to account for opportunity differences
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tural equation modeling. Sociological Methodology, 25, 267–316. https://doi.org/10.2307/271070. Veenstra, R., Lindenberg, S., Munniksma, A., & Dijkstra, J. K. (2010). The complex relation between bullying, victimization, accep-
tance, and rejection: Giving special attention to status, affection, Rivera, M. T., Soderstrom, S. B., & Uzzi, B. (2010). Dynamics of
dyads in social networks: Assortative, relational, and proximity 692 Journal of Youth and Adolescence (2020) 49:678–692 Jan Kornelis Dijkstra is an Associate Professor at the University of
Groningen. His major research interests include peer relations, peer
status, social networks, delinquency, and criminal networks. Jan Kornelis Dijkstra is an Associate Professor at the University of
Groningen. His major research interests include peer relations, peer
status, social networks, delinquency, and criminal networks. and sex differences. Child Development, 81, 480–486. https://doi. org/10.1111/j.1467-8624.2009.01411.x. and sex differences. Child Development, 81, 480–486. https://doi. org/10.1111/j.1467-8624.2009.01411.x. g
j
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bullying perpetration: a meta-analysis. Journal of Research on
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major research interests include computational sociology, polarization,
segregation, and cooperation. Marianne Hooijsma is a PhD Candidate at the University of
Groningen. Her
major
research
interests
include
youth’s
peer
relationships, ethnicity, and diversity. René Veenstra is a full Professor at the University of Groningen. His
major
research
interests
include
the
theoretical
and
empirical
elaboration
of
a
social
network
approach
to
bullying
and
victimization and pro- and antisocial behavior. Gijs Huitsing is an Assistant Professor at the University of
Groningen. His major research interests include a social network
approach to bullying and anti-bullying interventions.
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https://openalex.org/W1508825410
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https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/1471-2105-6-248
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English
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Better prediction of protein contact number using a support vector regression analysis of amino acid sequence
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BMC bioinformatics
| 2,005
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cc-by
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BioMed Central BioMed Central Better prediction of protein contact number using a support vector
regression analysis of amino acid sequence Address: Institute for Molecular Bioscience and ARC Centre in Bioinformatics, The University of Queensland, St. Lucia, 4072, Australia
Email: Zheng Yuan* - z.yuan@imb.uq.edu.au
* Corresponding author Received: 04 July 2005
Accepted: 13 October 2005 © 2005 Yuan; 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. © 2005 Yuan; 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. Abstract Background: Protein tertiary structure can be partly characterized via each amino acid's contact
number measuring how residues are spatially arranged. The contact number of a residue in a folded
protein is a measure of its exposure to the local environment, and is defined as the number of Cβ
atoms in other residues within a sphere around the Cβ atom of the residue of interest. Contact
number is partly conserved between protein folds and thus is useful for protein fold and structure
prediction. In turn, each residue's contact number can be partially predicted from primary amino
acid sequence, assisting tertiary fold analysis from sequence data. In this study, we provide a more
accurate contact number prediction method from protein primary sequence. Results: We predict contact number from protein sequence using a novel support vector
regression algorithm. Using protein local sequences with multiple sequence alignments (PSI-BLAST
profiles), we demonstrate a correlation coefficient between predicted and observed contact
numbers of 0.70, which outperforms previously achieved accuracies. Including additional
information about sequence weight and amino acid composition further improves prediction
accuracies significantly with the correlation coefficient reaching 0.73. If residues are classified as
being either "contacted" or "non-contacted", the prediction accuracies are all greater than 77%,
regardless of the choice of classification thresholds. Conclusion: The successful application of support vector regression to the prediction of protein
contact number reported here, together with previous applications of this approach to the
prediction of protein accessible surface area and B-factor profile, suggests that a support vector
regression approach may be very useful for determining the structure-function relation between
primary protein sequence and higher order consecutive protein structural and functional
properties. Open Acc
Research article
Better prediction of protein contact number using a support vector
regression analysis of amino acid sequence
Zheng Yuan* Open Access Page 1 of 9
(page number not for citation purposes) Background Approaches include comparative modelling, threading
and fold recognition methods. One protein structural fea-
ture is of particular interest here, namely, residue contact
number (CN) which can be used to enhance protein fold
recognition [1]. This measure has also been regarded as
the conserved solvent exposure descriptor of similar folds g
Prediction of protein three-dimensional structure from
primary sequence is the central problem in structural bio-
informatics. One approach is to use known structur∈ho-
molog proteins as templates to determine the tertiary
structures of novel proteins of unknown structure. Page 1 of 9
(page number not for citation purposes) Page 1 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/6/248 BMC Bioinformatics 2005, 6:248 Contact number distributions according to different
definitions
Figure 1
Contact number distributions according to different
definitions. The radius cutoffs are selected as 8 Å, 10 Å, 12
Å and 14 Å, represented by dotted, slashed, solid and dot-
and-slashed lines, respectively. A is for discrete contact
number while B is for consecutive contact number. without a common evolutionary origin [2]. Furthermore,
contact number may be used to determine the energy
function allowing molecular dynamics simulations of
protein structures [3]. Here, we seek to use protein contact
number to assist with the tertiary fold prediction of novel
proteins for which an accurate functional relationship
between a protein's primary sequence and its residues'
contact numbers must be determined. To fulfil the task,
we use a new method, the so-called support vector regres-
sion, to approximate the sequence-contact number rela-
tionship. We demonstrate that, as a result, we achieve
more accurate predicted contact numbers than have been
achieved to date. The contact number, or coordination number, of a given
residue of a folded protein is defined as the number of Cβ
(or Cα) atoms in other residues within a sphere around
the Cβ (or Cα) atom of that given residue. Previous
approaches to the prediction of protein contact number
fall into two categories: classification and regression. In
the classification approach, residue contact numbers were
first classified into two populations allowing a subsequent
use of machine learning methods such as recurrent neural
networks to perform predictions [4,5]. Unfortunately,
decomposing contact numbers into two states via an arbi-
trary threshold oversimplifies the problem and much
original information is lost. Contact n
definitions
Figure 1 Contact number distributions according to different
definitions
Figure 1
Contact number distributions according to different
definitions. The radius cutoffs are selected as 8 Å, 10 Å, 12
Å and 14 Å, represented by dotted, slashed, solid and dot-
and-slashed lines, respectively. A is for discrete contact
number while B is for consecutive contact number. g
g
Contact number distributions according to different
definitions. The radius cutoffs are selected as 8 Å, 10 Å, 12
Å and 14 Å, represented by dotted, slashed, solid and dot-
and-slashed lines, respectively. A is for discrete contact
number while B is for consecutive contact number. Thus, in this present work, we focus on multiple sequence
inputs. For completeness, we examine a range of different
definitions of contact number ("consecutive" and "dis-
crete"), and also examine whether including further infor-
mation such as protein molecular weight and amino acid
composition allows improved predictions. As a result, we
are able to make predictions which match observed values
with a correlation coefficient of 0.73, a significant
improvement on earlier studies. Page 2 of 9
(page number not for citation purposes) Background In contrast, the regression
approach provides a direct and more accurate way to
determine a functional relationship matching contact
numbers and protein sequence and thus to provide more
accurate contact number predictions. A recent study of
Kinjo et al. [3], followed this approach but used a simple
linear regression scheme to determine the functional rela-
tionship. They reported that the predicted and observe
contact numbers had a correlation coefficient (CC) of
0.627. However, most functions in nature are non-linear
and cannot be accurately approximated by linear formu-
las. Under the reasonable expectation that the sequence-
contact number is indeed nonlinear, we use a more com-
plicated machine learning method to determine the rela-
tionship and expect thereby to obtain more accurate
predictions. In particular, we adopt a support vector
regression (SVR) algorithm fully capable of determining a
non-linear sequence-contact number relationship. Contact nu
definitions
Figure 1 http://www.biomedcentral.com/1471-2105/6/248 BMC Bioinformatics 2005, 6:248 The accessible surface area as a function of contact number
Figure 2
The accessible surface area as a function of contact
number. Discrete contact numbers are used with a radius
cutoff of 12 Å. Error bars represent the standard deviations. group and examine the estimated function using the
remaining two groups. The correlation coefficient and
root mean square error (RMSE) are computed for each test
group, and their averages are shown in Table 2. For all radius cutoffs, predictions using consecutive con-
tact numbers are slightly better than predictions using
their discrete counterparts. However, when the larger
thresholds (e.g. 12 Å and 14 Å) are used, the accuracy
difference decreases to insignificance. Previous work has
shown that CNs with a radius cutoff of 12 Å or 14 Å are
more useful for protein fold recognition [1]. Likewise, in
this work, we also find that these cutoffs give better predic-
tions. But, compared with the discrete contact numbers,
the consecutive contact numbers give only a very slight
improvement in predictions. The best accuracies are for
consecutive contact numbers with thresholds of 12 Å and
14 Å, in which case the correlation coefficient between
predicted and observed values can reach 0.70. If we con-
vert the normalized contact numbers to their original
absolute ones, the RMSE of 0.72 is equal to an actual error
of 7.5 for a threshold of 12 Å, and an actual error of 11.1
for a threshold of 14 Å. The access
Figure 2 The accessible surface area as a function of contact number
Figure 2
The accessible surface area as a function of contact
number. Discrete contact numbers are used with a radius
cutoff of 12 Å. Error bars represent the standard deviations. g
The accessible surface area as a function of contact
number. Discrete contact numbers are used with a radius
cutoff of 12 Å. Error bars represent the standard deviations. We also calculate the CC and RMSE for each individual
protein using discrete contact numbers with a radius cut-
off of 12 Å. The average CC and RMSE are then 0.67 and
7.31, respectively. More than half of the proteins are pre-
dicted with CCs greater than 0.70, and more than half are
predicted with RMSEs less than 6.93. http://www.biomedcentral.com/1471-2105/6/248 To illustrate the
meaning of the CC and RMSE measures in this study, two
comparisons of predicted and observed values are given in
Figure 3. This figure shows the better agreement between
the predicted and observed values in GP130 (PDB: 1bj8)
as the CC is 0.75 and the RMSE is 6.07. In contrast, the
prediction for human chorionic gonadotropin (PDB:
1dz7, chain A) yields a correlation coefficient of 0.58 and
a RMSE of 9.73 with the region between position 40 and
50 being worst predicted. Table 1: The mean (
) and standard deviation (SD) of contact
numbers according to different radius (rd) cutoffs. All results are
expressed as (
, SD). rd = 8 Å
rd = 10 Å
rd = 12 Å
rd = 14 Å
Discrete
6.14, 3.29
12.90, 6.19
23.50, 10.46
35.39,15.39
Consecutive
6.27, 3.25
13.07, 6.14
23.56, 10.41
35.53, 15.39
N
N To study the relationship between CN and ASA of a resi-
due, we obtained the ASA for each residue in the 945 pro-
teins using the DSSP program [9]. Using discrete
definition of CN with a radius cutoff of 12 Å, we calculate
the mean and standard deviation of ASA for each contact
number and show the results in Fig. 2. A strong negative
correlation between the two solvent exposure descriptors
can be observed as indicated by a correlation coefficient of
-0.734. Sequence weight, as a feature, can improve prediction
accuracy significantly Prediction accuracy can be improved by taking account of
protein size as measured by its weight. Given a sequence,
the weight is the sum of individual weights of consisting
amino acids. We use discrete contact numbers (radius cut-
off = 12 Å) and divide all proteins into three groups with
equal number of proteins, according to their weights. For
the three groups, the weights (mean ± standard deviation)
are 10611 ± 1637, 17421 ± 2666 and 40073 ± 5952 Dal-
tons. Their average correlation coefficient between pre-
dicted and observed contact numbers are 0.61, 0.67 and
0.72, while their average RMSEs are 7.49, 7.09 and 7.35,
respectively. These results suggest that smaller molecules
are worst predicted. Page 3 of 9
(page number not for citation purposes) Contact numbers according to different rd values d
We give 8 definitions of contact number and show their
CN distributions in Fig. 1. For each definition, we com-
pute the mean and standard deviation (Table 1). For the
same radius cutoff rd, the discrete and consecutive defini-
tions have very similar distributions with nearly the same
mean and standard deviation. Their correlations are
greater than 0.99 for all values of rd (8 Å, 10 Å, 12 Å and
14 Å). The contact numbers defined by different radius
cutoffs have CCs greater than 0.83. Distributions with
larger radius cutoffs more closely approximate normal
distributions as their left-hand tails are almost all present. Since absolute contact numbers are normalized by a lin-
ear transformation (Equation 3), the general characteris-
tics of their distributions will still be kept even after the
normalization. In our former work, we studied the dependence of protein
accessible surface area (ASA) [6,7] and B-factor [8] on pri-
mary sequence. These works established that ASAs can be
predicted and match observed values with a correlation
coefficient of 0.69, while B-factors can be predicted and
match observed values with a correlation coefficient of
0.53. These approaches established that multiple
sequence inputs outperform single sequence inputs signif-
icantly. The importance of using multiple sequences was
also observed in prior predictions of contact numbers [3]. Page 2 of 9
(page number not for citation purposes) Page 2 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/6/248 http://www.biomedcentral.com/1471-2105/6/248 Estimating the sequence-contact number relationship For the separate groups of small chains,
median chains, large chains and all chains, we calculate
the mean and median values of the correlation coefficient
between predicted and observed values of contact
number, and their RMSEs, according to each set of differ-
ent input information: local sequence ("LS"), local
sequence plus sequence weight ("LS+W"), local sequence
plus amino acid composition ("LS+AA") and local
sequence plus sequence weight and amino acid composi-
tion ("LS+W+AA"). All results are shown in Table 3. To measure the prediction performance for residues with
different contact numbers, we compute the absolute
errors for the residue with contact numbers from 0 to 60. The mean absolute errors for certain contact numbers are
shown in Fig. 5, partitioned according to the four infor-
mation inputs. Clearly, "LS+W+AA" gives the least abso-
lute errors and therefore perform the best. Residues with
about 20 contacting Cβ atoms are predicted with the least
mean absolute error (4.1) and are the best predicted. This
is because they have the largest number of samples in the
dataset. Greater errors are found at each tail-end of the dis-
tribution corresponding to the residues with smaller or
greater contact numbers. This is due to the small number
of data points in each tail fed into support vector
machines, and their representation is not adequate. Fur-
thermore, this can be used to explain why the region from
residue 40 to residue 50 of protein 1dz7 in Fig 3B is the
worst predicted. This region mostly contains exposed res-
idues with smaller contact numbers and thus, the residues
cannot be well predicted by our method. An improvement
on this part may be achieved by using other predicted fea-
tures such as accessible surface area. For all cases, it was determined that amino acid composi-
tion information can improve prediction performance. However, sequence weight can give yet more significant
improvements. For example, in the group of small mole-
cules, data about amino acid composition can increase
the CC mean to 0.62 and decrease the RMSE mean to
7.24, while sequence weight data can increase the CC
mean to 0.64 and decrease the RMSE mean to 6.68. When
information about both sequence weight and amino acid
composition is used together, we find still further
improvement compared with using each data individu-
ally, although this may not be reflected by all measures. Estimating the sequence-contact number relationship When we train the SVR algorithm, normalized CNs are
used instead of absolute CNs because the normalized val-
ues are always located between -3.0 and 3.0 for all radius
cutoffs. Therefore, in all cases, the same set of SVR learn-
ing control parameters can be applied. Among the three
groups of proteins (each has 315 chains), we estimate the
sequence-contact number function in turn using one Page 3 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/6/248 BMC Bioinformatics 2005, 6:248 Table 2: Correlation coefficient (CC) and root mean square error (RMSE) for different contact number predictions. All results are
expressed as mean ± standard deviation. rd = 8 Å
rd = 10 Å
rd = 12 Å
rd = 14 Å
Discrete
CC
0.64 ± 0.01
0.66 ± 0.01
0.69 ± 0.01
0.69 ± 0.01
RMSE
0.77 ± 0.01
0.75 ± 0.01
0.72 ± 0.01
0.72 ± 0.02
Consecutive
CC
0.66 ± 0.01
0.67 ± 0.01
0.70 ± 0.01
0.70 ± 0.01
RMSE
0.75 ± 0.01
0.74 ± 0.01
0.72 ± 0.01
0.72 ± 0.02 cient (CC) and root mean square error (RMSE) for different contact number predictions. All results are
ard deviation. Table 2: Correlation coefficient (CC) and root mean square error (RMSE) for different contact number p
expressed as mean ± standard deviation. In addition to the above analyses based on individual pro-
teins, we also measure the accuracies on protein residues
in the whole data set. We calculate CCs and normalized
RMSEs for six testing groups, and express them as mean ±
standard deviation, as given in Table 4. The CCs for "LS",
"LS+W", "LS+AA" and "LS+W+AA" are 0.69, 0.733, 0.708
and 0.734, respectively. The normalized RMSEs are 0.72,
0.68, 0.71 and 0.68, respectively. Therefore, an obvious
improvement can be found even if we measure it at the
residue level. To consider this effect, we calculate weight for each pro-
tein sequence and include this data as an additional input
to the machine learning algorithm and re-run the training
and testing procedures. Additional information, that of
protein amino acid composition, was also included as an
input, either individually or together with sequence
weight data. Estimating the sequence-contact number relationship Particularly,
the
difference
between "LS+W"
and
"LS+W+AA" is very minor. However, all the results clearly
show that sequence weight is more important than amino
acid composition in the prediction of contact numbers. Examining performance by formulating regression as a
two-class problem Fig. 4 gives the overall distributions of CCs and RMSEs for
945 proteins. Compared with the CC, the RMSE is more
sensitive in that its distribution more clearly reflects the
improvement, while the distributions of "LS+W" and
"LS+W+AA" are nearly identical. For all cases, the peak
values of CC and RMSE are around 0.70 and 6.0,
respectively. CN prediction has previously been examined as a two-
class classification problem through use of a threshold
with the accuracy being defined as the percentage of the
correctly predicted residues on the overall residues [3-5]. However, this is not a good measure because the accuracy
is susceptible to changes in the selected threshold that Page 4 of 9
(page number not for citation purposes) Page 4 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/6/248 BMC Bioinformatics 2005, 6:248 The predicted and observed contact numbers for proteins GP130 (PDB: 1bj8) and Human chorionic gonadotropin (PDB: 1dz7,
chain A)
Figure 3
The predicted and observed contact numbers for proteins GP130 (PDB: 1bj8) and Human chorionic gonado-
tropin (PDB: 1dz7, chain A). Discrete contact numbers are used with a radius cutoff of 12 Å. Observed and predicted con-
tact numbers are represented by solid and dashed lines, respectively. A) GP 130 is predicted with a correlation coefficient of
0.75 and a root-mean-squar∈error of 6.07; B) Human chorionic gonadotropin is predicted with a correlation coefficient of
0.58 and a root mean square error of 9.73. Th
d
d
d b
d
b
f
GP130 (PDB 1b 8)
d H
h
d
(PDB 1d 7
h
A)
Fi
3 The predic
chain A)
Figure 3 The predicted and observed contact numbers for proteins GP130 (PDB: 1bj8) and Human chorionic gonadotropin (PDB: 1dz7,
chain A)
Figure 3
The predicted and observed contact numbers for proteins GP130 (PDB: 1bj8) and Human chorionic gonado-
tropin (PDB: 1dz7, chain A). Discrete contact numbers are used with a radius cutoff of 12 Å. Observed and predicted con-
tact numbers are represented by solid and dashed lines, respectively. A) GP 130 is predicted with a correlation coefficient of
0.75 and a root-mean-squar∈error of 6.07; B) Human chorionic gonadotropin is predicted with a correlation coefficient of
0.58 and a root mean square error of 9.73. Table 3: Correlation coefficients (CCs) and root mean square errors (RMSEs) for individual proteins in different weight groups. The
results are given as (mean, median). Examining performance by formulating regression as a
two-class problem LS
LS+W
LS+AA
LS+W+AA
W≤3485
CC
0.61, 0.65
0.64, 0.67
0.62, 0.66
0.64, 0.67
RMSE
7.49, 6.95
6.68, 6.41
7.24, 6.82
6.71, 6.45
13485<W≤22750
CC
0.67, 0.70
0.68, 0.71
0.68, 0.71
0.69, 0.72
RMSE
7.09, 6.79
6.76, 6.54
7.05, 6.79
6.76, 6.60
W>22750
CC
0.72, 0.73
0.72, 0.74
0.72, 0.73
0.73, 0.74
RMSE
7.35, 7.07
7.12, 6.95
7.24, 6.94
7.10, 6.90
All
CC
0.67, 0.70
0.68, 0.71
0.68, 0.71
0.68, 0.71
RMSE
7.31, 6.94
6.86, 6.66
7.18, 6.86
6.86, 6.66 http://www.biomedcentral.com/1471-2105/6/248 BMC Bioinformatics 2005, 6:248 Distributions of correlation coefficients and root mean square errors given different input information
Figure 4
Distributions of correlation coefficients and root mean square errors given different input information. Discrete
definition is used with a radius cutoff of 12 Å. The four inputs "LS", "LS+W", "LS+AA" and "LS+W+AA" are represented by
dotted, slashed, dot-and-slashed and solid lines, respectively. A is for correlation coefficients while B is for root mean square
errors. Distributio
Figure 4 Distributions of correlation coefficients and root mean square errors given different input information
Figure 4
Distributions of correlation coefficients and root mean square errors given different input information. Discrete
definition is used with a radius cutoff of 12 Å. The four inputs "LS", "LS+W", "LS+AA" and "LS+W+AA" are represented by
dotted, slashed, dot-and-slashed and solid lines, respectively. A is for correlation coefficients while B is for root mean square
errors. The mean absolute errors for residues of different contact
numbers
Figure 5
The mean absolute errors for residues of different
contact numbers. The four inputs "LS", "LS+W", "LS+AA"
and "LS+W+AA" are represented by dotted, slashed, dot-
and-slashed and solid lines, respectively. Table 4: Correlation coefficients (CCs) and root mean square
errors (RMSEs) are calculated for all residues when performing
support vector regression algorithm using different input
information. CC
RMSE
LS
0.69 ± 0.01
0.72 ± 0.01
LS+W
0.733 ± 0.005
0.68 ± 0.01
LS+AA
0.708 ± 0.009
0.71 ± 0.01
LS+W+AA
0.734 ± 0.006
0.68 ± 0.01 splits the data set. If the data set is heavily unbalanced,
accuracy is always very high [10]. In this study, we use a
different measure, and adopt the least (worst) prediction
accuracy for each case to reflect its performance. Table 5
gives the least two-class prediction accuracies for eight def-
initions of contact number when using only local
sequence information. Note that all the accuracies are the
average of six tests. Residue contact number We take two definitions of contact number in this study,
namely, that of "discrete" and "consecutive" contact
number. The "discrete" contact number, Nd, is defined by
the number of Cβ atoms on other residues located within
a sphere of radius rd centred on the Cβ atom of the residue
of interest. The discrete contact number for i-th residue in
a sequence with M residues is given by Examining performance by formulating regression as a
two-class problem rd = 8 Å
rd = 10 Å
rd = 12 Å
rd = 14 Å
Discrete
74.1
74.5
75.8
75.2
Consecutive
75.6
75.7
76.1
75.6 Conclusion We provide a new method for the prediction of protein
contact number. Using protein local sequence informa-
tion generated by multiple sequence alignments, the cor-
relation coefficient between predicted and observed
contact numbers can reach 0.70, with normalized root
mean square error less than 0.72. The addition of infor-
mation about sequence weight and amino acid composi-
tion as input features can increase the correlation
coefficient to 0.734 and decrease the root mean square
error to 0.68. This improvement is mainly attributed to
the information about sequence weight while the infor-
mation about amino acid composition only contributes
slightly. Moreover, more than half of the proteins are pre-
dicted with correlation coefficients greater than 0.71. The
prediction accuracies in the two-class problems, regardless
of the cutoff thresholds, are greater than 77.0%. The suc-
cessful application of SVR approach in this study suggests
that it can more accurately describe the relationship
between protein contact numbers and primary sequence. Prediction
two-class p
thresholds
Figure 6 p
p
g
g
Prediction accuracies when predictions are formu-
lated as two-class problems using different contact
number thresholds. The four inputs "LS", "LS+W",
"LS+AA" and "LS+W+AA" are represented by dotted,
slashed, dot-and-slashed and solid lines, respectively. Examining performance by formulating regression as a
two-class problem All accuracies are found to be greater
than 74%, and in particular, when rd = 12 Å and a consec-
utive contact number definition is adopted, the least
prediction accuracy is around 76.1%, which is compara-
ble with the accuracy 76.3% recently reported based on
choosing a particular threshold in linear models also
using the same consecutive contact number definition [3]. The mean
numbers
Figure 5 g
The mean absolute errors for residues of different
contact numbers. The four inputs "LS", "LS+W", "LS+AA"
and "LS+W+AA" are represented by dotted, slashed, dot-
and-slashed and solid lines, respectively. either "contacted" or "non-contacted" and calculate their
accuracies. All accuracies are plotted in Fig. 6, according to
different information input. The least accuracies for "LS",
"LS+W", "LS+AA" and "LS+W+AA" are 75.8%, 77.2%,
76.1% and 77.1%, respectively. Using sequence weight is
much better than using amino acid composition. We use discrete definitions of contact number and let rd =
12 Å. Using all the SVM outputs from six tests, we choose
a number of thresholds to classify the data points as being Page 6 of 9
(page number not for citation purposes) Page 6 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/6/248 http://www.biomedcentral.com/1471-2105/6/248 http://www.biomedcentral.com/1471-2105/6/248 BMC Bioinformatics 2005, 6:248 Table 5: The least prediction accuracy (%) for two-class
problems according to different contact number definitions. Only local sequence information is used. rd = 8 Å
rd = 10 Å
rd = 12 Å
rd = 14 Å
Discrete
74.1
74.5
75.8
75.2
Consecutive
75.6
75.7
76.1
75.6
Prediction accuracies when predictions are formulated as
two-class problems using different contact number
thresholds
Figure 6
Prediction accuracies when predictions are formu-
lated as two-class problems using different contact
number thresholds. The four inputs "LS", "LS+W",
"LS+AA" and "LS+W+AA" are represented by dotted,
slashed, dot-and-slashed and solid lines, respectively. Table 5: The least prediction accuracy (%) for two-class
problems according to different contact number definitions. Only local sequence information is used. From our work here, we confirm the utility of a regression
analysis, and more specifically, establish that allowing for
non-linearity via support vector regression allows a more
accurate determination of the sequence-contact number
relation which further illuminates relationships between
protein structural and functional properties and their pri-
mary sequence and other features. Table 5: The least prediction accuracy (%) for two-class
problems according to different contact number definitions. Only local sequence information is used. Sequence coding We predict contact number from protein local sequence. For a given residue, the local sequence contains its N-ter-
minal and C-terminal seven nearest-neighbour residues. Thus, the local sequence makes a window of fifteen amino
acids. We code each residue in the window using the PSI-
BLAST position-specific scoring matrix [12]. The matrices
are obtained by querying the input sequence using PSI-
BLAST against the NCBI non-redundant protein sequence
database with three rounds, masking coil-coiled and low-
complexity regions [13]. The elements in the row of the
matrix reflect the probabilities for 20 amino acids occur-
ring at this position. All the elements are divided by 10 for
normalization and thus each residue is represented by a
20-dimesional vector. Since the residues in coil-coil and
low-complexity regions do not have meaningful scores,
we encode the residue with an orthogonal scheme. In the
20-dimensional vector coding a given residue, only the
entry representing this type of amino acid is assigned as
0.5 with all other entries set as zeros. To consider the
terminal residues, we expend the 20-dimensional vector
to being 21-dimensional for all residues. When the last
entry is set as 0.5 and other entries have zeros, it represents
a blank residue added to the N-terminal or the C-terminal
to make a local sequence of 15-residue length. For all
other residues, the 21-st entries are set to zero. In sum-
mery, a residue is coded by a 315-dimensional vector. The solution of the above problem was given by the
authors of ∈SVR [14,15] as follows, f X
X
X
b
i
i
i
i
M
( )
(
)
(
),
( )
,
*
=
−
+
( )
=∑α
α
Φ
Φ
6
1 ( )
6 where αi and
are Lagrange multipliers. We can replace
Φ(Xi), Φ(X), the inner product of Φ(Xi) and Φ(X), by a
kernel function K(Xi, X), if K(Xi, X) = Φ(Xi), Φ(X). The
radial basis function are used in our study, as given by
αi
* K(Xi, X) = exp(-γ||Xi - X||2), (7) K(Xi, X) = exp(-γ||Xi - X||2), (7) where γ is a parameter to be tuned by the user. where γ is a parameter to be tuned by the user. We constantly set ε as 0.01, γ as 0.01 and C as 5.0, because
this set of parameters yielded the best performance in our
previous work [6,8]. Discussion Protein structural properties such as secondary structure,
solvent accessibility and contact number provide valuable
information for prediction of protein tertiary structures. How to improve the prediction accuracy of these
parameters is still a challenging problem. Following Rost
and Sander's pioneering work [11] on how to find a con-
served and useful prediction index, Hamelryck [2] exam-
ined the conservation of nine solvent-exposure measures
and found that contact number is the most conserved
(correlation coefficient 0.72). His study suggested that CN
is more suitable for fold recognition than other descrip-
tors such as ASA. However, difficulties in accurately
expressing the prediction problem (for example, it was
previously framed as a two class problem using an arbi-
trary threshold) limited its further application. Recent
work on contact number [3] formulating the problem for
a regression analysis has enhanced studies in this area. Nd
i
r
r
r
r
r
i j
i j
i j
d
i j
=
=
<
=
σ
σ
σ
(
)
(
)
(
)
,
,
,
,
1
0
if
if r
r
j :| j
i |
M
i j
d
,
,
≥
( )
−
>
∑
1
2 ( )
1 where ri,j is the distance between the Cβ atoms of the ith
and jth residues which are understood to be separated in
sequence by at least two amino acids. Note that
is a
discrete integer. By replacing the step function σ(ri,j) with
Nd
i where ri,j is the distance between the Cβ atoms of the ith
and jth residues which are understood to be separated in sequence by at least two amino acids. Note that
is a
discrete integer. By replacing the step function σ(ri,j) with
Nd
i a sigmoid function,
becomes a real number. This pro-
cedure was previously adopted by Kinjo et al. [3] to
smooth the discrete contact numbers. A particular sig-
moid function is given by
Nd
i σ(ri,j) = 1/{1 + exp [3(ri,j - rd)]}. (2) σ(ri,j) = 1/{1 + exp [3(ri,j - rd)]}. Dataset preparation and prediction evaluation p
p
p
To test our approach, we selected 945 unique protein
chains, which were previously used for prediction of pro-
tein ASA, and were prepared by PDB-REPRDB [17]. The
structures solved by X-ray crystallography were with reso-
lution less than 2.0 Å and with an R-factor less than 0.2. All chains are at least 60 amino acids or longer, and the
pair-wise identity is less than 25%. The protein names can Minimize ( )
3 At the first step, we predict the normalized contact
number because 1) it is easy to handle the data, and 2) it
is easy to compare the results for different rd thresholds. At
the second step, we recover the absolute contact numbers
from their predicted normalized values using this
equation. subject to
f X
y
y
f X
i
M
i
i
i
i
i
i
i
i
(
)
(
)
,
,
,
*
*
−
≤
+
−
≤
+
≥
=
ε
ξ
ε
ξ
ξ ξ
0
1
for
…
( )
5 ( )
5 where C is the regularization constant that determines the
trad∈off between the norm and the error penalty. Discussion (2) (2) Page 7 of 9
(page number not for citation purposes) (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/6/248 http://www.biomedcentral.com/1471-2105/6/248 http://www.biomedcentral.com/1471-2105/6/248 BMC Bioinformatics 2005, 6:248 f(Xi) = W, Φ(Xi) + b, (4) We have tried four values of rd (8 Å, 10 Å, 12 Å and 14 Å)
with discrete and consecutive definitions and thus have 8
combinations all of which will be used in our SVR
approach. where W is the weight and b is the bias. Φ(Xi) is a non-lin-
ear function mapping a data point from the input space to
the feature space, so consequently, SVR is able to perform
non-linear regression. The goal of the regression is to find
the optimal W and b using some optimisation criteria. In
ε-SVR, errors greater than ε are penalized, where two pos-
itive variables ξ and ξ* are used to measure the deviation
of samples outside the ε-insensitive tube. The optimisa-
tion problem can be expressed as Sequence coding A number of software packages can
be used to find the solution such as SVMlight [16]. Normalization of contact number The distributions of contact numbers can be approxi-
mated by normal distributions, as shown in Fig. 1. With
respect to a certain rd, we calculate the mean (
) and
standard deviation (SD). So, the normalized contact
number Nnorm is determined by the following formula:
N The distributions of contact numbers can be approxi-
mated by normal distributions, as shown in Fig. 1. With respect to a certain rd, we calculate the mean (
) and
standard deviation (SD). So, the normalized contact
number Nnorm is determined by the following formula:
N 1
2
2
1
W
C
i
i
i
M
+
+
=∑(
),
*
ξ
ξ Minimize
1
2
2
1
W
C
i
i
i
M
+
+
=∑(
),
*
ξ
ξ N
N
N
SD
norm =
−
( )
. 3 Minimize Additional material Additional File 1
The names of 945 protein chains. The first four characters are their PDB
names. The fifth is the chain name and "_" means single chain. Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2105-6-248-S1.doc] Additional File 1
The names of 945 protein chains. The first four characters are their PDB
names. The fifth is the chain name and "_" means single chain. Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2105-6-248-S1 doc] http://www.biomedcentral.com/1471-2105/6/248 BMC Bioinformatics 2005, 6:248 be found in the additional file 1 (supplementary
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59:38-48. Publish with BioMed Central and every
scientist can read your work free of charge
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(page number not for citation purposes) Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
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BioMedcentral Publish with BioMed Central and every
scientist can read your work free of charge 3. Kinjo AR, Horimoto K, Nishikawa K: Predicting absolute contact
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4. Pollastri G, Baldi P, Fariselli P, Casadio R: Prediction of coordina-
tion number and relative solvent accessibility in proteins. Proteins 2002, 47:142-153. 5. Pollastri G, Baldi P, Fariselli P, Casadio R: Improved prediction of
the number of residue contacts in proteins by recurrent neu-
ral networks. Bioinformatics 2001, 17 Suppl 1:S234-42. f
pp
6. Yuan Z, Huang B: Prediction of protein accessible surface areas
by support vector regression. Proteins 2004, 57:558-564. by support vector regression. Proteins 2004, 57:558- 7. Yuan Z, Bailey TL: Prediction of protein solvent profile using
SVR. Proceedings of the 26th Annual International Conference of the IEEE
EMBS 2004:2889-2892. 7. Acknowledgements The author thanks Dr. Rohan Teasdale for kind assistance and Dr. Michael
Gagen for critical reading of this article and some helpful suggestions. The
work was supported by funds from the Australian Research Council (ARC)
and The University of Queensland Early Research Grant. The computer
simulations were performed at the High Performance Computing Facility at
The University of Queensland. References Yuan Z, Bailey TL: Prediction of protein solvent profile using
SVR. Proceedings of the 26th Annual International Conference of the IEEE
EMBS 2004:2889-2892. 8. Yuan Z, Bailey TL, Teasdale RD: Prediction of protein B-factor
profiles. Proteins 2005, 58:905-912. 8. Yuan Z, Bailey TL, Teasdale RD: Prediction of protein B-factor
profiles. Proteins 2005, 58:905-912. Support vector regression To find the function between protein local sequence and
normalized contact number, we use ∈-insensitive support
vector regression (∈-SVR) [14,15]. The expected function
can be formulated as Page 8 of 9
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Trabajadores indígenas en el Chaco argentino: algunos sentidos estigmatizadores
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*
Las consideraciones aquí vertidas fueron presentadas anteriormente en mi tesis doctoral, que analiza el proceso
de producción de los qom del este de la provincia argentina de Formosa como trabajadores, ciudadanos e
indígenas (Iñigo Carrera, V., 2008). La investigación fue financiada por el Consejo Nacional de Investigaciones
Científicas y Técnicas (Conicet) y la Universidad de Buenos Aires. Agradezco a los evaluadores anónimos y a la
editora invitada, cuyos comentarios enriquecieron la versión final del presente trabajo.
**
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ti Artículo recibido: 1° de marzo de 2013 | aceptado: 17 de septiembre de 2013 | modificado: 5 de octubre de 2013
Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 ,
y
q
** Doctora en Antropología, Universidad de Buenos Aires. Argentina. Trabajadores indígenas
en el Chaco argentino: algunos
sentidos estigmatizadores* Valeria Iñigo Carrera**
v.inigocarrera@conicet.gov.ar. Consejo Nacional de Investigaciones Científicas y Técnicas (Conicet). Universidad de Buenos Aires,
Argentina Valeria Iñigo Carrera**
v.inigocarrera@conicet.gov.ar. Consejo Nacional de Investigaciones Científicas y Técnicas (Conicet). Universidad de Buenos Aires,
Argentina Valeria Iñigo Carrera**
v.inigocarrera@conicet.gov.ar. Consejo Nacional de Investigaciones Científicas y Técnicas (Conicet). Universidad de Buenos Aires,
Argentina R e s u m e n Los qom del Chaco centro-oriental (en el extremo
noreste de Argentina) constituyen una población trabajadora
desplazada de la producción agropecuaria. El objetivo del
presente trabajo es avanzar sobre las formas que la materialidad
de este proceso asume en la conciencia de los sujetos, a través
del despliegue de los siguientes sentidos: la tensión entre
“marisca” y “trabajo”, la supuesta ausencia de una “cultura
productiva” y la “dependencia” en que se encontrarían sumidos
los indígenas chaqueños. P a l a b r a s c l a v e : P a l a b r a s c l a v e :
Qom, marisca, trabajo, producción, Chaco argentino. DOI: http://dx.doi.org/10.7440/antipoda17.2013.13 DOI: http://dx.doi.org/10.7440/antipoda17.2013.13 A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 Key words: Qom, marisca, labor, production, Argentinean Chaco. Trabalhadores indígenas no Chaco argentino:
alguns sentidos estigmatizadores* Resumo Os qom do Chaco centro-oriental (no extremo nordeste da Argentina) constituem uma
população trabalhadora deslocada da produção agropecuária. O objetivo do presente trabalho é
avançar sobre as formas que a materialidade desse processo assume na consciência dos sujeitos,
por meio da implementação dos seguintes sentidos: a tensão entre “marisca” e “trabalho”,
a suposta ausência de uma “cultura produtiva” e a “dependência” em que se encontrariam
submersos os indígenas do Chaco. Indigenous Workers in El Chaco, Argentina:
Some Stigmatizing Concepts Abstract The indigenous Qom people of the central-eastern Chaco (in northeastern Argentina) are
workers who are being expelled from agrarian production. This work seeks to help us understand
the materiality that this process assumes in peoples’ minds, teasing out the following meanings: the
tension between “marisca” and “labour”, the supposed absence of a “productive culture”, and the
“dependency” in which Chaco indigenous people allegedly are immersed. 1 Se trata de aquella porción del Chaco argentino que se extiende entre los ríos Bermejo y Pilcomayo (límite fron-
terizo, allí, entre Argentina y Paraguay), comprendiendo la totalidad de la provincia de Formosa y el noreste de
Salta. Me refiero, en este trabajo, a los qom de Misión Tacaaglé, Potae Napocna Navogoh (ambas, comunidades
rurales ubicadas sobre la ruta nacional N° 86, cercanas al río Pilcomayo y distantes, respectivamente, unos 195 y
137 km de la ciudad capital de Formosa) y Namqom (barrio periurbano ubicado a 10 km de la capital formoseña). Palavras-chave: Qom, marisca, trabalho, produção, Chaco argentino. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 Trabajadores indígenas
en el Chaco argentino: algunos
sentidos estigmatizadores V a l e r i a I ñ i g o C a r r e r a Introducción Por cierto, de las formas que tomó el avance del capital en el Chaco cen-
tral, fue el cultivo del algodón el que, desde que inició su ciclo sostenido de
expansión en la década de los treinta, absorbió en mayor proporción la masa
de brazos indígenas de la porción oriental de dicha región (Bartolomé, 1972;
Gordillo, 2004); aquella separada, de manera repentina y violenta, de sus condi-
ciones materiales de existencia originarias3. La incorporación de los qom a esa
producción fue bajo la forma de trabajadores asalariados de temporada en las
labores de carpida (desmalezamiento) y cosecha del cultivo y como pequeños
productores independientes de algodón en bruto. Pero su absorción o su expul-
sión se vieron determinadas por los sucesivos momentos de fuerte expansión,
intercalados con otros de igualmente fuerte contracción, que han caracterizado
históricamente la escala de la producción algodonera; la tendencia decreciente
en la superficie implantada y en el volumen de la producción, que se detecta
detrás de esos movimientos de vaivén; el cambio en las condiciones técnicas
de la producción de algodón, a partir de la década de 1980, a través de la intro-
ducción del control químico y mecánico de las malezas y de la cosecha mecá-
nica; el proceso de concentración y centralización del capital algodonero y su
expresión en la creciente diferenciación entre los agentes de la producción, que
aceleró su tendencia en los noventa. El conjunto de estas determinaciones ha generado la progresiva muti-
lación de los atributos productivos de la fuerza de trabajo indígena, la cual
encuentra múltiples expresiones: la también progresiva limitación en la par- A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 evidenciar que quien la protagonizaba era una población trabajadora que
se encontraba cada vez más en exceso para los requerimientos del capital
agropecuario en general y algodonero en particular2. evidenciar que quien la protagonizaba era una población trabajadora que
se encontraba cada vez más en exceso para los requerimientos del capital
agropecuario en general y algodonero en particular2. No es mi propósito aquí avanzar sobre las mencionadas formas de acción
política en sí mismas, cuyo análisis ha sido objeto de otros escritos (Iñigo
Carrera, V., 2012a y 2012b). 2 El algodón es el principal, si no único, cultivo comercial de medianos y pequeños productores, entre estos últi-
mos, los qom.
3 Cordeu y Siffredi (1971) y Miller (1979) reseñaron la importancia de la agroindustria del algodón para los qom
de la provincia de Chaco (en términos regionales, el Chaco austral). Introducción E E
n los últimos años, las formas de acción polí-
tica de los qom de la porción oriental del Chaco central
(en el extremo noreste de Argentina)1 revelan una particu-
laridad: su implicación en un proceso de lucha desplegado
a mediados de la década de 2000 por pequeños producto-
res de mercancías agrarias y trabajadores rurales criollos (es
decir, descendientes de aquellos procedentes de las provincias argentinas
de Chaco y Corrientes y de la vecina República del Paraguay). Esta partici-
pación encontró su expresión en los reclamos contenidos en los petitorios
del Movimiento Campesino de Formosa –Mocafor–, cuyo destinatario era
la administración provincial: subsidios compensatorios para pequeños pro-
ductores algodoneros, apoyo tecnológico y precios justos para producto-
res campesinos, trabajo para desocupados mediante la agroindustria, agua
potable y energía eléctrica para las comunidades rurales, becas para estu-
diantes de pocos recursos, mayor presupuesto e inversión en salud y educa-
ción públicas, regulación de las producciones transgénicas, devolución de
tierras expropiadas a las comunidades indígenas, tierra para trabajo a fami-
lias campesinas sin tierra, defensa de los recursos naturales ante su con-
centración, extranjerización y destrucción (Mocafor, 2005). Que la lucha
fuera por derechos considerados básicos (educación, salud, trabajo), por
la tierra (su acceso, devolución, titularización, no concentración ni extran-
jerización) y por la asistencia en la producción, comercialización e indus-
trialización de las mercancías agrarias, y luego, también, por el acceso a los
programas sociales de asistencia a la pobreza y al desempleo, no hace sino Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 2 3 2
A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3
evidenciar que quien la protagonizaba era una población trabajadora que
se encontraba cada vez más en exceso para los requerimientos del capital
agropecuario en general y algodonero en particular2. No es mi propósito aquí avanzar sobre las mencionadas formas de acción
política en sí mismas, cuyo análisis ha sido objeto de otros escritos (Iñigo
Carrera, V., 2012a y 2012b). Antes bien, la finalidad del presente trabajo es exa-
minar el proceso que se encuentra en su base: el desplazamiento de la produc-
ción (me refiero, en particular, a la producción algodonera) del que son objeto
los qom del Chaco centro-oriental. q
3 Cordeu y Siffredi (1971) y Miller (1979) reseñaron la importancia de la agroindustria del algodón para los qom
de la provincia de Chaco (en términos regionales, el Chaco austral). 2 El algodón es el principal, si no único, cultivo comercial de medianos y pequeños productores, entre estos últ
mos, los qom. T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o posterior asalarización como carpidores y cosecheros en sus propias tierras; la
caída del salario obtenido como cosechero, que apenas representa la reproduc-
ción física durante el período de trabajo; la venta de los productos del trabajo
artesanal y de los subproductos de la “marisca” (caza de animales silvestres,
pesca, recolección de frutos silvestres y miel) por debajo del valor al que luego
se realiza normalmente en el mercado, con lo que quien adquiere la mercancía
del productor directo indígena se apropia de una parte del valor que éste pro-
dujo; la constitución generalizada de los qom en beneficiarios de programas
sociales de asistencia a la pobreza y al desempleo. Decía que el propósito de este trabajo es examinar el proceso de desplaza-
miento de la producción del que son objeto los qom del Chaco centro-oriental. Pero, por cuanto abordé su materialidad en otro sitio (Iñigo Carrera, V., 2008 y
2009), lo que aquí me interesa es atender a las formas que asume esa materia-
lidad en la conciencia de los sujetos4. La tensión entre “marisca” y “trabajo”, la
supuesta ausencia –o, en el mejor de los casos, pérdida– de una “cultura pro-
ductiva” y la “dependencia” en que se encontrarían sumidos los indígenas cha-
queños constituyen los ejes de construcción de sentidos (y prácticas) cotidia-
nos que habitan –de maneras encontradas y no fijas– la conciencia y asumen
expresión en enunciaciones o concepciones (de los qom, agentes del Estado,
criollos), cuya presentación atraviesa este trabajo. Mi argumento, que sintetizo
en el apartado conclusivo, es que se trata de unas construcciones de sentido que
evidencian una estigmatización de las formas en que los trabajadores indígenas
(re)producen su vida. 4 Atendiendo a lo planteado por Menéndez (2002), en el sentido de que la mayoría de los antropólogos, a pesar
de preocuparse casi inéditamente en la actualidad por recuperar el sujeto, no explicitan ni reflexionan sobre la
concepción de subjetividad que manejan, quisiera realizar al respecto dos señalamientos de manera por demás
sintética, pues se trata de una cuestión que excede ampliamente los límites de este trabajo. Primero, que todo
sujeto existe socialmente como configuración resultante de una multiplicidad de determinaciones (históricas,
estructurales, institucionales, cotidianas) (Trinchero, 1994). Segundo, que, en cuanto lo anterior, el sujeto cons-
truye sentidos –o produce representaciones– de manera condicionada por un determinado desarrollo de las
fuerzas productivas de la sociedad (Marx y Engels, 1973) o, lo que es lo mismo, que la conciencia es la expresión
en ideas de las condiciones materiales de la vida humana (Iñigo Carrera, J., 2008).
5 Según los resultados del Censo Nacional de Población, Hogares y Viviendas 2010, habitan Formosa
32.216 personas que se autorreconocen como descendientes (porque tienen algún antepasado) o per-
tenecientes (porque se declaran como tales) a algún pueblo indígena. Representan, así, un 6% de la
población total de la provincia. 4 Atendiendo a lo planteado por Menéndez (2002), en el sentido de que la mayoría de los antropólogos, a pesar
de preocuparse casi inéditamente en la actualidad por recuperar el sujeto, no explicitan ni reflexionan sobre la
concepción de subjetividad que manejan, quisiera realizar al respecto dos señalamientos de manera por demás
sintética, pues se trata de una cuestión que excede ampliamente los límites de este trabajo. Primero, que todo
sujeto existe socialmente como configuración resultante de una multiplicidad de determinaciones (históricas,
estructurales, institucionales, cotidianas) (Trinchero, 1994). Segundo, que, en cuanto lo anterior, el sujeto cons-
truye sentidos –o produce representaciones– de manera condicionada por un determinado desarrollo de las
fuerzas productivas de la sociedad (Marx y Engels, 1973) o, lo que es lo mismo, que la conciencia es la expresión
en ideas de las condiciones materiales de la vida humana (Iñigo Carrera, J., 2008). Introducción Antes bien, la finalidad del presente trabajo es exa-
minar el proceso que se encuentra en su base: el desplazamiento de la produc-
ción (me refiero, en particular, a la producción algodonera) del que son objeto
los qom del Chaco centro-oriental. Por cierto, de las formas que tomó el avance del capital en el Chaco cen-
tral, fue el cultivo del algodón el que, desde que inició su ciclo sostenido de
expansión en la década de los treinta, absorbió en mayor proporción la masa
de brazos indígenas de la porción oriental de dicha región (Bartolomé, 1972;
Gordillo, 2004); aquella separada, de manera repentina y violenta, de sus condi-
ciones materiales de existencia originarias3. La incorporación de los qom a esa
producción fue bajo la forma de trabajadores asalariados de temporada en las
labores de carpida (desmalezamiento) y cosecha del cultivo y como pequeños
productores independientes de algodón en bruto. Pero su absorción o su expul-
sión se vieron determinadas por los sucesivos momentos de fuerte expansión,
intercalados con otros de igualmente fuerte contracción, que han caracterizado
históricamente la escala de la producción algodonera; la tendencia decreciente
en la superficie implantada y en el volumen de la producción, que se detecta
detrás de esos movimientos de vaivén; el cambio en las condiciones técnicas
de la producción de algodón, a partir de la década de 1980, a través de la intro-
ducción del control químico y mecánico de las malezas y de la cosecha mecá-
nica; el proceso de concentración y centralización del capital algodonero y su
expresión en la creciente diferenciación entre los agentes de la producción, que
aceleró su tendencia en los noventa. El conjunto de estas determinaciones ha generado la progresiva muti-
lación de los atributos productivos de la fuerza de trabajo indígena, la cual
encuentra múltiples expresiones: la también progresiva limitación en la par-
ticipación del cultivo del algodón, como productores independientes del textil
y como trabajadores estacionales; el arrendamiento de sus parcelas de tierra a
terceros, por falta de herramientas e insumos para ponerla en producción, y su Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. (
g
, ,
)
5 Según los resultados del Censo Nacional de Población, Hogares y Viviendas 2010, habitan Formosa
32.216 personas que se autorreconocen como descendientes (porque tienen algún antepasado) o per-
tenecientes (porque se declaran como tales) a algún pueblo indígena. Representan, así, un 6% de la
población total de la provincia. Introducción 229-251 T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o ¿“Marisca” versus “trabajo”? “Así como mariscan en el monte, mariscan en la ciudad” constituye una expre-
sión frecuente entre los agentes de la administración pública formoseña. Se
refieren, claro está, a los pobladores indígenas de la provincia5. A principios Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 2 3 4
A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3
del año 2000, quien presidía el Instituto de Comunidades Aborígenes (ICA), la
agencia provincial para la administración de los asuntos indígenas, enriquecía
aquella expresión en los siguientes términos: “Viste que ellos le dicen mariscar. Salís a mariscar al monte y como que también el mariscar se traslada a este
monte que es la ciudad: visitar el Ministerio de Acción Social, una gran depen-
dencia de pedir todo. Entonces, bueno, si consigo, consigo. Así como cuando
vas a cazar, si cazás, cazás, si no…”. Pero la proyección de la práctica de la caza, pesca y recolección al espacio
urbano para explicar la forma de (re)producir la vida en este último constituye
un supuesto fuertemente arraigado que no se revela exclusivo de los agentes
del Estado provincial. Un pequeño productor criollo de la localidad de Misión
Tacaaglé, integrante del Mocafor, no sólo reproducía aquella proyección, sino
que también explicaba la productividad del trabajo indígena en la producción
algodonera sobre la base de aquella especificidad cazadora-recolectora:
Son lentos para cosechar algodón. Hacen ochenta kilos, mientras que el
blanco hace ciento veinte kilos. Pero son más limpios6. Son menos pro-
ductores que los criollos, producen en menor cantidad. Los aborígenes no
tienen esa cultura. Sólo algunos son buenos productores. Pero ellos tienen
otra cultura. Preparan el suelo, tiran la semilla y dejan, no se ocupan. Viste
que históricamente han sido más cazadores-recolectores. […] Antes maris-
caban en el monte, ahora en el pueblo7. A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 del año 2000, quien presidía el Instituto de Comunidades Aborígenes (ICA), la
agencia provincial para la administración de los asuntos indígenas, enriquecía
aquella expresión en los siguientes términos: “Viste que ellos le dicen mariscar. ¿“Marisca” versus “trabajo”? Salís a mariscar al monte y como que también el mariscar se traslada a este
monte que es la ciudad: visitar el Ministerio de Acción Social, una gran depen-
dencia de pedir todo. Entonces, bueno, si consigo, consigo. Así como cuando
vas a cazar, si cazás, cazás, si no…”. Pero la proyección de la práctica de la caza, pesca y recolección al espacio
urbano para explicar la forma de (re)producir la vida en este último constituye
un supuesto fuertemente arraigado que no se revela exclusivo de los agentes
del Estado provincial. Un pequeño productor criollo de la localidad de Misión
Tacaaglé, integrante del Mocafor, no sólo reproducía aquella proyección, sino
que también explicaba la productividad del trabajo indígena en la producción
algodonera sobre la base de aquella especificidad cazadora-recolectora: Son lentos para cosechar algodón. Hacen ochenta kilos, mientras que el
blanco hace ciento veinte kilos. Pero son más limpios6. Son menos pro-
ductores que los criollos, producen en menor cantidad. Los aborígenes no
tienen esa cultura. Sólo algunos son buenos productores. Pero ellos tienen
otra cultura. Preparan el suelo, tiran la semilla y dejan, no se ocupan. Viste
que históricamente han sido más cazadores-recolectores. […] Antes maris-
caban en el monte, ahora en el pueblo7. 6 Las cualidades del indígena como cosechero son compartidas por quien se desempeñaba en 1999 como subse-
cretario de Empleo de la provincia: “El cosechero aborigen produce un algodón de una muy buena calidad, un
algodón limpio. Pero su rendimiento es bajo. Cosecha entre veinticinco y treinta kilos por día contra setenta kilos
del criollo. […] hacen el cálculo de cuánto tienen que cosechar para conseguir la comida para subsistir durante
el tiempo que dura la cosecha. No tienen el concepto de acumulación ni el de reserva”. 7 La producción académica sobre estos pueblos no ha sido ajena a este tipo de argumentación. Más adelante
hago referencia a la etnología fenomenológica –que conoció su apogeo durante los años de la última dictadura
militar en Argentina– y su contribución a la producción de contenidos de esencialismo y ahistoricidad en torno a
aquéllos. Más acá en el tiempo, idénticos contenidos han visto la luz bajo la forma de otra expresión: la atribución
a la “cultura de cazadores-recolectores” de un rol determinante en sus actuales formas productivas. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 6 Las cualidades del indígena como cosechero son compartidas por quien se desempeñaba en 1999 como subse-
cretario de Empleo de la provincia: “El cosechero aborigen produce un algodón de una muy buena calidad, un
algodón limpio. Pero su rendimiento es bajo. Cosecha entre veinticinco y treinta kilos por día contra setenta kilos
del criollo. […] hacen el cálculo de cuánto tienen que cosechar para conseguir la comida para subsistir durante
el tiempo que dura la cosecha. No tienen el concepto de acumulación ni el de reserva”. ¿“Marisca” versus “trabajo”? Por cierto, “la
cultura de los indígenas del Gran Chaco explicaría la importancia entre ellos del trabajo asalariado, la dependencia
a programas asistenciales y de desarrollo, la mendicidad, la búsqueda de residuos en las ciudades y la relativa-
mente débil gravitación de la producción agrícola mercantil” (Gordillo, 2006: 279). Distintos antropólogos han
formulado este tipo de argumentación. Von Bremen (1987 y 2000) sostiene que el ajuste de los ayoreo del Chaco
paraguayo a las condiciones modernas de vida encierra en sí una continuidad, en cuanto sociedad cazadora-
recolectora que no producía mediante una acción intencional y consciente los bienes necesarios para satisfacer
sus necesidades sino que tomaba los recursos ya existentes en la naturaleza, en su adaptación a las condiciones
actuales de vida el interés sigue siendo el mismo: la adquisición de objetos ya existentes y dados para su propio
consumo, y la caza y recolección (y la conducta y la conciencia en ellas implicadas), como medio para alcanzarla,
asumen nuevas bases, tales como el trabajo asalariado y los proyectos de desarrollo. Por su parte, Braunstein y
Miller (1999) y Alvarsson (1999) señalan, de manera respectiva, que la gente del Gran Chaco (Argentina, Bolivia
y Paraguay), en general, y los ‘Weenhayek bolivianos, en particular, han transferido sus capacidades recolectoras
a la ciudad dándole forma a la práctica de la mendicidad. T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o Lo que es más importante aún, la apropiación más o menos directa de
recursos ya dados –antes, de la naturaleza, ahora, mayormente de la asistencia
del Estado– encarnada en la “marisca” se asociaría a una forma “no productiva”
de transformación de la naturaleza. Por cierto, “no hacen producción” es otra
de las expresiones habituales. Quien en 2004 era funcionaria de la Secretaría
de Planeamiento y Desarrollo provincial se explayaba así: “Para ellos, la tierra
no es para trabajarla, sino para habitarla. En un sentido, es similar a una posi-
ción ecologista. El primer gobernador era proindígena y les dio las tierras más
lindas8. Se han hecho programas, se les han entregado cosas, herramientas,
pero han hecho desastres, todo han vendido”. 8 Floro Bogado ejerció la gobernación entre 1983 y 1987. En 1984, con la sanción de la Ley Integral del Aborigen N°
426, se inició la regularización de la situación de dominio de las tierras que ocupan las comunidades indígenas.
Según datos del Instituto Provincial de Colonización y Tierras Fiscales (2012), la superficie rural en manos de esas
comunidades (ya fuera bajo la forma predominante de títulos comunitarios y también de títulos individuales)
ascendía en 2012 a 307.424 hectáreas. No obstante, por cuanto la transferencia de derechos se ha limitado a áreas
reducidas, la movilización indígena es aún en defensa del territorio y en torno al manejo de los recursos naturales
presentes en el ámbito del frente expansivo regional. p
p
g
9 Se trató de un programa de empleo para la emergencia ocupacional ejecutado durante los noventa. Sus
beneficiarios (desocupados que no contaran con ningún tipo de prestación social) recibían una ayuda eco-
nómica no remunerativa, debiendo realizar a cambio tareas en proyectos desarrollados por organismos de la
administración pública u organizaciones no gubernamentales sin fines de lucro. Del conjunto de programas
implementados en ese decenio, en la mayoría de los casos bajo la órbita del Ministerio de Trabajo, Empleo y
Seguridad Social de la Nación, se destacó el Programa Trabajar, tanto por su duración efectiva (desde fines de
1995 hasta 2001) como por el número de beneficiarios que tuvo (Golbert, 2004). Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 ¿“Marisca” versus “trabajo”? En una palabra, la funcionaria
transmitía la actitud pasiva que en apariencia tendrían los indígenas hacia la
naturaleza. Y aun cuando se reconoce que trabajan, el carácter productivo de
su trabajo resulta constantemente tensionado: Ellos son cazadores-recolectores. Históricamente, y todavía mantienen. Enton-
ces yo creo que nosotros nos hemos equivocado en querer implantar un modelo
productivo que son modelos nuestros: ladrillería, carpintería, agricultura. [...]
Entonces, hemos transferido lo que nosotros conocemos, lo que creemos
válido, y ahí es el choque porque [...] el sistema que funciona no es el de ellos. [...] Pero también entra el concepto cultural, ¿no? Es decir, un poco aquello de
que ¿qué hacía el paisano? El paisano recolectaba, iba a pescar [...] Ahí es donde
lo económico no funciona. Tienen la tierra, por ejemplo, todas las comunidades
tienen la propiedad de la tierra. Y, bueno, creo que es un paso importante. Pero
vos no ves tierras productivas, que se encare algo desde la tierra. [...] No es que
el aborigen es vago porque si no se deforma, se dice “el aborigen no trabaja”. Yo
creo que salir a cazar, salir a las ocho de la mañana a cazar y volver a las seis,
siete de la tarde, que tenés que caminar en el monte con el calor y todo, es un
trabajo. Si hacen ése pueden hacer otro, entonces no es que la gente no tra-
baja, cuesta insertarse. [...] El problema del Plan Trabajar9, otro inconveniente. Nosotros hemos planteado que el Plan Trabajar no responde a las necesidades p
p
g
9 Se trató de un programa de empleo para la emergencia ocupacional ejecutado durante los noventa. Sus
beneficiarios (desocupados que no contaran con ningún tipo de prestación social) recibían una ayuda eco-
nómica no remunerativa, debiendo realizar a cambio tareas en proyectos desarrollados por organismos de la
administración pública u organizaciones no gubernamentales sin fines de lucro. Del conjunto de programas
implementados en ese decenio, en la mayoría de los casos bajo la órbita del Ministerio de Trabajo, Empleo y
Seguridad Social de la Nación, se destacó el Programa Trabajar, tanto por su duración efectiva (desde fines de
1995 hasta 2001) como por el número de beneficiarios que tuvo (Golbert, 2004). 10 Se sabe que la representación del Chaco como un territorio prácticamente inexpugnable, tanto por la naturaleza
de su suelo como por aquella atribuida a sus pobladores originarios, fue corriente en los inicios del curso histórico
concreto que siguió el proceso de acumulación de capital en la región. Aún a fines del siglo XIX, ésta emergía
como un espacio que se resistía a ser sometido al dominio del capital y de su proyecto geopolítico. Pero también
se erigía, a partir de las riquezas de su ambiente y del carácter de límite fronterizo con otros Estados-nación
sudamericanos, en un espacio de potencial económico e importancia política, destinado a estar sujeto, desde y
con los inicios de su colonización, a la renovada expansión de relaciones capitalistas de producción. Se sabe, ade-
más, que el desarrollo de las distintas agroindustrias en la región (obrajes madereros, explotaciones ganaderas,
ingenios azucareros, plantaciones algodoneras) tuvo por condición de posibilidad no sólo la apropiación privada
de la tierra, sino también la disponibilidad de mano de obra indígena. Por cierto, próximo a finalizar el siglo XIX,
el indígena, en su carácter de fuerza de trabajo aclimatada y barata, era visto como el único brazo apropiado
para el Chaco (Beck, 1994; Lagos, 2000). La necesidad de su sometimiento pero también de la reproducción de
su existencia fue lo suficientemente clara para quienes expresaron política, militar, científica y espiritualmente el
interés del capital agroindustrial (Iñigo Carrera, N., 1984). ¿“Marisca” versus “trabajo”? A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 de las comunidades. Por lo general, el Plan Trabajar se le da a la municipalidad
de un lugar, entonces, ¿qué pasa? Vos te imaginás, nosotros tenemos comuni-
dades que en vez de poder, queremos alambrar, tenemos el alambre y queremos
que el Plan Trabajar pueda cubrir la mano de obra, y eso no está contemplado. Entonces vos tenés que al aborigen lo llevan a arreglar la plaza, hacer trabajos
en el pueblo, porque eso está contemplado. Lo que nosotros planteamos es que
para las comunidades no haya un Plan Trabajar especial pero se tenga en cuenta
que es una problemática distinta, que tiene que responder a las necesidades
de la comunidad. Bueno, ésa es una discusión. Cuando se habla de programas
Trabajar productivos, ¿cuál es la concepción de lo productivo? […] Nosotros
presentamos programas de alambrado de tierras y no se lo considera produc-
tivo en el Plan Trabajar. Ahora nosotros creemos que alambrando las tierras
es productivo, por cuanto la conservación del monte, mejoran las tierras, y es
el aspecto de una economía propia, la gente indígena vive de la caza, la pesca,
la recolección de frutos, recolección para hacer su artesanía. Entonces eso es
productivo. En el sistema capitalista eso no se considera. Más adelante avanzo sobre la productividad del trabajo, que, en la concep-
ción de quien se desempeñó por cerca de diez años como presidente del ICA,
se presenta como esencialmente indígena. Por el momento, no resulta baladí
señalar que, con sus palabras, reconoce en la caza, pesca y recolección prácti-
cas productivas ajenas a la organización capitalista de la producción social. Es
cierto que la “marisca” no constituye la expresión más evidente de la falsedad
de tal ajenidad. Pero es largamente sabido que, ya sea empuñando el hacha en
el obraje maderero, el machete en la zafra en los ingenios azucareros o la azada
en los algodonales, los indígenas chaqueños se constituyeron tempranamente
en brazos baratos para la expansión del capitalismo regional10. La tensión entre “marisca” (recordemos, caza, pesca y recolección) y “tra-
bajo” (agricultura, cría de animales, artesanía, trabajo asalariado estacional, empleo
en el sector público), presente de manera recurrente en las enunciaciones incluidas Antipod. Rev. Antropol. Arqueol. No. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-25 ¿“Marisca” versus “trabajo”? 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o en este trabajo, es un tópico repetido en la literatura antropológica referida a los
indígenas chaqueños (Citro, 2009; Gordillo, 2004; Vivaldi, 2010; Wright, 2008). Lo
es también, según Gordillo (2004), en la experiencia histórica de los qom. El autor
asume que se trata de una tensión entre diferentes relaciones sociales de produc-
ción11. Comparto su preocupación por analizarla, antes que de una manera bipolar
(es decir, como expresión de una “economía dual”), como una práctica social sin-
gular en cuyo seno se desenvuelven múltiples contradicciones (Gordillo, 2004: 5;
mi traducción). En su expresión metodológica, tal preocupación lo conduce a cen-
trar el análisis en los significados culturales producidos en torno a ambos términos
(“marisca” y “trabajo”). Es cierto que, al hacerlo, la “marisca” se erige en un “símbolo
crucial de aboriginalidad” (Gordillo, 2004: 24; mi traducción). Quiero decir: en mi
propia experiencia antropológica se repetía la evocación de los qom a la “marisca”
como “la esencia de la cultura aborigen”. No obstante, también era frecuente su
alusión como una práctica de “los que son pobres”. Cierta vez, un cazador asiduo
en los campos que circundan Namqom explicaba así la práctica de la “marisca”: Entrevistado: Toda la gente aborigen marisca. […] porque por supuesto que
somos pobres. ¿Adónde vamos a sacar la platita para comprar puchero? Si
entra, bueno, puede ser que vas a comer. Pero a través de este momento, en
esta época, andamos sin trabajo, si tenés un arma por ahí, podés mariscar
charata, tatú, carpincho, guazuncho, es carne todo. [...] Pero hay veces dicen
la gente: “¿por qué cazan ustedes?”, como esta mañana fuimos en la Fauna
[Dirección de Fauna y Parques de la provincia de Formosa]. Y, bueno, nosotros
estamos mariscando acá por nuestra, por nuestra raza, qué va a ser de nuestro
viejo, salió de, y bueno salimos de pobres, algunos, y bueno, no dejamos, nunca
vamos a dejar nuestra cultura. Parte de la cultura. Y hay que mantener. Le
dijimos esta mañana, “¿qué trabajo […]?”. 11 Es éste el contenido que asumía la mencionada tensión en el contexto de la acción de las misiones francis-
canas instaladas en Formosa a principios del siglo XX. Por cierto, las formas de trabajo comprendidas en el
ámbito sedentario de la misión entraban en clara tensión con las formas en que históricamente organizaban
la producción de su vida social quienes se querían convertir en cristianos, asalariados y colonos. Era ésta
una tensión que se les aparecía a los misioneros como indolencia y falta de apego al trabajo, y que los
indígenas resolvían en las retiradas estacionales al monte circundante para cazar, pescar y recolectar. La
movilidad implicada en la “marisca”, a la vez que forma de resistencia cotidiana al trabajo agrícola, actuali-
zaba una excesiva proximidad con la naturaleza que debía quedar en el pasado. Wright (2008) dice que en
los intentos franciscanos por lograr la conversión de los qom –en productores mercantiles y trabajadores
asalariados–, trabajo, misión y colono se constituyeron en la contraparte de “marisca”, desierto e indio. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 ¿“Marisca” versus “trabajo”? Gordillo
sostiene que “esta aboriginalidad [la simbolizada por la “marisca”] es tam-
bién un marcador de clase, y mucha gente considera la marisca como una
práctica que separa a los tobas [qom] pobres de los acomodados” (2004: 24;
mi traducción). Pero en más de una oportunidad he escuchado a los qom
afirmar que “el criollo también caza”. A este respecto, el qarashe (es decir, el
líder junto a su pueblo) de Potae Napocna Navogoh decía:
Es común. Pero la diferencia es que el criollo a veces abusa de esto, porque
como ellos tienen esa costumbre de buscar cosas es para vender. Los indíge-
nas se van en busca de eso es para consumir. Entonces ahí es donde está la
diferencia. Entonces cuando el criollo se va al campo cualquier animal que
encuentra lo mata, es para poder vender en la ciudad, pescar la misma cosa,
piel de animal silvestre la misma cosa. Todo es economía el tema de la marisca
para el criollo. El indígena la única cosa que hace es buscar el ñandú, traer la
carne y las plumas vender. Pero no así depredar. A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 La “marisca” se evidencia –para los qom– como una práctica inmuta-
ble y eterna de los indígenas. Imposibilitados de abandonarla, los diferen-
cia. Pero no sólo lo hace, en cuanto indígenas, del resto, sino que también
diferencia a “los que son pobres” de quienes no lo son. Cuando no se tiene
“trabajo”, la “marisca” es una práctica a la cual se puede recurrir. Gordillo
sostiene que “esta aboriginalidad [la simbolizada por la “marisca”] es tam-
bién un marcador de clase, y mucha gente considera la marisca como una
práctica que separa a los tobas [qom] pobres de los acomodados” (2004: 24;
mi traducción). Pero en más de una oportunidad he escuchado a los qom
afirmar que “el criollo también caza”. A este respecto, el qarashe (es decir, el
líder junto a su pueblo) de Potae Napocna Navogoh decía: Es común. Pero la diferencia es que el criollo a veces abusa de esto, porque
como ellos tienen esa costumbre de buscar cosas es para vender. Los indíge-
nas se van en busca de eso es para consumir. Entonces ahí es donde está la
diferencia. 12 Sapkus (2002), en su clasificación en estratos de los criollos integrantes del Mocafor, atribuye a los “campesinos
semiproletarizados” o “productores de infrasubsistencia” –la gran mayoría, por otra parte, de quienes conforman
la base del movimiento–, la recurrencia regular a la caza de animales silvestres y la recolección de miel para el
autoconsumo y la venta como parte de las tareas extraprediales realizadas para la supervivencia. ¿“Marisca” versus “trabajo”? Y nada, no trabajo, no tengo trabajo,
como el otro mi hermano también no tiene. Valeria: ¿Lo hacen porque no tienen trabajo o porque es parte de su cultura? E: Parte de la cultura y parte es que no hay trabajo. Porque estando en la casa
qué es lo que podemos mantener para nuestros hijos. Tenés suerte traés miel
de abeja, todo, se consigue. [...] Siendo aborigen vamos a seguir siendo, porque
vos mismo no podés cambiar. Teniendo plata podés cambiar. 11 Es éste el contenido que asumía la mencionada tensión en el contexto de la acción de las misiones francis-
canas instaladas en Formosa a principios del siglo XX. Por cierto, las formas de trabajo comprendidas en el
ámbito sedentario de la misión entraban en clara tensión con las formas en que históricamente organizaban
la producción de su vida social quienes se querían convertir en cristianos, asalariados y colonos. Era ésta
una tensión que se les aparecía a los misioneros como indolencia y falta de apego al trabajo, y que los
indígenas resolvían en las retiradas estacionales al monte circundante para cazar, pescar y recolectar. La
movilidad implicada en la “marisca”, a la vez que forma de resistencia cotidiana al trabajo agrícola, actuali-
zaba una excesiva proximidad con la naturaleza que debía quedar en el pasado. Wright (2008) dice que en
los intentos franciscanos por lograr la conversión de los qom –en productores mercantiles y trabajadores
asalariados–, trabajo, misión y colono se constituyeron en la contraparte de “marisca”, desierto e indio. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 2 3 8
A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3
La “marisca” se evidencia –para los qom– como una práctica inmuta-
ble y eterna de los indígenas. Imposibilitados de abandonarla, los diferen-
cia. Pero no sólo lo hace, en cuanto indígenas, del resto, sino que también
diferencia a “los que son pobres” de quienes no lo son. Cuando no se tiene
“trabajo”, la “marisca” es una práctica a la cual se puede recurrir. ¿“Marisca” versus “trabajo”? Entonces cuando el criollo se va al campo cualquier animal que
encuentra lo mata, es para poder vender en la ciudad, pescar la misma cosa,
piel de animal silvestre la misma cosa. Todo es economía el tema de la marisca
para el criollo. El indígena la única cosa que hace es buscar el ñandú, traer la
carne y las plumas vender. Pero no así depredar. La práctica de la “marisca”, común a indígenas y criollos, encuentra en las
anteriores palabras una distinción fundada en un sentido armónico de la trans-
formación del medio físico atribuido a los primeros y en otro sentido contra-
puesto al anterior ligado al saqueo de los recursos de la naturaleza imputado a
los segundos. Esto no me impide, sin embargo, sugerir que, en cuanto marcador
de clase, la “marisca” establece la separación sin distinciones étnicas –aunque,
quiero decir, con un peso histórico e identitario ciertamente distinto– entre
quienes logran obtener de manera regular sus medios de vida y quienes no12. Por otra parte, si continúo con la propuesta de centrar la mirada en
los sentidos construidos en torno a la “marisca” y el “trabajo”, uno y otra
constituyen prácticas, en principio, mutuamente excluyentes. La referen-
cia más clara en este sentido remite al tiempo de “los antiguos” (es decir,
los antepasados de los actuales qom). En el recuerdo de un qom de Misión
Tacaaglé, de unos 35 años: Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o E: Porque ese tiempo, yo no lo viví, pero no conseguían trabajo. O digamos no
conocían trabajo. V: ¿Cómo no conocían trabajo? E: Y, claro, porque ellos vivían en el monte. Y ellos lo que conocían más es
mariscando. Los antiguos no trabajan, mariscan en el monte. [...] Pero ahora
con éste que estamos, nosotros estamos modernizados, conocemos trabajo. Conocemos lo que es como construcción, así, conocemos todo eso. Estamos
modernizados, ya no existe casi la marisca ahora. ¿“Marisca” versus “trabajo”? En este contrapunto entre “marisca” y “trabajo”, el tiempo marca una
divisoria de aguas: mientras que el de “los antiguos” fue uno en el que sólo se
cazaba, pescaba y recolectaba en el monte, la modernidad ha traído consigo
el trabajo asalariado (aquí, en un rubro característico del medio urbano: la
construcción). Por su parte, desde su casa en Namqom, un frecuente maris-
cador y trabajador estacional en la producción de algodón rememoraba su
experiencia más inmediata: Y mi papá trabajaba en la zafra algodonera y a la vez se iba a mariscar. Entra-
mos en la carpida algodonera y después de ahí empieza la cosecha algodonera,
que es ahora, justamente esta es la época [mes de marzo]. Si terminaba eso ya
nosotros no teníamos trabajo, empezamos a vivir en la caza solamente. O sea,
a partir de terminar la caza, a partir de mayo, finaliza todo ese mes de mayo,
junio, julio, agosto, septiembre hasta que llega el trabajo, todos esos meses
nosotros vivíamos de la caza. En cuanto a su carácter estacional, “marisca” y “trabajo” se suceden tem-
poralmente: al fin de uno, sobreviene la otra. Sin embargo, la tensión entre
ambas prácticas deja, por momentos, de ser tal. La “marisca” aparece en la con-
ciencia como un “trabajo”; por cierto, uno que tiene al sacrificio como forma
necesaria. Una artesana de Namqom me decía al respecto: Es algo para vivir también, porque se van y consiguen algo de..., en vez
de traer esos dos, tres pesos a la casa, traen carne del monte; porque uno
trabaja en el centro, le dan plata, ¿no es cierto? En cambio, uno se va en el
monte, trae carne. Pero no es que sólo la carne traen, porque todo lo que
es del animal ñandú servía. Sirve. Porque hay gente todavía que se van al
monte y traen las plumas, la carne, todo. Entonces con esa platita que jun-
tás de las plumas, del cuero, con eso se compra algo de ropa para los chicos. Y así, es una especie de trabajo, y así, todas esas cositas del monte sirven. La marisca “es una especie de trabajo”, por cuanto constituye una pro-
ducción para el consumo directo de lo obtenido: la carne de los animales del
monte alimenta. Pero lo es también, y en mayor medida, según el énfasis de Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. 13 Cabe aclarar que la caza comercial presenta limitaciones vinculadas al carácter de propiedad privada de los
campos donde se realiza. Se trata de limitaciones que, en el mejor de los casos, se sortean a través de permisos
concedidos de manera informal por los propietarios de las explotaciones agropecuarias, que da como resultado,
en ocasiones, la confiscación del arma de fuego portada, por la falta de su registro ante la Dirección de Fauna y
Parques de la provincia e, incluso, situaciones que pueden adoptar un carácter altamente conflictivo. ¿“Marisca” versus “trabajo”? De ahí en más, es el movimiento
del capital total de la sociedad el que determina qué es un trabajador productivo,
atribuyendo ese carácter sólo al que trabaja para adicionar más valor al capital, o
sea, para la autovalorización del capital (Iñigo Carrera, J., 2004). Por cierto, las actividades de caza, pesca y recolección, realizadas bajo los propios qom, por cuanto constituye una producción de mercancías: las
plumas y el cuero del ñandú se venden13. Resulta oportuno, a esta altura del análisis de las formas de la conciencia,
detenerme en la materialidad que expresan: quiero decir, en el carácter productivo
o improductivo del trabajo. Observan Marx y Engels (1973) que el hombre se dife-
rencia del resto de los animales por su capacidad para transformar al medio en un
medio para sí mediante el trabajo, es decir, por la capacidad de actuar consciente y
voluntariamente sobre la naturaleza, a fin de producir los medios de vida para su
reproducción social. Es decir, que el trabajo se revela como condición de la vida
humana: hay una producción intencional del medio, a través del uso de instrumen-
tos de trabajo que, junto con la materia prima objeto del trabajo, constituyen los
medios que es necesario consumir en el proceso de trabajo, con el fin de producir
los valores de uso necesarios para satisfacer las necesidades humanas. Pero esto es
así desde un punto de vista abstracto general (es decir, de manera independiente de
las modalidades que asume en las distintas formas sociales). En la producción capi-
talista ya no alcanza con que el obrero produzca en términos generales: “Dentro del
capitalismo, sólo es productivo el obrero que produce plusvalía para el capitalista
o que trabaja para hacer rentable el capital” (Marx, 2001: 425-426; subrayado en
el original). Desde el momento en que el objeto deja de ser simplemente la pro-
ducción de valores de uso para pasar a ser la producción de plusvalía, el trabajo
del individuo que aplicaba su fuerza de trabajo de manera directa en el proceso
de producción va a perder contenido en sí mismo para pasar a constituirse en una
forma subsumida en la reproducción del capital. ¿“Marisca” versus “trabajo”? ISSN 1900-5407, pp. 229-251 A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 2 4 0
A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3
los propios qom, por cuanto constituye una producción de mercancías: las
plumas y el cuero del ñandú se venden13. Resulta oportuno, a esta altura del análisis de las formas de la conciencia,
detenerme en la materialidad que expresan: quiero decir, en el carácter productivo
o improductivo del trabajo. Observan Marx y Engels (1973) que el hombre se dife-
rencia del resto de los animales por su capacidad para transformar al medio en un
medio para sí mediante el trabajo, es decir, por la capacidad de actuar consciente y
voluntariamente sobre la naturaleza, a fin de producir los medios de vida para su
reproducción social. Es decir, que el trabajo se revela como condición de la vida
humana: hay una producción intencional del medio, a través del uso de instrumen-
tos de trabajo que, junto con la materia prima objeto del trabajo, constituyen los
medios que es necesario consumir en el proceso de trabajo, con el fin de producir
los valores de uso necesarios para satisfacer las necesidades humanas. Pero esto es
así desde un punto de vista abstracto general (es decir, de manera independiente de
las modalidades que asume en las distintas formas sociales). En la producción capi-
talista ya no alcanza con que el obrero produzca en términos generales: “Dentro del
capitalismo, sólo es productivo el obrero que produce plusvalía para el capitalista
o que trabaja para hacer rentable el capital” (Marx, 2001: 425-426; subrayado en
el original). Desde el momento en que el objeto deja de ser simplemente la pro-
ducción de valores de uso para pasar a ser la producción de plusvalía, el trabajo
del individuo que aplicaba su fuerza de trabajo de manera directa en el proceso
de producción va a perder contenido en sí mismo para pasar a constituirse en una
forma subsumida en la reproducción del capital. 14 Lejos de haber sido expresada únicamente por quienes personificaron el avance del capital sobre la región,
la negación de la condición de trabajador ha sido encarnada, asimismo, y de manera mucho más reciente,
por quienes monopolizaron en los setenta la producción de conocimiento antropológico sobre los indíge-
nas del Chaco: la etnografía fenomenológica. Su representante más conspicuo sostenía que es en el mito
donde reside la esencia de la cultura del hombre etnográfico y es, por tanto, en él donde debe referenciarse
la explicación de su comportamiento (Bórmida, 1969 y 1976). A partir de esta argumentación, primero, se
atribuyen el desarrollo de las técnicas implicadas en las actividades económicas de producción, así como
la creación de los instrumentos utilizados en esas tareas, y aun la división sexual del trabajo y las prácticas
de consumo involucradas, a la acción de personajes tesmofóricos y no a la desplegada por los hombres; y
segundo, aquellas actividades, a la vez que restringidas a la caza, pesca y recolección, no son concebidas
como trabajo, el cual, por su parte, es identificado con la agricultura y el trabajo asalariado y presentado
como ajeno a la naturaleza del hombre etnográfico (Mashnshnek, 1975). Sobre la base de la atribución de
estos contenidos a la conciencia indígena –recordemos, presentada como mítica, irracional y ajena a los
principios de la razón occidental–, lo que caracterizaría a los indígenas, antes que el estigma de impro-
ductivo, es la posesión de una cualidad distintiva: su incapacidad radica en que el trabajo es ajeno a su
naturaleza –una inalterable con la historia–. La etnología fenomenológica evidenció su cinismo al consagrar
la naturalidad de la pobreza en que esos pueblos producían su vida. Como si la pobreza estuviera portada
en su misma condición de indígena (Bórmida, 1969). Por cierto, los propios pueblos indígenas terminaron
imputados con la responsabilidad por la persistencia en condiciones de vida miserables. Imputación que, a
la vez, ponía en duda la necesidad, por ejemplo, de intervenir en pos de la mejora de esas condiciones de
vida (Bórmida, 1976). Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 De pérdidas, ausencias y dependencias De pérdidas, ausencias y dependencias
Productivas por donde se las mire –las formas de trabajo implicadas en la
caza, pesca y recolección–, no obstante, la condición de trabajador produc-
tivo de los indígenas ha sido paradójica y sistemáticamente obliterada, en
pos del diseño del estigma de improductivo como justificador del proceso
de creación de las condiciones para la valorización capitalista de su trabajo
(Trinchero y Leguizamón, 1995)14. Es de notar que, aún a mediados de la 14 Lejos de haber sido expresada únicamente por quienes personificaron el avance del capital sobre la región,
la negación de la condición de trabajador ha sido encarnada, asimismo, y de manera mucho más reciente,
por quienes monopolizaron en los setenta la producción de conocimiento antropológico sobre los indíge-
nas del Chaco: la etnografía fenomenológica. Su representante más conspicuo sostenía que es en el mito
donde reside la esencia de la cultura del hombre etnográfico y es, por tanto, en él donde debe referenciarse
la explicación de su comportamiento (Bórmida, 1969 y 1976). A partir de esta argumentación, primero, se
atribuyen el desarrollo de las técnicas implicadas en las actividades económicas de producción, así como
la creación de los instrumentos utilizados en esas tareas, y aun la división sexual del trabajo y las prácticas
de consumo involucradas, a la acción de personajes tesmofóricos y no a la desplegada por los hombres; y
segundo, aquellas actividades, a la vez que restringidas a la caza, pesca y recolección, no son concebidas
como trabajo, el cual, por su parte, es identificado con la agricultura y el trabajo asalariado y presentado
como ajeno a la naturaleza del hombre etnográfico (Mashnshnek, 1975). Sobre la base de la atribución de
estos contenidos a la conciencia indígena –recordemos, presentada como mítica, irracional y ajena a los
principios de la razón occidental–, lo que caracterizaría a los indígenas, antes que el estigma de impro-
ductivo, es la posesión de una cualidad distintiva: su incapacidad radica en que el trabajo es ajeno a su
naturaleza –una inalterable con la historia–. La etnología fenomenológica evidenció su cinismo al consagrar
la naturalidad de la pobreza en que esos pueblos producían su vida. Como si la pobreza estuviera portada
en su misma condición de indígena (Bórmida, 1969). Por cierto, los propios pueblos indígenas terminaron
imputados con la responsabilidad por la persistencia en condiciones de vida miserables. T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o mente improductivas, aquellas formas de trabajo resultaban productivas desde
el punto de vista material de la producción social, por cuanto producían los
valores de uso necesarios. Aún hoy, lo son en idéntico sentido; a pesar, claro
está, de la ya mencionada restricción en la posibilidad de su realización por
la creciente discontinuidad de la cobertura boscosa ante el avance de las tie-
rras agropecuarias y por el progresivo acorralamiento territorial producto de la
apropiación privada individual de los campos. Ahora bien, estas mismas formas
de trabajo resultan productivas también desde el punto de vista de la organiza-
ción de la producción social a través del mercado, por cuanto producen valor,
y, aun, desde el punto de vista de la organización capitalista de la producción
social, cuando de lo que se trata es de la producción de plusvalía. Por cierto, la
mercantilización de los recursos obtenidos de la caza conlleva una apropiación
de plustrabajo, por cuanto el productor directo vende la mercancía por debajo
del valor al que luego se realiza normalmente en el mercado. ¿“Marisca” versus “trabajo”? De ahí en más, es el movimiento
del capital total de la sociedad el que determina qué es un trabajador productivo,
atribuyendo ese carácter sólo al que trabaja para adicionar más valor al capital, o
sea, para la autovalorización del capital (Iñigo Carrera, J., 2004). Por cierto, las actividades de caza, pesca y recolección, realizadas bajo
la organización de la producción social fundada en las relaciones directas de
dependencia personal, implicaban formas de trabajo relativamente simples. Esto, en el sentido de que se trataba de tareas para cuya ejecución bastaba con
el desarrollo natural de la fuerza de trabajo, es decir, que el individuo hubiera
desarrollado tanto la aptitud física como la conciencia y voluntad requeri-
das para trabajar, y con el desarrollo de cierta pericia adquirida en el ejercicio
mismo del trabajo (Iñigo Carrera, J., 2004). Sin embargo, lejos de ser abstracta- Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 De pérdidas, ausencias y dependencias Imputación que, a
la vez, ponía en duda la necesidad, por ejemplo, de intervenir en pos de la mejora de esas condiciones de
vida (Bórmida, 1976). Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 década de 2000, y como beneficiarios de programas sociales (nacionales
y provinciales) en materia de empleo y alimentación, los indígenas eran
presentados como portadores de la necesidad de la asistencia. Por cierto,
la carencia de bienes y servicios básicos, fundamentalmente alimentarios,
y de trabajo, fue institucionalizada como carencia particular o del sujeto
individual, obliterando así la relación social que da lugar a la asistencia
(Grassi, 2003). Y, en su modalidad, las acciones asistenciales y compen-
satorias primaron sobre las de promoción de actividades destinadas a la
producción de mercancías que entraran en el consumo social general, es
decir, sobre el impulso al desarrollo de los atributos productivos de los qom
(Iñigo Carrera, V., 2008). Quiero decir: también las modalidades de la asis-
tencia a la pobreza y al desempleo (que comprendían ayudas económicas
no remunerativas, asistencia alimentaria directa, pensiones asistenciales no
contributivas) construían como su objeto a un sujeto a quien negaban su
condición de trabajador. ¿Bajo qué forma se les presenta esto a quienes se
encuentran en la gestión de esas modalidades de la asistencia? Como la sim-
ple ausencia o, en el mejor de los casos, pérdida de una “cultura productiva”
que alguna vez el trabajador indígena supo conseguir a través del aprendi-
zaje de la agricultura. Por cierto, esta forma específica de la actividad productiva ha sido eri-
gida, de manera histórica, en la quintaesencia del trabajo verdaderamente
productivo. La radicación del indígena en la tierra y su introducción, edu-
cación y entrenamiento en las labores agrícolas, como formas de su con-
versión en trabajadores productivos para el capital, han sido parte de un
proyecto civilizador cuyos coletazos es posible advertir en el presente. 15 Briones y Delrío (2002) señalan que a las primeras dos formas de radicación (misiones y reducciones) subyacía
la construcción de los indígenas chaqueños según parámetros de máxima alteridad, y la concepción de la nece-
sidad de su civilización como momento previo a su argentinización; y, a la radicación en colonias agrícolas, la
conclusión de esa argentinización. En el caso de las comunidades referidas en este trabajo, Tacaaglé fue, en sus
orígenes, una misión católica de la orden de los franciscanos, fundada en 1901, en el marco del Art. 100 de la Ley
N° 817 de Inmigración y Colonización de 1876, que disponía la creación de misiones con el objeto de procurar la
atracción gradual de las tribus indígenas a la vida civilizada, estableciéndolas por familias en lotes de cien hectá-
reas (Consejo Agrario Nacional, 1945). Por su parte, Potae Napocna Navogoh (anteriormente, La Primavera) fue,
primero, una misión de la Iglesia protestante británica Emmanuel, que, establecida en 1937, procuró su civiliza-
ción a partir de la prédica evangélica, la alfabetización y la transformación en trabajadores agrícolas, y luego, en
1952, una colonia bajo la jurisdicción de la Dirección de Protección al Aborigen (creada en 1946, en el ámbito de
la Secretaría de Trabajo y Previsión, en reemplazo de la Comisión Honoraria de Reducciones de Indios, y disuelta
en 1955 con el golpe militar a Juan D. Perón), encargada de la adquisición de implementos agrícolas y ganado
con destino a las colonias existentes. De pérdidas, ausencias y dependencias Es
sabido el papel encarnado por el Estado (en sus distintos niveles, de forma
directa o mediada por el accionar de otros actores, de manera solidaria pero
también en tensión) en el desarrollo de un trabajador con determinados
atributos productivos; esto, desde el momento mismo de expropiación –de
manera violenta– de la población indígena de sus condiciones materiales de
existencia originarias. En la colonización de la subjetividad de los indígenas –tanto en sus atribu-
tos materiales como mentales– desempeñaron un papel insoslayable las misiones
religiosas y las reducciones estatales establecidas en las primeras décadas del siglo
XX, en cuanto “formas legales de espacialización y organización de colectivos indí-
genas” (Briones y Delrio, 2002: 45). Aquélla encontró una continuidad –sobre otras
bases– en la creación de colonias agrícolas hacia la década de 1940, cuando la ges-
tión estatal de los asuntos indígenas –encarnada por el Consejo Agrario Nacional–
pasó a consistir, antes que en su colocación, en su integración (Briones y Carrasco, Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o Es de notar que, en la actualidad, la dedicación de los qom de Misión
Tacaaglé y Potae Napocna Navogoh a la producción de algodón sólo es posible por
la asistencia brindada por el ICA y el Ministerio de la Producción y Ambiente de la
provincia, consistente en la preparación del suelo, la provisión de las semillas nece-
sarias, el suministro de los insumos utilizados (herbicidas, insecticidas, regulador
de crecimiento) y la entrega de mercaderías (harina, azúcar, yerba, sal, grasa) para
el sostenimiento inmediato de las familias durante el período de ocupación en las
tareas agrícolas16. N° 1363). Es de notar que, en la actualidad, la dedicación de los qom de Misión
Tacaaglé y Potae Napocna Navogoh a la producción de algodón sólo es posible por
la asistencia brindada por el ICA y el Ministerio de la Producción y Ambiente de la
provincia, consistente en la preparación del suelo, la provisión de las semillas nece-
sarias, el suministro de los insumos utilizados (herbicidas, insecticidas, regulador
de crecimiento) y la entrega de mercaderías (harina, azúcar, yerba, sal, grasa) para
el sostenimiento inmediato de las familias durante el período de ocupación en las
tareas agrícolas16. En síntesis: los indígenas fueron orientados hacia la labor agrícola, disci-
plinados en la organización de los procesos de trabajo, adiestrados en la comer-
cialización de su producción, administrados en su subsistencia e instruidos
escolar, moral y religiosamente. Todo lo cual, sostiene Arengo (1996), erosionó,
cuando no socavó por completo, su autonomía para garantizar la misma sub-
sistencia. Una de las manifestaciones señaladas de manera corriente aún hoy
es la necesidad de contar con instancias de coordinación externas a la hora de
poner en marcha sus atributos productivos. Así lo refería quien se desempeñó
como presidente del ICA: E: Nosotros formamos cooperativas de hacheros, […] tuvimos aserraderos. Pero vimos que todos esos programas económicos funcionaban en la medida
en que el mercado funcionaba y que estábamos nosotros administrando eso. ¿Por qué? Porque la gente su trabajo lo hace, pero ellos siempre tienen una
palabra que a nosotros no nos gusta pero que refleja. Es decir, nosotros, ¿qué
pasa?, les decimos “les vamos a dar, vamos a hacer un proyecto de carpinte-
ría, son diez, bueno, les vamos a dar los medios, todo”, pensando que ellos ya
están en condiciones de funcionar. Después fracasa. Entonces atribuimos que
fracasa porque la gente no trabaja. 16 Claro está que detrás de la aparente continuidad en el rol del Estado en lo referido a la promoción de la produc-
ción agrícola a través del despliegue de mecanismos similares a los de antaño asoman mutaciones propias del
curso histórico concreto que siguió el proceso de acumulación de capital en la región. T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o 229-251 2 4 4
A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3
N° 1363). Es de notar que, en la actualidad, la dedicación de los qom de Misión
Tacaaglé y Potae Napocna Navogoh a la producción de algodón sólo es posible por
la asistencia brindada por el ICA y el Ministerio de la Producción y Ambiente de la
provincia, consistente en la preparación del suelo, la provisión de las semillas nece-
sarias, el suministro de los insumos utilizados (herbicidas, insecticidas, regulador
de crecimiento) y la entrega de mercaderías (harina, azúcar, yerba, sal, grasa) para
el sostenimiento inmediato de las familias durante el período de ocupación en las
tareas agrícolas16. En síntesis: los indígenas fueron orientados hacia la labor agrícola, disci-
plinados en la organización de los procesos de trabajo, adiestrados en la comer-
cialización de su producción, administrados en su subsistencia e instruidos
escolar, moral y religiosamente. Todo lo cual, sostiene Arengo (1996), erosionó,
cuando no socavó por completo, su autonomía para garantizar la misma sub-
sistencia. Una de las manifestaciones señaladas de manera corriente aún hoy
es la necesidad de contar con instancias de coordinación externas a la hora de
poner en marcha sus atributos productivos. Así lo refería quien se desempeñó
como presidente del ICA:
E: Nosotros formamos cooperativas de hacheros, […] tuvimos aserraderos. Pero vimos que todos esos programas económicos funcionaban en la medida
en que el mercado funcionaba y que estábamos nosotros administrando eso. ¿Por qué? Porque la gente su trabajo lo hace, pero ellos siempre tienen una
palabra que a nosotros no nos gusta pero que refleja. Es decir, nosotros, ¿qué
pasa?, les decimos “les vamos a dar, vamos a hacer un proyecto de carpinte-
ría, son diez, bueno, les vamos a dar los medios, todo”, pensando que ellos ya
están en condiciones de funcionar. Después fracasa. Entonces atribuimos que A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 N° 1363). Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o 1996)15. El objetivo seguía siendo, aún entonces, su incorporación a la civilización
–a través de la instrucción elemental, agrícola y religiosa–, y las maneras evidencia-
ban la persistencia del paternalismo estatal, a través de la entrega de tierras, semi-
llas, implementos agrícolas y alimentos. En una misma dirección, en los inicios de
la segunda presidencia de Juan D. Perón, se implementó el Segundo Plan Quinque-
nal (1953-1957), que establecía como un objetivo la protección del indígena por
la acción directa del Estado mediante su incorporación progresiva al ritmo y nivel
de vida general de la nación (Dirección de Información Parlamentaria, 1991), y se
sancionó una ley de creación de colonias granjas de adaptación y educación en,
entre otras jurisdicciones, el Territorio Nacional de Formosa. Con posterioridad a
su provincialización en 1955, la colonización con indígenas fue también contem-
plada en el marco de una ley de carácter general y en la órbita de un organismo
específico. La Ley N° 113/60 de Colonización y Tierras Fiscales señalaba que el
ciudadano aborigen podía ser adjudicatario de la tierra fiscal, previa acreditación
de su capacidad para ello, y preveía planes especiales de habilitación, con el objeto
de conseguir el aprovechamiento integral, racional e intensivo de la tierra, invir-
tiéndose su producto en la adquisición de máquinas y herramientas para uso de
los indígenas. Por su parte, la Dirección de Colonias Aborígenes –creada en 1963
bajo la égida del Ministerio de Asuntos Sociales, con el fin de que el Estado provin-
cial tomara a su cargo lo atinente a la población indígena– tenía como funciones:
colonizar con aborígenes las reservas existentes, sobre la base de la subdivisión en
unidades económicas, de acuerdo con la Ley N° 113; incrementar las fuentes de tra-
bajo existentes y propiciar la instalación de nuevas explotaciones agrícolas e indus-
trias; planificar y dirigir la ejecución de los trabajos en chacras y talleres; tomar a su
cargo lo relativo a la comercialización e industrialización de las producciones de las
colonias; otorgar préstamos en herramientas, animales, mercaderías y controlar su
amortización; garantizar a los indígenas la posesión de sus propiedades (Decreto Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o Entonces ellos dicen: “lo que necesitamos
es un capataz”, que es como lo formulan, como que necesitan la presencia del
blanco para que esté encima y dirija. [...] Yo creo que es cuestionable, no es lo
correcto, no es lo que tendría que ser, pero cuando ellos te dicen, ellos tienen
el concepto de que en el trabajo necesitan una conducción, un responsable. V: ¿Ellos mismos? E: Ellos mismos. El capataz es como aquel que estaba en el ingenio, que les
marcaba “hay que hacer esto, esto es lo que hay que hacer, hoy se cosecha
tanto, acá termina la tarea, hoy se limpia tantos surcos”, era el capataz que
hacía el seguimiento. Nosotros hemos encontrado como que entre ellos no se
respeta esa autoridad [la de un indígena sobre los otros], viste que es un poco
como algo familiar. […] es como que el blanco, entre ellos es positivo eso, ¿no? 16 Claro está que detrás de la aparente continuidad en el rol del Estado en lo referido a la promoción de la produc-
ción agrícola a través del despliegue de mecanismos similares a los de antaño asoman mutaciones propias del
curso histórico concreto que siguió el proceso de acumulación de capital en la región. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o A la hora de hacer referencia a la presentación de proyectos por ser eje-
cutados en el marco del Plan Nacional de Desarrollo Local y Economía Social
“Manos a la Obra”17, el coordinador del Mocafor en la localidad de Misión
Tacaaglé me comentaba: “Lo que necesitan [los indígenas] es una buena coordi-
nación. Muchas veces han vendido los animales o herramientas que han tenido. Pero eso es por la falta de un buen trabajo. Si se les baja bien la información,
ellos entienden”. 17 Se trató de un programa de asistencia al desempleo lanzado en 2003 que procuró transcender el subsidio
transitorio al ingreso de los hogares a través de estrategias que promovieran la creación y consolidación de
emprendimientos productivos y comunitarios de pequeña escala con criterio de autosustentabilidad, estra-
tégicos en el marco del desarrollo local. En agosto de 2004 eran 73 los proyectos aprobados en la provincia
de Formosa. Aquellos presentados por comunidades indígenas (entre ellas, Namqom) se restringían a la
confección de artesanías.
18 Ya mencioné los análisis antropológicos que hablan de la “dependencia” respecto de la asistencia brindada
por los organismos estatales, y la explican sobre la base de formular la existencia de una “cultura cazadora-
recolectora” natural y esencialmente propia. T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o En su argumentación acerca de la “dependencia” en que se encuentran
sumidos los indígenas chaqueños, Arengo llega a sostener, incluso, que “Cierta-
mente, contribuyó a crear y mantener una cultura de la dependencia que prác-
ticamente ha definido la vida económica de los aborígenes en la porción este del
Chaco, hasta el presente” (1996: 212; mi traducción). Es indudable la produc-
ción de dependencias en la que, a lo largo de la historia, se han visto implicados
los indígenas, por un lado, y, en el mejor de los casos, un Estado proteccionista
y paternalista, por otro. Sin embargo, suponer, con la autora, que se trata de una
“cultura” que, con origen en el “proceso civilizatorio”, se encontraría en la base
de –aún más, definiría– la naturaleza misma de su lógica actual de producción
–una lógica, dice, relacionada con el crédito y los subsidios– implicaría soslayar
la determinación general de los qom como una población trabajadora cuyos
atributos productivos se han visto progresivamente mutilados. Por cierto, la asociación de la práctica de asistencia del Estado con la gene-
ración de lazos de “dependencia”, más aún, con la pérdida paulatina y continua
de la “cultura productiva”, se revela generalizada en la administración pública
formoseña. Diversos funcionarios se expresan en este sentido. El subsecretario
de Empleo de la provincia aludía, a fines de la década de 1990, a la “pérdida de
la cultura productiva como consecuencia de las políticas implementadas por el
Estado basadas en el asistencialismo”. Por su parte, el ya citado presidente del
ICA refería, como mencioné al inicio de este trabajo, “una gran dependencia de
pedir todo”, ilustrando la adopción de formas pasivas de receptividad18. La “dependencia” –en principio, pero no de manera única– para la
obtención de los medios de vida se hace presente también en las formas de Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 la conciencia indígenas. T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o Es cierto que la “ayuda del Gobierno”, identificada
con las distintas formas de la asistencia estatal pero de manera predomi-
nante con la entrega de mercaderías, es valorada de modo positivo por diri-
gentes políticos indígenas con vínculos con la estructura de poder local, a
partir de su empleo en la administración pública provincial o municipal, su
pertenencia a facciones del Partido Justicialista, históricamente dominante
en Formosa, o a asociaciones comunitarias. Pero no es menos cierto que,
reconocida como de larga data, la “dependencia” es también concebida como
claramente intencionada. Al respecto, me decía un qom de Misión Tacaaglé:
“Hemos sido sometidos de esa forma. Se le busca depender del otro para
poder seguir recibiendo las migajas que el otro le da. Todo el tiempo esta-
mos dependiendo, ‘¿cuándo voy a tener una olla popular, cuándo voy a tener
una caja, cuándo me van a dar pensión?’”. Esta concepción no está aislada;
por el contrario, muchos de sus elementos resultan recurrentes: El tema es que acá a la gente le hicieron, le hicieron acostumbrar en el
comedor. Vino la política ésta, crearon comedores comunitarios, la gente
se van ahí, retiran comida, le dan bolsita. Y entonces la gente ya están acos-
tumbrados, ya están en pleno, o sea, ya le metieron la mentalidad de mos-
trar a la bolsita de pan. O sea, al pueblo aborigen lo enseñaron de mirar a la
olla. Porque viste que acá, allá está el comedor ése, y está el otro comedor
comunitario y acá se está por levantar otro comedor. Es decir, nosotros, yo
a mi hija yo ya le enseño que a ella le mantengan. En vez de enseñarle a mis
hijos que ellos trabajen, que ellos sean algo más que yo. La verdad nosotros
necesitamos, yo necesito para comer al día, pero necesito de vivir de un
trabajo digno y, bueno, lo que gano compro para mi mercadería y con eso
me mantengo. Pero no es lo que me malacostumbren, ya que ellos me man-
tienen. […] O sea, que la gente ya no, ya nos hicieron acostumbrar, le hicie-
ron acostumbrar totalmente. Nunca usted vas a escuchar un aborigen de
que te dice “yo necesito el título definitivo de la propiedad de la tierra, yo
quiero trabajar, necesito el apoyo técnico para trabajar en la tierra, nece-
sito que me entreguen semilla en tiempo y forma”. Nunca. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 19 En su conocido ensayo sobre el estigma, Goffman distingue tres tipos de atributos indeseables devenidos este-
reotipos –tal la definición que brinda de estigma–: “las abominaciones del cuerpo”, “los defectos del carácter del
individuo” y –los que aquí nos interesan– “los estigmas tribales de la raza, la nación y la religión, susceptibles de
ser transmitidos por herencia” (2003: 14). T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o Si vos hablás con
uno, dice: “nosotros queremos caja [alimentaria]”, empiezan a […] porque
eso le hicieron, le hicieron meter en la cabeza de que les regalan cosas. Y
eso es la mentalidad de ellos. No sé, hay muchos hermanos que están en la
conformidad porque viven en la dependencia pero no en la independencia. Lo que le dice el Gobierno, bueno, agradecen. Y muchos hermanos que hoy
se mueren de hambre porque dependen mucho del Gobierno, de la religión,
de los partidos políticos, o sea son gente que dependen, y no de sí mismos
para poder salir a rebuscar estas cosas. Yo creo que eso lo que tiene que
cambiar el pueblo indígena, de no vivir en la dependencia […] Entonces el
pueblo indígena no queremos ser dependientes, sino queremos ser inde-
pendientes y nosotros seamos los protagonistas de lo que vamos a hacer. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o Dos son las cuestiones por subrayar respecto de estas palabras de un qom
de Namqom. Primera: resulta significativa, en cuanto implicancia de la “depen-
dencia”, y ya no sólo causa o manifestación, la pérdida progresiva de atributos
productivos (materiales pero también mentales). Segunda: resulta igualmente
significativa, en contraposición con aquellas construcciones de sentido que
hablaban de la ajenidad del indígena al trabajo, la reivindicación de una trayec-
toria como trabajador. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 Conclusión Dije en la introducción que el propósito del presente trabajo era examinar
el proceso de desplazamiento de la producción del que son objeto los qom
del Chaco centro-oriental, atendiendo a las formas que asume su materiali-
dad en la conciencia de los sujetos. Para ello, desplegué la construcción de
unos sentidos cotidianos (producidos por los qom, agentes del Estado, crio-
llos) que expresan, de manera paradigmática, esas formas: la tensión entre
“marisca” y “trabajo”, la supuesta ausencia –o, en el mejor de los casos, pér-
dida– de una “cultura productiva”, y la “dependencia” en que se encontrarían
sumidos los indígenas chaqueños. Y, al hacerlo, invité a realizar un contra-
punto entre aquéllos –y su correlato en la academia– y las formas en que los
indígenas (re)producen su vida. De resultas, se trata de unas construcciones
de sentido que evidencian una estigmatización de estas últimas19. A lo largo
de la historia, son raras las veces en que el Estado, tanto nacional como pro-
vincial, no construyó representaciones estigmatizadoras sobre los pueblos
indígenas (Vázquez, 2002); agrego: narrativas hegemónicas incorporadas,
internalizadas y disputadas por los mismos sujetos. En el Chaco central, el
proceso de estigmatización –en su constitución en condición de posibili-
dad de formas de legitimación política de prácticas de poder concretas– ha
tenido por objeto uno evidente: el que nos muestra Trinchero (2000) en su
análisis de las trayectorias sociales sobre las que el Estado diseña sus moda-
lidades de intervención. Allí, el autor despliega el proceso de estigmatiza-
ción, sobre la base de la naturalización, de las racionalidades (re)producti-
vas indígenas. Por cierto, las construcciones de sentido desplegadas en este
trabajo no hacen sino naturalizar las formas productivas de los trabajadores
indígenas, en cuanto configuradoras de una “cultura cazadora-recolectora” Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 2 4 8
A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3
a la que se borra de toda especificidad histórica, desde el momento en que
se busca con ella explicar la forma de su inserción actual en la organización
capitalista de la producción social. Frente a esto, los qom son elocuentes
respecto de la forma en que sus prácticas (re)productivas históricamente
específicas son objeto de la producción de estigmas sociales. Conclusión 2 4 8 T r a b a j a d o r e s i n d í g e n a s e n e l C h a c o a r g e n t i n o | V a l e r i a I ñ i g o R e f e r e n c i a s 1. Alvarsson, Jan-Åke. 1999. Foraging in Town: Survival Strategies among the ‘Weenhayek of Bolivia and
Argentina. En Peoples of the Gran Chaco, ed. Elmer Miller, pp. 23-36. Westport, Bergin y Garvey. 2. Arengo, Elena. 1996. “Civilization and Its Discontents”. History and Aboriginal Identity in the Argentine
Chaco. Tesis de Doctorado, Departamento de Antropología, New School for Social Research, Nueva York. 3. Bartolomé, Miguel Alberto. 1972. La situación de los indígenas en la Argentina: área chaqueña y
provincia de Misiones. En La situación del indígena en América del Sur (aportes al estudio de la
fricción inter-étnica en los indios no-andinos), coord. Georg Grünberg, pp. 309-352. Montevideo,
Tierra Nueva. 4. Beck, Hugo. 1994. Relaciones entre blancos e indios en los territorios nacionales de Chaco y Formosa. 1885-1950. Resistencia. Instituto de Investigaciones Geohistóricas, Conicet, Fundanord. 5. Bórmida, Marcelo. 1976. Etnología y fenomenología. Ideas acerca de una hermenéutica del
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tautegórica. Runa: Archivo para las Ciencias del Hombre 12, pp. 9-52. 7. Braunstein, José y Elmer Miller. 1999. Ethnohistorical Introduction. En Peoples of the Gran Chaco, ed. Elmer Miller, pp. 1-22. Westport, Bergin y Garvey. 8. Briones, Claudia y Morita Carrasco. 1996. “La tierra que nos quitaron”. Reclamos indígenas en
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Mónica Lacarrieu y Omar Jerez, pp. 45-78. Córdoba, Alción. 0. Citro, Silvia. 2009. Cuerpos significantes. Travesías de una etnografía dialéctica. Buenos Aires, Biblos. 10. Citro, Silvia. 2009. Cuerpos significantes. Travesías de una etnografía dialéctica. Buenos Aires, Biblos. 11. Consejo Agrario Nacional. 1945. El problema indígena en la Argentina. Buenos Aires, Secretaría de
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es por las posibles implicancias de esa construcción en las formas de acción
política de los qom de la porción oriental del Chaco central, y en particular, en
los límites y posibilidades del proceso de lucha mencionado en la introducción. Pero éste será el objeto de futuros trabajos. . A N T Í P O D A N º 1 7 | j u l i o - d i c i e m b r e 2 0 1 3 a la que se borra de toda especificidad histórica, desde el momento en que
se busca con ella explicar la forma de su inserción actual en la organización
capitalista de la producción social. Frente a esto, los qom son elocuentes
respecto de la forma en que sus prácticas (re)productivas históricamente
específicas son objeto de la producción de estigmas sociales. Ya Marx (1973), refiriéndose a los economistas clásicos y su afirmación
del carácter natural y, por tanto, eterno de las instituciones y relaciones de
producción burguesas, advertía que la atribución de relaciones sociales, y del
producto de esas relaciones sociales, a la naturaleza hace olvidar su génesis y
carácter históricos. Y de los peligros que ello encierra. Reconstruidos entonces
los términos en que son construidos los sentidos acerca de los indígenas como
trabajadores, la pregunta –que se abre a partir de lo hasta aquí desarrollado–
es por las posibles implicancias de esa construcción en las formas de acción
política de los qom de la porción oriental del Chaco central, y en particular, en
los límites y posibilidades del proceso de lucha mencionado en la introducción. Pero éste será el objeto de futuros trabajos. . Pero éste será el objeto de futuros trabajos. . R e f e r e n c i a s Chaco, 1870-
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Hirsch, pp. 101-121. Buenos Aires, La Crujía. 43. Von Bremen, Volker. 2000. Dynamics of Adaptation to Market Economy among the Ayoréode of
Northwest Paraguay. En Hunters and Gatherers in the Modern World: Conflict, Resistance, and
Self-Determination, eds. Peter Schweitzer, Megan Biesele y Robert K. Hitchcock, pp. 275-286. Nueva York, Berghahn Books. 43. Von Bremen, Volker. 2000. Dynamics of Adaptation to Market Economy among the Ayoréode of
Northwest Paraguay. En Hunters and Gatherers in the Modern World: Conflict, Resistance, and
Self-Determination, eds. Peter Schweitzer, Megan Biesele y Robert K. Hitchcock, pp. 275-286. Nueva York, Berghahn Books. 44. Von Bremen, Volker. 1987. Fuentes de caza y recolección modernas. Proyectos de ayuda al desarrollo
destinados a los indígenas del Gran Chaco Argentina, Paraguay, Bolivia. Stuttgart, Servicios de
Desarrollo de las Iglesias. 44. Von Bremen, Volker. 1987. Fuentes de caza y recolección modernas. Proyectos de ayuda al desarrollo
destinados a los indígenas del Gran Chaco Argentina, Paraguay, Bolivia. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 R e f e r e n c i a s Stuttgart, Servicios de
Desarrollo de las Iglesias. 45. Wright, Pablo. 2008. Ser-en-el-sueño. Crónicas de historia y vida toba. Buenos Aires, Biblos. 45. Wright, Pablo. 2008. Ser-en-el-sueño. Crónicas de historia y vida toba. Buenos Aires, Biblos. 45. Wright, Pablo. 2008. Ser-en-el-sueño. Crónicas de historia y vida toba. Buenos Aires, Biblos. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251
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Managing clustering effects and learning effects in the design and analysis of randomised surgical trials: a review of existing guidance
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Managing clustering effects and learning
effects in the design and analysis of randomised
surgical trials: a review of existing guidance zabeth J. Conroy1,2* , Jane M. Blazeby3 , Girvan Burnside1 , Jonathan A. Cook2 and Car Abstract Background: The complexities associated with delivering randomised surgical trials, such as clustering effects, by
centre or surgeon, and surgical learning, are well known. Despite this, approaches used to manage these complexi-
ties, and opinions on these, vary. Guidance documents have been developed to support clinical trial design and
reporting. This work aimed to identify and examine existing guidance and consider its relevance to clustering effects
and learning curves within surgical trials. Methods: A review of existing guidelines, developed to inform the design and analysis of randomised controlled
trials, is undertaken. Guidelines were identified using an electronic search, within the Equator Network, and by a
targeted search of those endorsed by leading UK funding bodies, regulators, and medical journals. Eligible documents
were compared against pre-specified key criteria to identify gaps or inconsistencies in recommendations. Results: Twenty-eight documents were eligible (12 Equator Network; 16 targeted search). Twice the number of guid-
ance documents targeted design (n/N=20/28, 71%) than analysis (n/N=10/28, 36%). Managing clustering by centre
through design was well documented. Clustering by surgeon had less coverage and contained some inconsistencies. Managing the surgical learning curve, or changes in delivery over time, through design was contained within several
documents (n/N=8/28, 29%), of which one provided guidance on reporting this and restricted to early phase stud-
ies only. Methods to analyse clustering effects and learning were provided in five and four documents respectively
(N=28). Conclusions: To our knowledge, this is the first review as to the extent to which existing guidance for designing
and analysing randomised surgical trials covers the management of clustering, by centre or surgeon, and the surgi-
cal learning curve. Twice the number of identified documents targeted design aspects than analysis. Most notably,
no single document exists for use when designing these studies, which may lead to inconsistencies in practice. The
development of a single document, with agreed principles to guide trial design and analysis across a range of realistic
clinical scenarios, is needed. Keywords: Trials, Clinical trial, Randomised controlled trial, Complex intervention, Surgical intervention, Trial design,
Trial analysis, Summary, Review, Clustering, Learning Background Randomised controlled trials (RCTs) are recognised
as providing the highest level of evidence, second only
to systematic reviews of such trials [1]. There are many
practical and methodological difficulties that a medical
researcher must overcome to deliver successful RCT. In © The Author(s) 2022. 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. Conroy et al. Trials (2022) 23:869
https://doi.org/10.1186/s13063-022-06743-6 Open Access *Correspondence: elizabeth.conroy@ndorms.ox.ac.uk 2 Oxford Clinical Trials Research Unit, Centre for Statistics in Medicine, Nuffield
Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences,
University of Oxford, Windmill Road, Oxford OX3 7LD, UK
Full list of author information is available at the end of the article © The Author(s) 2022. 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. Conroy et al. Trials (2022) 23:869 Page 2 of 8 trials with a surgical intervention, these difficulties are
often magnified [2–5]. Surgical interventions, delivered
as an intervention or as a setting, consist of many inter-
acting components — such as the procedure itself, sur-
geon expertise, and pre- or postoperative care [4]. Because of the nature of the search, full texts of iden-
tified guidelines were obtained to determine eligibility. Documents that provided guidance such that RCTs and
statistical aspects were covered within their scope were
included and reasons for exclusion were recorded. Patient outcomes often depend on the treatment pro-
vider delivering the intervention. Due to the nature of
surgical interventions, RCTs within this field can be vul-
nerable to criticism if concerns over variability in treat-
ment delivery are raised. Variability can arise between
intervention providers (clustering) or due to change in
delivery over time, often as a result of increased experi-
ence (learning) [6–8]. Therefore, when designing these
trials, it is important to consider the homogeneity of the
treatment effect and therefore the potential existence and
impact of both clustering and learning, by centre and sur-
geon. This should be done as early as possible during trial
design to avoid issues arising that violate the validity of
the trial results [9].hf Key criteria relevant to the design and analysis of surgi-
cal trials, or trials of complex interventions, were iden-
tified a priori (see Table 1). Identifying eligible documents The search within the EQUATOR website identified 80
documents: 36 (45%) were identified using the search
term ‘statistic’ and 44 (55%) using the search term ‘sur-
gery’. The search was conducted on 21 October 2021. Figure 1 presents the flowchart of eligibility, with rea-
sons for exclusion where necessary. An additional 16
documents were manually identified from the tar-
geted search (funders: 2; regulators: 6; journals: 8, see
Supplementary Material 1 and Fig. 1). There were no
duplicates between the two searches leaving a total
of 28 eligible documents for review. Supplementary
Material 1 provides the list of included documents. Details as to the background and justification for the
documents included as part of the targeted search are
provided in Supplementary Material 2. The importance of managing these effects within these
trials is well known, but the methods used to do so in
practice, and opinions on these, vary [10–12]. Guid-
ance documents exist to support clinical trial design and
reporting, but the majority target generic aspects of clini-
cal trials and originate specifically from medicinal trials. Therefore, whilst their relevance to all trials is indisput-
able, the extent to which they cover clustering and learn-
ing may be limited.h The aim of this review is to identify and examine exist-
ing guidance and consider its relevance to clustering
effects and learning curves within surgical trials. Methodsh This work sought to include guidance documents devel-
oped to inform the design and analysis of randomised
controlled trials (RCTs). Guidelines for inclusion in this
review were identified by undertaking: Summary of identified guidance Seven of the 28 eligible documents (25%) were developed
specifically for surgery. Of those that were not (n=21), • An electronic search within the Equator Network
(http://www.equator-network.org), an online library
containing a comprehensive searchable database of
reporting guidelines, using each of the search terms
‘surgery’ and ‘statistic’. Documents that provided
guidance specific to non-randomised studies, aspects
of trial methodology or medical specialties that were
not applicable, or focussed on applicable medical
specialties, such as surgery, with no statistical scope
were excluded. Table 1 Key criteria to be considered within design and analysis Eligible documents were
compared against these to identify gaps or inconsisten-
cies in recommendations. Guidelines for reporting the
aspects of design and analysis were also assessed against
these criteria. Specific methods within the guidelines
related to analysing clustering or learning, at the centre
or treatment provider level, were also collected. Docu-
ments were examined using NVivo qualitative data analy-
sis software [13]. Design
• The appropriate trial design, such as an expertise-based design
• Delivery of the intervention in terms of:
◦ The health professionals delivering treatment
◦ The extent to which treatments are to be standardised
◦ The potential for change in delivery over time
• Adjusting the sample size
• Balancing treatment within centres and treatment providers
Analysis
• When the randomisation has been stratified
• When analysing the primary outcome, such as adjustment
• When there are multiple centres and/or treatment providers Design • A targeted search of guidelines endorsed by leading
UK funding bodies, regulators, and medical journals
such that they covered aspects of trial design, analy-
sis, and reporting. Analysis Conroy et al. Trials (2022) 23:869 Page 3 of 8 Fig. 1 Flowchart of identification of guidelines two were written for complex interventions, two for
devices, and the remaining 17 for general medicine. of inter-rater reliability, between individual centres and
surgeons, can provide an understanding of any impact
due to expertise. This type of analysis can be useful when
considering rolling out the interventions into routine
healthcare, see the ‘Analysing a trial with clustering and
learning’ section [15]. Designing a trial with clustering and learning
Choosing a trial design Eleven out of 28 documents (39%) provided guidance
relating to trial design. See D1 in Supplementary Table 1. In cluster randomised trials, groups of patients are the
unit of randomisation. These designs are less common
and are generally less efficient than individually ran-
domised studies. They require more surgeons and intro-
duce the potential for the treatment comparison to be
confounded by the delivery, despite inflating the sample
size to account for the intraclass correlation coefficient
(ICC) [3, 4, 17]. The options of trial design depend on the unit of ran-
domisation and the intervention of interest. The key
aspects of relevant designs are briefly summarised here. Many design options, and associated limitations, were
discussed and no single document provided a single com-
prehensive summary. In individually randomised trials, patients are the unit
of randomisation [3]. When conducting these trials in
surgery, differential expertise between the treatments
being investigated can raise issues that can be alleviated
by defining eligibility criteria for centres and surgeons,
such as years in practice or the number of interven-
tions performed previously [14, 15]. However, applying
criteria that are too strict may reduce the generalis-
ability of trial results [16]. Instead, a statistical analysis Expertise-based designs are a half-way house between
individual and cluster randomised trials. Patients are
individually randomised to surgeon, who treats all
patients with a single intervention. This can be the sur-
geon’s preferred technique or an unavoidable feature in
trials comparing interventions delivered by different
specialties [4, 14]. This design has the same limitations
as cluster trials, and when a surgeon is only performing Conroy et al. Trials (2022) 23:869 Page 4 of 8 prepare investigators and standardise performance were
suggested by one document [24]. their preferred technique, shared waiting lists [4] and
understanding how the treatment can be rolled out into
routine healthcare can be a challenge. Resultantly, this
design is relatively uncommon [10, 11]. Monitoring treatment adherence was an important
aspect across documents [5, 9, 14, 24, 25]. Suggested
methods included reviewing case report forms, vide-
otapes, and audiotapes, extending to decertifying and
excluding surgeons not submitting a videotape rated
acceptable by an independent committee [14]. Tracker designs, proposed by Ergina et al., where new
or evolving interventions can theoretically be developed
within a single randomised study, and the incremental
changes to the intervention tracked within the analysis,
would be very challenging in practice [4]. Considering who will deliver the interventionh Thirteen out of 28 documents (46%) discussed the impor-
tance of deciding who will deliver the intervention. See
D2 in Supplementary Table 1. Some variation in delivery, in part, will depend on the
skill and training of those delivering the intervention [4,
14, 18]. As such, the selection of centres and treatment
providers was a critical element of design discussed by a
number of guidance documents [9, 17, 19, 20]. Any eligi-
bility criteria for participating centres and treatment pro-
viders, and a description such as the degree to which they
are typical, should be reported [14, 16, 21]. Anticipating changes over time Eight out of 28 documents (29%) discussed considering
changes in the delivery of the intervention over time. See
D4 in Supplementary Table 1. Delivery may still vary irrespective of training, expe-
rience, and other steps to enforce standardisation. The
amount of variation will depend on the stage and tech-
nicality of intervention development [3, 5, 14, 26]. An
important aspect of surgical evaluation across the guide-
lines was that delivery may change over time for prag-
matic reasons, changes in external factors, or as a result
of expertise developing during the study [3–5]. Two guidelines suggested criteria by which recruiting
centres should be chosen, such as caseload for the proce-
dure under investigation and ensuring sufficient numbers
of the target population [14, 20]. No guidelines provided advice on selecting treatment
providers. Treatment providers could be a limited group
or all professionals offering the intervention [22]. If it is a
limited group, guidance on selecting centres, and report-
ing requirements, may be looked upon as a proxy for tri-
alists when deciding how to select treatment providers,
for example caseload and ensuring specific qualifications
[14, 20, 21].h Expertise can develop over a very long time and so
requiring a set expertise level can slow the delivery of
surgical trials [4]. Some guidelines discussed evaluating
the learning curve within the trial [5] and highlighted this
was particularly important in earlier phase trials [26]. In
trials comparing more established techniques, the statis-
tical advantages and gain in ‘internal validity’ need to be
considered against the loss of generalisability or ‘external
validity’ of applying too much emphasis on the learning
curve [3]. The results of the main trial should report on the num-
ber of centres and treatment providers performing each
intervention [21]. Designing a trial with clustering and learning
Choosing a trial design Reporting in-depth details of the intervention, and
comparator, was required by a number of documents. Aspects required included technical procedures; full
details on preoperative, intraoperative, and postoperative
care; and the extent to which delivery was permitted to
vary between participants, treatment providers, and cen-
tres [14, 16, 25]. Ensuring that the intervention is standardised Reporting learning curve assessment results was
required by one document but this was limited to early
phase studies [26]. Fifteen out of 28 documents (54%) discussed the impor-
tance of standardising the intervention. See D3 in Sup-
plementary Table 1. When analysing the primary outcome Two out of 28 documents (18%) provided guidance on
adjusting the primary outcome analysis. See A2 in Sup-
plementary Table 1. Unexplained differences between treatments, for exam-
ple between adjusted and unadjusted analyses, can jeop-
ardise the trial results [27]. For this reason, when the
primary outcome is expected to be influenced by centre
or treatment provider, an adjustment should be planned. When the potential value of an adjustment is in doubt,
such as little existing prior knowledge, the primary analy-
sis should be an unadjusted analysis, supported by an
adjusted analysis [9, 27]. In general, larger datasets gener-
ally support more factors than smaller ones and results
based on simpler models are generally numerically stable,
the assumptions underpinning the statistical model eas-
ier to validate and improve generalisability [27]. Estimating the sample size Variation in delivery can be reduced by standardising
all, or aspects of, the intervention of interest. Limiting
variation in treatment delivery may be more desirable
in an efficacy trial than a pragmatic, effectiveness study
[3, 5]. In pragmatic trials, standardisation might con-
sist of simply informing treatment providers to perform
the treatment as usual [14]. Regardless of the stage, trial
delivery should be similar at all centres [9] and designed
such that a clear description of the procedures per-
formed can be provided [16, 23]. Investigator meetings to Eight out of 28 documents (29%) discussed sample size. See D5 in Supplementary Table 1. A number of guidance documents highlighted the
impact of failing to reduce variation within trial arms by
standardising the intervention on the sample size and
power calculation, where typical estimates assume that
differences between the treatments across centres, or
treatment provider, are unbiased estimates of the same
quantity [3, 9]. In the presence of multilevel data struc-
tures, where variability in individual-level outcomes can Conroy et al. Trials (2022) 23:869 Page 5 of 8 also include treatment provider in surgical trials, but no
guidance specifically made this point. reflect higher-level processes, calculations are more com-
plicated [7, 9, 18]. To avoid associated imprecision in
results, the sample size should adjust for any clustering
effects as estimated by the intraclass correlation coeffi-
cient (ICC) and this should be reported in the main result
paper [14, 21]. Conversely, two documents that discussed
sample size did not comment on adjusting for clusters
[15, 20]. Analysing multi‑centre trials Balance can be achieved by stratifying the randomisa-
tion and stratifying by centre was a common topic, par-
ticularly when centre is expected to be confounded with
other prognostic factors [9, 20, 27]. When there are too
few patients per centre, stratifying by a larger unit, such
as country or region, may be warranted [27]. Despite
stratifying by treatment provider not being specifically
addressed within the documents, in some circumstances,
it may be desirable to stratify for more than just both
centre and treatment provider, or treatment provider
alone, where numbers allow [27]. The use of more than
two stratification factors is rarely necessary [9]. Six out of 28 documents (21%) provided guidance on
analysing multi-centre trials. See A3 in Supplementary
Table 1. Investigations into the heterogeneity of the main treat-
ment effect across centre and/or treatment provider were
covered by a number of documents [5, 9, 14, 25, 26]. Fur-
thermore, the main trial publication should report meth-
ods to adjust for, and results into, clustering by centre or
treatment provider [14, 21]. These investigations are crit-
ical when a positive treatment effect is found and there
are appreciable numbers of subjects per centre [9]. In the
simplest multi-centre trial, a single investigator recruits
and is responsible for all patients within a single hospi-
tal, such that the centre is identified uniquely by hospi-
tal. When the definition of a centre is ambiguous, such as
a single investigator recruits from several hospitals or a
clinical team recruits from numerous clinics, the proto-
col should provide a definition [9, 25]. Analysing a trial with clustering and learning
When the randomisation was stratified Two out of 28 documents (18%) provided guidance on
adjusting the analysis following stratification. See A1 in
Supplementary Table 1. Stratifying randomisation and subsequently adjusting
the analysis are complementary methods of accounting
for prognostic factors, unless the stratification factor was
chosen for administrative reasons only [9, 27]. i
Quantitative approaches may comprise a graphical
display of the results of individual centres, such as forest
plots, or analytical methods, such as a significance test
although this generally has low power [9]. One stated that
investigations use a model which allows for centre differ-
ences but no interaction terms [9]. Fixed or mixed effects
models can be used, although mixed models are especially
relevant when there is a large number of centres [9, 25]. Two documents discussed the issue of adjusting for
too many, or too small, strata in the analysis, for which
there is no best solution [9, 27]. When included in the
randomisation scheme, ignoring centres or adjusting for
a large number of small centres might lead to unreliable
estimates of the treatment effect and p-values [27]. At
best, using an unadjusted analysis should be supported
by sensitivity analyses that indicate trial conclusions are
not affected because of this [27]. As above, the statistical
justifications for including centre could be considered to Ensuring balance of treatment within centre and treatment
provider Six out of 28 documents (21%) discussed ensuring that
treatment allocations are equally distributed within cen-
tre. See D6 in Supplementary Table 1. Balancing treatment groups with respect to prognos-
tic factors enhances trial credibility [20, 27]. Ensuring
balancing of patients within centre was highlighted as
important within many of the guidance documents [9,
20, 27], and similar reasoning would lead surgical trial-
ists to extend this to treatment provider which was not
discussed within any document. Methods for investigating the learning curve Four out of 28 documents (14%) provided guidance on
analysing the learning curve within centre and/or treat-
ment provider. See A4 in Supplementary Table 1. Conroy et al. Trials (2022) 23:869 Conroy et al. Trials (2022) 23:869 Page 6 of 8 Clustering, at the centre level, was well covered within
the design, analysis, and reporting guidance. However,
there were inconsistencies with regard to the treatment
provider coverage. For example, reporting required that
the eligibility of the treatment provider be covered, yet no
guidance on the design or analysis covered this [14, 21]. However, this may be due to the original guidance largely
not being written specifically for surgery, or indeed com-
plex interventions, where these effects may be more
prominent [10, 11]. The role of the centre within conven-
tional drug trials could be extended to provide guidance
on the role of the treatment provider in surgery trials [3,
9, 20]. Methods to monitor the quality of delivery of the
trial intervention through study conduct have been con-
sidered, recommending that stablished protocols that
determine prohibited, mandated, and flexible interven-
tion components and monitoring adherence are devel-
oped [28]. Yet a specific guidance document that covers
the design and analysis of randomised surgical trials, or
intervention trials, could address the discrepancies iden-
tified within this review to improve the quality of under-
standing and awareness of these issues [10–12]. Reporting of continuous quality control measures can
be useful for all phases of the trial, particularly early
phase surgical trials [5, 26]. Time series and longitudinal
models or multilevel models can be used to analyse long
and short sequences of data respectively [3, 18]. Simpler
exploratory methods such as cusum plots enable centres
or surgeons to be compared against themselves which
can be preferable to surgeons [5, 26]. Method for investigating clustering Five out of 28 documents (18%) provided guidance on
investigating clustering due to centre and/or treatment
provider. See A5 in Supplementary Table 1. Hierarchically structured data, such as patients within
surgeon, can be analysed using multilevel models or gen-
eralised estimating equations (GEEs) [3, 21]. Multilevel
models are subject-specific models whereas GEEs are
population average. For multilevel models, fixed, random,
or mixed effects can be specified to account for clustering
[21] and different types of these models allow for flexible
data structures [18].f For ordinary linear models, the treatment effect esti-
mate is likely to be similar but not necessarily iden-
tical for adjusted and unadjusted models. Adjusted
analyses are more efficient, and so a less significant result
for unadjusted should not be a concern. For generalised
linear or non-linear models, adjusted and unadjusted
treatment effects may not have the same interpretation
and may provide different results [27]. A number of guidance documents acknowledged the
importance of the surgical learning curve, or delivery
changing over time, within design and analysis, particu-
larly in early phase surgical trials or when the interven-
tions differ in their technicality [3–5, 14, 26]. Yet there
was little coverage within reporting standards to reflect
this, with the surgical learning curve analysis only neces-
sary in the early phase, and not necessarily randomised,
trials [26] and broader RCT reporting guidelines only
requiring differential expertise be addressed in the dis-
cussion [14]. Lack of clear standards, and guidance that
is too broad in scope, may lead to reporting how delivery
of intervention changes over time, despite its importance,
being generally under-recognised in the literature [10,
11]. Supplementary Information analysis of the trial. Being able to explore effects will be par-
ticularly beneficial if concerns of learning or clustering are
raised, or more generally will allow better understanding to
contextualise study findings to ultimately support the roll-
out of the interventions into routine practice. The online version contains supplementary material available at https://doi.
org/10.1186/s13063-022-06743-6. The online version contains supplementary material available at https://doi.
org/10.1186/s13063-022-06743-6. The online version contains supplementary material available at https://doi.
org/10.1186/s13063-022-06743-6. Additional file 1: Supplementary Material 1. List of eligible guidance
documents. Supplementary Material 2. Additional information on docu-
ments obtained by the targeted search. Supplementary Table 1. Key
criteria coverage across documents summary. Additional file 1: Supplementary Material 1. List of eligible guidance
documents. Supplementary Material 2. Additional information on docu-
ments obtained by the targeted search. Supplementary Table 1. Key
criteria coverage across documents summary. When reading this review, it is important to consider
its limitations. First, country-specific guidance beyond
the UK, such as US Food and Drug Administration,
was not included. However, international documents
that are applicable to other countries, including the UK,
were obtained, such as ICH which are followed globally
and EMA which are adopted within Europe. Second,
only four guidance documents, developed by the same
research group, were written specifically for surgery and
not written specifically for RCTs, which may explain the
lack of specific coverage of the surgeon in the wider set
[4, 5, 26, 32]. Third, very little of the guidance documents
covered statistical aspects, leaving a trialist to extend the
centre-drug connection to surgeon-intervention using
existing guidelines [9, 27]. The development of a statis-
tical guidance document that covers randomised surgi-
cal trials in more depth would help trialists, in particular
statisticians, and the IDEAL framework provide a good
basis for this development to be integrated [4, 5, 26, 32]. Abbreviations
GEE: Generalised estimating equation; ICC: Intraclass correlation coefficient;
RCT: Randomised controlled trial. Conclusionshi This is the first review, to our knowledge, to explore the
coverage of guidance for managing clustering effects and
the surgical learning curve within the design and analy-
sis of randomised surgical trials. Twice the number of
identified documents target design aspects than analysis. Furthermore, no single and complete guidance document
exists that covers aspects of learning and clustering leav-
ing trialists to have to access multiple documents to gain
full understanding of these considerations. Competing interests p
g
CG, JAC, and JMB co-authored some of the guidance documents included
within this review. The authors declare that they have no other competing
interests. Existing documents should therefore be extended to
incorporate statistical guidance on the management of
clustering and learning. The IDEAL framework aligns
perfectly with the focus of this work as it is developed
specifically for surgical trials and is already widely used
by surgical trialists [5]. Future work should address
integrating these statistical themes into this framework
as a priority. This would encourage better consistency
between trialists, improve awareness of these methodo-
logical challenges, and support the use of optimal meth-
ods within the surgical field. Availability of data and materials Datasets used and/or analysed during the current study, which are not already
included in this published article, are available from the corresponding author
on reasonable request. Authors’ contributions EJC participated in the study design, developed the data extraction form,
drafted the manuscript, and extracted and analysed the data. CG partici-
pated in the study design, developed the data extraction form, and drafted
the manuscript. GB, JMB, and JAC participated in the study design and
contributed to manuscript development. The authors read and approved
the final manuscript. Author details 1 Liverpool Clinical Trials Centre, University of Liverpool, Liverpool, UK. 2 Oxford Clinical Trials Research Unit, Centre for Statistics in Medicine, Nuffield
Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences,
University of Oxford, Windmill Road, Oxford OX3 7LD, UK. 3 Centre for Surgical
Research, Bristol Biomedical Research Centre, Population Health Sciences,
University of Bristol, Bristol, UK. Received: 28 February 2022 Accepted: 13 September 2022 Funding This research was funded by National Institute for Health Research (NIHR)
Doctoral Fellowship Programme (DRF-2015-08-082). EJC is funded through
this fellowship programme. Declarations Ethics approval and consent to participate
Not applicable. Department of Health disclaimer
h
d
h
f h The views expressed are those of the authors and not necessarily of the
National Health Service, the National Institute for Health Research (NIHR), or
the Department of Health and Social Care. Discussion Trialists should consider the impact of clustering and
learning when designing and analysing randomised sur-
gical trials. Considerations should be incorporated into
reporting to aid the interpretation and applicability of
trial results. This investigation is the first review as to the
extent that existing guidance within the UK covers these
important effects. Existing guidance documents are iden-
tified and summarised, with a focus on aspects relating to
clustering effects and the learning curve and their appli-
cation to surgical trials. Not all documents were written
specifically for surgery, yet all contain aspects that can
be applied to surgery, for example, the role of the cen-
tre in the delivery of treatments in drug trials has some
commonalities to the role of the surgeon in delivering a
surgical trial. Twice the number of identified documents
targeted design aspects than analysis. Whilst a good anal-
ysis cannot rescue a poor design, and this may have led
to a larger focus on the design on guidance for trialists,
there is a notable dearth of analysis guidance available
that requires addressing. In addition, there is also scope
for guidance on study conduct. When designing and analysing a randomised surgical
trial, there can be a view that clustering and learning are less
pronounced or irrelevant in more pragmatic large-scale tri-
als where the interventions are stabilised and in widespread
use. Measures to reduce variation in treatment effects are
often introduced into the trial design by defining a mini-
mum level of expertise or providing training for treatment
providers [12]. However, despite measures being taken,
variation in delivery may remain, and the need to account
for the breadth of the setting, learning curve, and experi-
ence of surgeons is an area for improvement in more prag-
matic RCTs [29–31]. Trialists should therefore be aware
of the potential for clustering and learning and routinely
consider their impact at the trial outset. Early and careful
consideration will improve data collection to ensure that, if
required, investigation can be integrated into the planned Conroy et al. Trials (2022) 23:869 Conroy et al. Trials (2022) 23:869 Page 7 of 8 Consent for publication Consent for publication
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update and a CONSORT extension for nonpharmacologic trial abstracts. Ann Intern Med. 2017;167(1):40–7. 22. Elias KM, Stone AB, McGinigle K, Tankou JI, Scott MJ, Fawcett WJ, et al. The Reporting on ERAS Compliance, Outcomes, and Elements Research
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CORDES statement. Hum Reprod. 2016;31(6):1219–23. 24. ICH Expert Working Group. ICH harmonised tripartite guidelines: general
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UvA-DARE (Digital Academic Repository) Link to publication Citation for published version (APA):
Martins, Z., Cottin, H., Kotler, J. M., Carrasco, N., Cockell, C. S., de la Torre Noetzel, R.,
Demets, R., de Vera, J. P., d'Hendecourt, L., Ehrenfreund, P., Elsaesser, A., Foing, B.,
Onofri, S., Quinn, R., Rabbow, E., Rettberg, P., Ricco, A. J., Slenzka, K., Stalport, F., ... Westall, F. (2017). Earth as a Tool for Astrobiology: A European Perspective. Space Science
Reviews, 209(1-4), 43-81. https://doi.org/10.1007/s11214-017-0369-1 Citation for published version (APA):
Martins, Z., Cottin, H., Kotler, J. M., Carrasco, N., Cockell, C. S., de la Torre Noetzel, R.,
Demets, R., de Vera, J. P., d'Hendecourt, L., Ehrenfreund, P., Elsaesser, A., Foing, B.,
Onofri, S., Quinn, R., Rabbow, E., Rettberg, P., Ricco, A. J., Slenzka, K., Stalport, F., ... Westall, F. (2017). Earth as a Tool for Astrobiology: A European Perspective. Space Science
Reviews, 209(1-4), 43-81. https://doi.org/10.1007/s11214-017-0369-1 General rights General rights
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DOI 10.1007/s11214-017-0369-1 SPECIAL COMMUNICATION Note by the editor: This is a Special Communication, supplementing the paper by Cottin et al. on
“Astrobiology and the Possibility of Life on Earth and Elsewhere...”, 2015, Space Science Reviews,
doi:10.1007/s11214-015-0196-1 and Cottin et al. on “Space as a Tool for Astrobiology: Review and
Recommendations for Experimentations in Earth Orbit and Beyond”, 2017, Space Science Reviews,
doi:10.1007/s11214-017-0365-5. Earth as a Tool for Astrobiology—A European
Perspective Zita Martins1 · Hervé Cottin2 · Julia Michelle Kotler3,4 · Nathalie Carrasco5 ·
Charles S. Cockell6 · Rosa de la Torre Noetzel7 · René Demets8 · Jean-Pierre de Vera9 ·
Louis d’Hendecourt10 · Pascale Ehrenfreund3,11 · Andreas Elsaesser12 ·
Bernard Foing8 · Silvano Onofri13 · Richard Quinn14 · Elke Rabbow15 ·
Petra Rettberg15 · Antonio J. Ricco16 · Klaus Slenzka17 · Fabien Stalport2 ·
Inge L. ten Kate18 · Jack J.W.A. van Loon19 · Frances Westall20 Received: 29 July 2016 / Accepted: 13 April 2017 / Published online: 20 June 2017
© The Author(s) 2017. This article is published with open access at Springerlink.com Abstract Scientists use the Earth as a tool for astrobiology by analyzing planetary field
analogues (i.e. terrestrial samples and field sites that resemble planetary bodies in our Solar
System). In addition, they expose the selected planetary field analogues in simulation cham-
bers to conditions that mimic the ones of planets, moons and Low Earth Orbit (LEO) space
conditions, as well as the chemistry occurring in interstellar and cometary ices. This paper Note by the editor: This is a Special Communication, supplementing the paper by Cottin et al. on
“Astrobiology and the Possibility of Life on Earth and Elsewhere...”, 2015, Space Science Reviews,
doi:10.1007/s11214-015-0196-1 and Cottin et al. on “Space as a Tool for Astrobiology: Review and
Recommendations for Experimentations in Earth Orbit and Beyond”, 2017, Space Science Reviews,
doi:10.1007/s11214-017-0365-5. B Z. Martins
z.martins@imperial.ac.uk An improvement of the coordinated logistics, infrastructures
and funding system supporting the combination of field work with planetary simulation in-
vestigations, as well as an optimization of the scientific return and data processing, data
storage and data distribution is also needed. Finally, a coordinated EU or ESA education
and outreach program would improve the participation of the public in the astrobiological
activities. Keywords Astrobiology · Exobiology · Astrochemistry · Planetary field analogues ·
Laboratory analogues · Field test campaigns Keywords Astrobiology · Exobiology · Astrochemistry · Planetary field analogues ·
Laboratory analogues · Field test campaigns B Z. Martins
z.martins@imperial.ac.uk B Z. Martins
z.martins@imperial.ac.uk B Z. Martins
z.martins@imperial.ac.uk 1
Department of Earth Science and Engineering, Imperial College London, SW7 2AZ, London, UK 2
Laboratoire Interuniversitaire des Systèmes Atmosphériques (LISA), UMR CNRS 7583,
Université Paris Est Créteil et Université Paris Diderot, Institut Pierre Simon Laplace, 61 avenue du
Général de Gaulle, 94010 Créteil Cedex, France 3
Leiden Observatory, P.O. Box 9513, 2300 Leiden, The Netherlands 4
Present address: Department of Chemistry, The University of Reading, Reading RG6 6UR, UK 5
Université Versailles St-Quentin, UPMC Univ. Paris 06, CNRS, LATMOS, 11 blv d’Alembert,
78280 Guyancourt, France 6
UK Centre for Astrobiology, SUPA, School of Physics and Astronomy, James Clerk Maxwell
Building, King’s Buildings, Edinburgh, EH9 3JZ, UK 7
Earth Observation, Remote Sensing and Atmosphere, INTA, Instituto Nacional de Técnica
Aeroespacial, Crta. Ajalvir, km. 4, 28850 Torrejón de Ardoz, Madrid, Spain 8
ESTEC (HRE-UB), Keplerlaan 1, 2201 AZ Noordwijk, The Netherlands 9
Institute of Planetary Research, Management and Infrastructure, Research Group Astrobiology
Laboratories, German Aerospace Center (DLR), Rutherfordstr. 2, 12489 Berlin, Germany 10
Institut d’Astrophysique Spatiale, UMR 8617 CNRS, Univ. Paris-Sud, Université Paris-Saclay,
Campus d’Orsay, Bat 121n, 91400 Orsay, France Z. Martins et al. 44 reviews the ways the Earth is used by astrobiologists: (i) by conducting planetary field ana-
logue studies to investigate extant life from extreme environments, its metabolisms, adapta-
tion strategies and modern biosignatures; (ii) by conducting planetary field analogue studies
to investigate extinct life from the oldest rocks on our planet and its biosignatures; (iii) by
exposing terrestrial samples to simulated space or planetary environments and producing a
sample analogue to investigate changes in minerals, biosignatures and microorganisms. The
European Space Agency (ESA) created a topical team in 2011 to investigate recent activities
using the Earth as a tool for astrobiology and to formulate recommendations and scientific
needs to improve ground-based astrobiological research. Space is an important tool for as-
trobiology (see Horneck et al. in Astrobiology, 16:201–243, 2016; Cottin et al., 2017), but
access to space is limited. Complementing research on Earth provides fast access, more
replications and higher sample throughput. The major conclusions of the topical team and
suggestions for the future include more scientifically qualified calls for field campaigns with
planetary analogy, and a centralized point of contact at ESA or the EU for the organization
of a survey of such expeditions. 1 Introduction One of the key goals of astrobiology is to explore planetary bodies of our solar system in
order to determine their potential for habitability and extra-terrestrial life. This is achieved
by partly analyzing materials from specific environments on Earth that exhibit similar con-
ditions as planets and moons in our Solar System (i.e. planetary field analogues), and by
using laboratory set-ups that mimic planetary and deep space conditions (i.e. laboratory
analogues). 11
Space Policy Institute, George Washington University, 20052 Washington, DC, USA 12
Experimental Molecular Biophysics, Free University of Berlin, Arnimallee 14, 14195 Berlin,
Germany 13
Università della Tuscia, Viterbo, Italy 14
NASA Ames Research Center MS 239-4, Moffett Field, CA 94035, USA 15
Radiation Biology Department, Research Group Astrobiology, DLR, Institute of Aerospace
Medicine, Koeln, Germany 16
NASA Ames Research Center, Mountain View, CA, USA 17
OHB SYSTEM AG, Universitätsallee 27-29, 28359 Bremen, Germany 17
OHB SYSTEM AG, Universitätsallee 27-29, 28359 Bremen, Germany 18
Department of Earth Sciences, Universiteit Utrecht, P.O. Box 8002, 3508 TA Utrecht,
The Netherlands 18
Department of Earth Sciences, Universiteit Utrecht, P.O. Box 8002, 3508 TA Utrecht,
The Netherlands 19
VU University, Amsterdam, The Netherlands 19
VU University, Amsterdam, The Netherlands 20
CBM, UPR 4301, CNRS, rue Charles Sadron, 45071 Orléans, France 45 Earth as a Tool for Astrobiology—A European Perspective There are many planetary field analogue environments on Earth with extreme ranges of
temperature, salinity, mineral content, pH, pressure, and water availability (Marlow et al. 2011). This diversity mimics in parts various extra-terrestrial environments that are of inter-
est to astrobiologists, such as the terrestrial planets (Venus and Mars), the Jovian moons (e.g. Europa), and the Saturn moons (Titan and Enceladus) (Preston and Dartnell 2014). Plane-
tary field analogues are used to (i) perform in-situ measurements during field campaigns,
allowing to test methodologies, protocols, and technologies, and (ii) collect samples, both
biotic (extant and extinct or fossilized) and abiotic for analyses by state-of-the-art meth-
ods in the laboratory, e.g. to investigate the relationship between biosignatures and their
rock/mineral context, and to identify microorganisms from extreme environments. These
kinds of activities inform astrobiologists about habitability and biosignatures present on
Earth and elsewhere, planetary processes, the most appropriate life-detection methods and
techniques, validation of space mission instrumentation and payload testing, selection of the
best landing sites for future life-detection missions, and finally, the mobility of rovers on
representative terrains. 1 Introduction Laboratory analogues on Earth provide laboratory access to selected parameters of extra-
terrestrial environments for a wide variety of experiments, e.g. the formation and preserva-
tion of biosignatures, the alteration of minerals and rocks, physical and chemical processes
or the survival and adaptation mechanisms of microorganisms, as well as their interaction
under simulated atmospheric composition, pressure, temperature and radiation of the space
environment of interest. In addition, simulation facilities are important tools for necessary
space hardware tests (e.g. Motamedi 2013). The European Space Agency (ESA) funded a Topical Team in 2011 to summarize the
most recent achievements in the field of astrobiology (Cottin et al. 2015). Reviews on exper-
imental studies of space as a tool for astrobiology (Cottin et al. 2017) and on the Earth as a
tool for astrobiology (current manuscript) have also been compiled. The present manuscript
is a review of recent activities using Earth as a tool for astrobiology, reporting on plane-
tary field analogues sites on Earth (Sect. 2), field test campaigns (Sect. 3), and simulation
facilities in Earth laboratories (Sect. 4). A synthesis of all relevant information, as well as
recommendations from the Topical Team to ESA is also presented (Sect. 5). This study thus
perfectly complements the EU-funded Astrobiology Roadmap, a very broad overview of the
state of the art of, and challenges for, astrobiology in general (Horneck et al. 2016). 2 Planetary Field Analogue Environments on Earth Planetary field analogue environments are places on Earth with geological or environmental
conditions that are similar to those that exist on an extra-terrestrial planetary body (Léveillé
2009). They do not replicate all the conditions of another planetary body, but instead mimic
specific parameter(s) (Marlow et al. 2008, 2011). The purpose of using terrestrial analogue
sites for planetary missions can be divided into five categories: (i) learning about planetary
processes (both geological and biological) on Earth and elsewhere; (ii) testing protocols,
strategies, methodologies, operations, technologies, and payload instrumentation; (iii) train-
ing highly-qualified personnel (e.g. PhD students and engineers), as well as science and op-
eration teams; (iv) engaging the public, space agencies, media, and educators; and (v) defin-
ing detectable biosignatures from extant/extinct life, the limits of life and habitability on
Earth (Lee 2007; Léveillé 2009). There are currently a few on-line databases for planetary
field analogue rocks and sites, such as: Z. Martins et al. 46 (i) the International Space Analogue Rockstore (ISAR), which is a collection of well-
characterized rocks used for testing and calibrating instruments to be flown on space
missions (Bost et al. 2013) (http://www.isar.cnrs-orleans.fr/isar/). The ISAR is lo-
cated in the Centre de Biophysique Moléculaire (CBM) of the Centre National de la
Recherche Scientifique (CNRS) in Orléans, and is supported by the Observatoire des
Sciences de l’Univers en Région Centre (OSUC), the Centre National d’Etudes Spa-
tiales (CNES), and ESA; (ii) the Concepts for Activities in the Field for Exploration (CAFE), which was commis-
sioned by ESA (Preston et al. 2013a), and resulted in a catalogue of all planetary field
analogue sites used and currently in use (Preston et al. 2013b). This catalogue describes
in detail each of these field sites (e.g. location, geological and environmental informa-
tion) and is the most extensive and up-to-date catalogue available; (iii) the Europlanet Planetary Field Analogues (PFA), which offers access to five well-
characterized
terrestrial
field
sites
(http://www.europlanet-2020-ri.eu/research-
infrastructure/field-lab-visits/ta1-pfa). The PFA has been selected to provide the most
realistic analogues of the surfaces of Mars, Europa and Titan. Access is provided for
scientists to perform high quality scientific research and test instrumentation for space
missions under realistic planetary conditions and undertake comparative planetology
research. 2 Planetary Field Analogue Environments on Earth The Committee on Space Research (COSPAR) Panel on Exploration (PEX) in cooper-
ation with the European Science Foundation (ESF) has elaborated a report on an “Inter-
national Earth-based research program as a stepping stone for global space exploration—
Earth-X” (Ehrenfreund et al. 2011). Analogue research is an international endeavor since
the sites of interest are distributed around the globe, and potential technology and scientific
developments are of interest to many fields of study and within many areas of exploration
both on the Earth and in space. In 2003 the ESF initiated a new research support activity called Investigating Life in
Extreme Environments (ILEE). This initiative showed the need for a coordinated, interdis-
ciplinary approach to improve future opportunities for funding research on “Life in extreme
environments” (LEXEN). The coordination action for research on study of life in extreme
environments (CAREX) was a Seventh Framework Programme (FP7) project funded by the
European Commission (EC) and coordinated by the British Antarctic Survey and the Nat-
ural Environment Research Council (NERC). The project operated from 1 January 2008
to 30 June 2011 and addressed the enhanced coordination of LEXEN research in Europe
by providing networking and exchange of knowledge opportunities to the scientific com-
munity. It was an interdisciplinary initiative covering all life forms existing in extreme en-
vironments on Earth, as well as addressing the use of extreme environments as planetary
field analogues in the search for extra-terrestrial life. The four priority areas identified in
the CAREX strategic roadmap included: (1) contributions of life in extreme environments
(LEXEN) to biogeochemical cycles and responses to environmental change, (2) stressful
environments—responses, adaptation and evolution, (3) biodiversity, bioenergetics and in-
teractions in extreme environments, and (4) life and habitability (final CAREX report sum-
mary available at http://cordis.europa.eu/result/rcn/55055_en.html). The resulting strategic
roadmap for European research on LEXEN is a basis for strategic programme planning for
science organizations such as the EC, COSPAR, and other institutions outside Europe such
as the National Aeronautics and Space Administration (NASA) Astrobiology Institute and
the Japan Agency for Marine-Earth Science and Technology (JAMSTEC). One example of
a European follow-on research project funded by the EC in the Framework Program 7 is
the Mars Analogues for Space Exploration (MASE, 2014–2017, http://mase.esf.org/). 2 Planetary Field Analogue Environments on Earth It has Earth as a Tool for Astrobiology—A European Perspective 47 the aim (i) to isolate and characterise anaerobic microorganisms from selected sites that
closely match environmental conditions that might have been habitable on early Mars, (ii)
to study their responses to realistic combined environmental stresses that might have been
experienced in habitable environments on Mars, and (iii) to investigate their potential for
fossilization on Mars and their detectability by carrying out a systematic study of the de-
tectability of artificially fossilized organisms exposed to known stresses. y
y
g
p
The selection of astrobiologically-relevant field sites is most often dependent on envi-
ronmental conditions that favor our current understanding of the limits of life and that could
support metabolic activity and growth, either currently or in the geologic past (see Cot-
tin et al. 2017). One example used in ecological field campaigns are the measurements on
metabolic activity such as measuring methane production by methanogens (Wagner et al. 2005, 2007) or photosynthetic activity by phototrophs like cyanobacteria, alga and lichens
(Lange 1969; Lange et al. 1970; Schroeter et al. 1994; Schroeter and Scheidegger 1995;
Pannewitz et al. 2003). Also, reproduction expressed by the growth rate may be recorded,
which might take several years of continuously monitoring the selected field sites in order
to obtain measurement results over longer time scales. This time-consuming observation is
necessary because of the dominance of very slow growing species present, for example, in
extreme Mars-analogue field sites. In parallel, measurements on irradiation, temperature,
relative humidity, methane/CO2 production or consumption and oxygen production are also
performed (Lange et al. 1970, 1996). Such investigation on planetary analogue field sites on
Earth provides fundamental information on life’s behavior and its capacity to influence or
change its environment. The interchange between the environment and the life forms might
also provide insight into search strategies and objectives for the search for life on other
planets or moons. The amount of measured gases released by microorganisms might serve
as references for potential gas traces produced by life on another planet. In addition, bio-
leaching of rocks and soils (Cockell 2010) due to metabolic activity and release of acidic
substances as well as possible deposits of secondary metabolites can change mineral compo-
sition or enrich soil with organic material. 2 Planetary Field Analogue Environments on Earth The analysis of such environmental patterns as a
biosignature of life can be studied in the field as well as in the laboratory, which would lead
to the identification of fossilization processes on Earth and support the search for extinct life
on other planets (Orange et al. 2011; Westall et al. 2011, 2015a, 2015b). Terrestrial microorganisms adapted to the most extreme environments on Earth (i.e. ex-
tremophiles) are potential models for understanding long-term survival in specific extra-
terrestrial conditions, in particular in the Martian surface/subsurface, and in the icy moons
of Jupiter and Saturn surface/subsurface environments. Environmental conditions, such as
extremes of dry, cold, with relatively high radiation background and weak seasonal fluc-
tuations (e.g. in the planetary field analogue environment of permafrost in Antarctica),
extreme dry and relatively hot or cold (e.g. in the planetary field analogue environments
of the Atacama Desert in Chile, and the Antarctic Dry Valleys), extreme pH conditions
(e.g. in the planetary field analogue environment of the Río Tinto), and extreme salinity
(e.g. in the planetary field analogue environment of the saturated salt areas of the Dead
Sea), make these environments similar to space- and planetary environments. Prokaryotes
(e.g. bacteria, bacterial spores, cyanobacteria) have been studied in such extreme planetary
field analogue environments due to the fact that early life forms on Earth were prokaryotes
and the assumption that if any extra-terrestrial life exists in the Solar System, it must be
simple cellular organisms (Hansen 2007; Westall et al. 2015a, 2015b). For example, mi-
crobial cryptoendolithic communities that colonize the pore spaces of sedimentary rocks
in Antarctica and other deserts to avoid stressful environmental conditions (e.g. ultravi-
olet (UV) radiation, very low or very high temperatures and very dry conditions) have 48 Z. Martins et al. been studied (Friedmann and Ocampo 1976; Friedmann 1982; Jänchen et al. 2014; de Vera
et al. 2014a). Eukaryotes (e.g. lichens, microcolonial fungi and tardigrades) have also been
studied, and shown a high resistance against the extreme conditions (Meeßen et al. 2015;
Jänchen et al. 2015, Onofri et al. 2004, 2008, 2012, 2015, Sancho et al. 2007, de la Torre
et al. 2010, de Vera et al. 2010, 2014b). In addition, fossil traces of primitive prokaryotes in
well-preserved rocks from the early Earth constitute ideal analogues for potential primitive
life forms on the early Mars (Westall et al. 2015a, 2015b). 2 Planetary Field Analogue Environments on Earth y
Characterization of the ecosystems where extremophiles will be collected during field
campaigns is necessary. Monitoring of the UV-climatology and microclimatology, as well
as parallel physiological activity determination is a fundamental step to compare the sea-
sonal adaptations of extremophile organisms to their natural habitat with the resistance to
simulated space- and planetary conditions (de la Torre 2002, de la Torre et al. 2004, de Vera
et al. 2014b). Community studies are performed by in situ exposure of the microbial popu-
lation in environmental samples (e.g. rock or soil; Olsson-Francis and Cockell 2010). The
community is characterized before exposure, which may include constructing a 16S rRNA
gene library and culturing. Environmental samples are generally diverse, and thus the num-
ber of exposed species is much higher than that of pure cultures. Exposing the community
will lead to the selection of microorganisms that are resistant. This will give information
regarding the physiological requirements of microbial survival in the conditions examined. In addition, the incubation of the microbial community in situ does not involve culturing
the microorganisms in the laboratory prior to exposure. Lichens have also been studied as
they have multiple protective mechanisms. These allow lichens to grow in some of the most
extreme environments on Earth (de Vera et al. 2004, 2008, 2010, 2014b). Using field cam-
paigns, lichens are collected as “fresh samples” directly from their natural habitat. For exam-
ple, samples of the lichen Pleopsidium chlorophanum and Buelia frigida were collected in
Mars-analogue dry, cold and UV-irradiated environments in the North Victoria Land Moun-
tains in Antarctica (de Vera et al. 2014b, Meeßen et al. 2015), or the vagrant lichen, such
as Circinaria fruticulosa originally from continental deserts and arid areas of Middle Asia,
Eurasia, North America and Northern Africa, and thus well adapted to harsh environmental
conditions (e.g. heat, drought, and high levels of solar UV radiation) have been collected on
clayey soils in continental high basins of Central Spain. After collection, they were kept dry
and protected from light under dark conditions for several months before the tests (Sancho
et al. 2000). In the next sub-section, we highlight diverse planetary field analogue sites. 2.1 Planetary Field Analogues Sites Identification is
as follow: Site 1—High-altitude atmosphere; 2—Yilgarn Craton (Western Australia); 3—Rio Tinto (Spain);
4—The Golden Deposit (Canadian High Arctic); 5—Yellowstone National Park (USA); 6—Haughton Im-
pact Structure (Devon Island); 7—Dongwanzi Ophiolite Complex (China); 8—Axel Heiberg Island (Cana-
dian High Arctic); 9—The Antarctic Dry Valleys (Antarctica); 10—Antarctic North Victoria Land Moun-
tains 11—Sub-glacial Volcanism (Iceland); 12—Kamchatka (Russian Federation); 13—Bockfjord Volcanic
Complex (Svalbard); 14—Kilimanjaro (Tanzania, Africa); 15—Atacama (South America); 16—The Mojave
Desert (USA); 17—Namib Desert (Namibia, Africa); 18—Ibn Battuta Centre Sites (Morocco); 19—Qaidam
Basin (Tibetan Plateau); 20—caves in Sardinia; 21—caves in Tenerife and Lanzarote; 22—Lake Vostok
(Antarctica); 23—Permafrost (Multiple Sites); 24—The Borup Fiord Pass (Ellesmere Island); 25—Lake
Tirez (Spain); 26—Orca Basin (Gulf of Mexico); 27—Mono Lake (California, USA); 28—The Dead Sea (Is-
rael); 29—Lost City (Mid-Atlantic Ridge); 30—The Mariana Trench (Pacific Ocean); 31—Lidy Hot Springs
(USA); 32—The Columbia River Basalts (USA); 33—Pitch Lake (Trinidad and Tobago); 34—Rancho La
Brea Tar Pits (California, USA); 35—Alaskan Oil Fields (USA); 36—Pilbara (north of Western Australia);
37—Barberton (South Africa). Number 1 broadly corresponds to high-altitude atmospheric analogues for
Venus, numbers 2 to 21 are analogue sites for Mars, numbers 22 to 32 are analogue site for Europa and Ence-
ladus, numbers 33 to 35 are analogue site for Titan, and numbers 36 to 37 are analogue sites for the early
Earth Fig. 1 Global map of the Earth with 33 analogue sites identified (adapted from Preston and Dartnell 2014). The map includes sites used by ESA state members and also the international community. 2.1 Planetary Field Analogues Sites Space missions to planetary bodies in our solar system are expensive and time-consuming,
taking decades to reach their target. In the meantime, and in order to prepare for these space
missions, scientists use locations on Earth that mimic some characteristics of those solar
system locations. The terrestrial materials used as planetary analogues provide a preview of
the properties of the solar system location they best mimic (e.g. Marlow et al. 2008, 2011): • Compositional analogues include properties such mineralogy, elemental abundances,
volatile content (water and dissolved gases), and organic content. • Compositional analogues include properties such mineralogy, elemental abundances,
volatile content (water and dissolved gases), and organic content. • Electrochemical analogues include similarities in terms of chemical properties such as
dielectric constant, redox potential, pH, water activity, and magnetism. • Electrochemical analogues include similarities in terms of chemical properties such as
dielectric constant, redox potential, pH, water activity, and magnetism. • Environmental analogues mimic the temperature, aridity, wind and radiation, or the con-
ditions existing on the early planets and moons in the Solar System. • Environmental analogues mimic the temperature, aridity, wind and radiation, or the con-
ditions existing on the early planets and moons in the Solar System. g
y p
y
• Physical analogues comprise thermo-physical (e.g. albedo and thermal inertia), mechan-
ical (e.g. bearing strength, cohesive strength, and the angle of internal friction), and bulk • Physical analogues comprise thermo-physical (e.g. albedo and thermal inertia), mechan-
ical (e.g. bearing strength, cohesive strength, and the angle of internal friction), and bulk Earth as a Tool for Astrobiology—A European Perspective 49 Fig. 1 Global map of the Earth with 33 analogue sites identified (adapted from Preston and Dartnell 2014). The map includes sites used by ESA state members and also the international community. 2.1 Planetary Field Analogues Sites Martins et al. 50 Fig. 2 Wright Valley, Dry Valleys, Antarctica Fig. 2 Wright Valley, Dry Valleys, Antarctica Acid-Saline Lakes of Western Australia
The acid-saline lakes of the Yilgarn Craton in
Western Australia were considered to have the most similar bulk physical and mineralogical
content to the Meridiani Planum on Mars (Grotzinger et al. 2005, Benison and Bowen 2006,
Bowen et al. 2008). In addition, microorganisms were found in the surface of the acid-saline
lakes (Hong et al. 2006; Mormile et al. 2007, 2009). Antarctic and Artic Ices
A diverse set of microorganisms has been detected in the ice-
sheet of the Lake Vostok in Antarctica (Priscu et al. 1999; Karl et al. 1999; Shtarkman et al. 2013). The Artic permafrost has also a high content of microorganisms (Jakosky et al. 2003;
Gilichinsky et al. 2003; Rivkina et al. 2000; Vorobyova et al. 1997; Vishnivetskaya et al. 2006), making both environments good analogues for the icy Moons Europa, Enceladus and
Ganymede. The Arctic ice may also be used as an analogue of Mars due to the observation
by the Phoenix Lander of the water ice-Martian surface interface (Smith 2009; Smith et al. 2009), the low water activity and limited organic content (Deming 2002; Vishnivetskaya
et al. 2006). Antarctic McMurdo Dry Valleys
The McMurdo Dry Valleys, with a surface area of
6861 km2 (Cary et al. 2010), are the largest ice-free area in Antarctica (Fig. 2) and the dri-
est cold place on Earth (Marchant et al. 1996). The precipitation, exclusively snow is often
less than 10 mm water-equivalent (weq) per year, ranging from 3 mm to less than 100 mm
in the location closer to the sea (Fountain et al. 2009). The Dry Valleys are located within
the Transantarctic Mountains in the Southern part of Victoria Land, close to the western
coast of McMurdo Sound (between 160°E and 164°E longitude and 76°S and 78°S lati-
tude). The winter air temperature fluctuates between −20 and −50°C (occasionally lower),
rising to mean daily values of about −15°C in the summer, up to 15°C or higher values at
ground surfaces. Surface soils seem sterile in large parts of the area, but rocks (Friedmann
1982), and permafrost (Gilichinsky et al. 2.1 Planetary Field Analogues Sites Identification is
as follow: Site 1—High-altitude atmosphere; 2—Yilgarn Craton (Western Australia); 3—Rio Tinto (Spain);
4—The Golden Deposit (Canadian High Arctic); 5—Yellowstone National Park (USA); 6—Haughton Im-
pact Structure (Devon Island); 7—Dongwanzi Ophiolite Complex (China); 8—Axel Heiberg Island (Cana-
dian High Arctic); 9—The Antarctic Dry Valleys (Antarctica); 10—Antarctic North Victoria Land Moun-
tains 11—Sub-glacial Volcanism (Iceland); 12—Kamchatka (Russian Federation); 13—Bockfjord Volcanic
Complex (Svalbard); 14—Kilimanjaro (Tanzania, Africa); 15—Atacama (South America); 16—The Mojave
Desert (USA); 17—Namib Desert (Namibia, Africa); 18—Ibn Battuta Centre Sites (Morocco); 19—Qaidam
Basin (Tibetan Plateau); 20—caves in Sardinia; 21—caves in Tenerife and Lanzarote; 22—Lake Vostok
(Antarctica); 23—Permafrost (Multiple Sites); 24—The Borup Fiord Pass (Ellesmere Island); 25—Lake
Tirez (Spain); 26—Orca Basin (Gulf of Mexico); 27—Mono Lake (California, USA); 28—The Dead Sea (Is-
rael); 29—Lost City (Mid-Atlantic Ridge); 30—The Mariana Trench (Pacific Ocean); 31—Lidy Hot Springs
(USA); 32—The Columbia River Basalts (USA); 33—Pitch Lake (Trinidad and Tobago); 34—Rancho La
Brea Tar Pits (California, USA); 35—Alaskan Oil Fields (USA); 36—Pilbara (north of Western Australia);
37—Barberton (South Africa). Number 1 broadly corresponds to high-altitude atmospheric analogues for
Venus, numbers 2 to 21 are analogue sites for Mars, numbers 22 to 32 are analogue site for Europa and Ence-
ladus, numbers 33 to 35 are analogue site for Titan, and numbers 36 to 37 are analogue sites for the early
Earth physical characteristics (e.g. particle size distribution, particle shape, density, and poros-
ity). An ideal analogue would mimic all the required compositional, electrochemical, physi-
cal and environmental properties of a specific solar system location. Note that some envi-
ronmental conditions cannot be found on Earth (e.g. high levels of unfiltered UV radiation,
altered gravity, different atmospheres/atmospheric composition, very low pressure) (Ret-
tberg et al. 2004; Prinn and Fegley 1987). While no present terrestrial analogue mimics
all properties, they play a crucial role in preparing for different aspects of future space mis-
sions. On the other hand, rocks from the early Earth record the corresponding environmental
conditions and are thus highly valuable samples. A summary of the representative locations studied as terrestrial analogue sites is present
in Fig. 1, and in the text below (Marlow et al. 2008, 2011; Preston and Dartnell 2014). Rep-
resentative terrestrial analogue sites used internationally are shown and supplemented by ter-
restrial analogues used by ESA-member states for astrobiological studies. Some are funded
by the EU (e.g. the CAFE study), while some are funded nationally (e.g. the GANOVEX-
expedition). Z. 2.1 Planetary Field Analogues Sites 2007) can be rich in microbial life; for instance,
soils collected in the upper part of the Dry Valleys, such as the University Valley at 1700 m
above sea level (mean annual air temperature −23°C, maximum −2.8°C), yielded only 51 Earth as a Tool for Astrobiology—A European Perspective 6 heterotrophic isolates on over 1000 agar plates in 2 years (Goordial et al. 2016). Microbial
diversity, investigated both by isolation cultural methods and by molecular analyses, resulted
much higher in milder sites (Cary et al. 2010). In particular, molecular tools demonstrated
a microbial diversity in soils higher than expected (Smith et al. 2006). Life in rocks is rich,
mainly represented by cryptoendolithic communities, thriving in air-spaces within porous
rocks (Friedmann 1982; Friedmann 1993), and biodiversity in cryptoendolithic communi-
ties has been studied also by molecular phylogenetic methods (de la Torre et al. 2003). Since
the NASA Viking Mars exploration in 1976, Antarctic Dry Valleys has been considered the
closest climatic terrestrial analogue of Mars (Horowitz et al. 1972; Wynn-Williams and Ed-
wards 2000). In fact, numerous similarities have been highlighted between the hyper-arid
cold desert Antarctic Dry Valleys microclimate zones and the latitudinal and local microcli-
mate zones observed on Mars (Marchant and Head 2007). Antarctic North Victoria Land Mountains
Like the Antarctic Dry Valleys, a number of
small valleys within the Antarctic Mountains of North Victoria Land are considered to be
Mars-analogue field sites. This is because of their isolated locations of about 100 to 300 km
away from the coastline where the mountains form barriers against the humid air streams
from the ocean. At altitudes of about 1500 to 2500 m high, the UV irradiation, very low
temperatures and the extreme dryness act on a variety of microorganisms, in particular on
lichens, microfungi and cyanobacteria (de Vera et al. 2014b; Meeßen et al. 2015). Since
2009 two Mars-analogue field campaigns were conducted in these areas during the German
Antarctic North Victoria Land Expeditions (GANOVEX) 10 (2009/2010) and GANOVEX
11 (2015/2016), by the German Aerospace Center (Deutsches Zentrum für Luft- und Raum-
fahrt e.V., DLR) in cooperation with the Federal Institute for Geosciences and Natural Re-
sources (Bundesanstalt für Geowissenschaften und Rohstoffe, BGR), and with the logistic
support of the Italian Antarctic Research Program (Programma Nazionale di Ricerche in
Antartide, PNRA). 2.1 Planetary Field Analogues Sites Results obtained from Mars simulation experiments with samples col-
lected in these habitats have shown that the organisms were able to retain water, survive
and even be active in a Mars-like environment (de Vera et al. 2014b; Jänchen et al. 2015;
Meeßen et al. 2015). Atacama Desert
The Yungay (Chile) and Arequipa (Peru) regions of the Atacama Desert
are the driest hot places on Earth, serving as a compositional Martian analogue because
of its sulphate and perchlorate mineralogy and low organic content (Catling et al. 2010;
Peeters et al. 2009; Buch et al. 2006; Amashukeli et al. 2007; Meunier et al. 2007; Skelley
et al. 2007). This is suggested to be the result of the highly oxidizing composition of the
Atacama Desert soil (Sutter et al. 2005; Ewing et al. 2006; Navarro-González et al. 2003;
Quinn et al. 2005; Lester et al. 2007). Deep Sea Vents
Extreme life forms live on Earth in the vicinity of deep sea hydrother-
mal vents, where no sunlight reaches, using, for example, sulphur compounds as source of
energy. In addition, lithoautotrophic methanogenesis (i.e. the conversion of CO2 and H2 to
methane) can be a source of metabolic energy for the production of biomass at hydrother-
mal systems (McCollom 1999). Similar environments might exist on Europa or Enceladus,
which have icy crusts and possibly liquid oceans beneath. In fact, magnesium sulphate salts
have been detected in Europa’s ice surface using the Keck II telescope, possibly providing
a sample of the ocean underneath (Brown and Hand 2013) and in the plumes of Enceladus
(Bouquet et al. 2015). 52 Z. Martins et al. Fig. 3 3.33 Ga-old volcanic sediments from the Barberton Greenstone Belt in South Africa, field view of
the terrain (left) and well-preserved tidal sediments (right) containing fossilized traces of early life, including
chemotrophs, lithified by hydrothermal, silica-rich fluids Fig. 3 3.33 Ga-old volcanic sediments from the Barberton Greenstone Belt in South Africa, field view of
the terrain (left) and well-preserved tidal sediments (right) containing fossilized traces of early life, including
chemotrophs, lithified by hydrothermal, silica-rich fluids Early Archaean Terrains
The early Earth represents an ideal environmental and micro-
bial analogue for early habitable bodies in the Solar System. The early Earth was a vol-
canically and hydrothermally active ocean planet, while Mars was basically a land-locked
volcanic planet with isolated pockets of habitability (Westall 2012; Westall et al. 2.1 Planetary Field Analogues Sites 2015a),
and the now icy moons (Enceladus, possibly Titan, Europa, and Callisto) of the outer So-
lar System could have been or are still similar. The reason for this is that, despite the great
distances of the various bodies from the early, fainter Sun, they all underwent melting, frac-
tionation and cooling. At a certain critical moment during their geological evolution, these
bodies would have had liquid water in contact with hot rock and hydrothermal edifices,
while there is indication that Enceladus has ongoing hydrothermal activity (Hsu et al. 2015)
and more recently, evidence of aqueous geysers has been observed on Europa (Roth et al. 2014). Hypothesizing that carbon-water-based life had a hydrothermal origin (Russell and
Hall 1997; Martin et al. 2008), life could have appeared on any of them, including Mars. Another important analogy is that the early Earth was anaerobic, as would have been (and
still are) the other bodies. Although tectonic recycling has destroyed terrestrial rocks of Hadean age (4.5–4.0 Ga)
that could have recorded the transition from prebiotic chemistry into biological chemistry,
there are two enclaves on Earth where well-preserved rocks dating back to 3.5 billion years
(Ga) are preserved. These locations are the Pilbara in Western Australia and the Barber-
ton Greenstone Belt in eastern South Africa (Fig. 3). Volcanoclastic sediments deposited
in shallow water—at depths similar to those of crater lakes on Mars—and hosting abun-
dant hydrothermal vents contain the remains of early, anaerobic life forms (Westall et al. 2011, 2015a, 2015b). Both chemotrophic and phototrophic colonies are preserved in the
strongly hydrothermally-influenced sediments. Thus, the local environmental conditions,
volcanic sediment type, hydrothermal environment, and the early life forms and their ex-
ceptional preservation, make the rocks of the Early Archaean terrains in the Pilbara and
Barberton ideal analogues for extra-terrestrial life, especially on Mars. On the other hand,
Deamer and colleague argue that the origin of life occurred in an evaporite pond in a pre-
biotic environment (Deamer 2014). According to these authors, in such geothermal setting
organic compounds would have interacted with mineral surfaces to promote self-assembly
and polymerization reactions. Lava Tubes and Caves
Lava tubes and caves are useful analogue sites since they pro-
vide both geological and biological data on subsurface environments. 2.1 Planetary Field Analogues Sites In particular, mi- Earth as a Tool for Astrobiology—A European Perspective 53 crobial metabolisms and biosignatures of organisms that persist independent of surface
photosynthesis such as methanogens and chemolithotrophs can be examined (Fernández-
Remolar et al. 2008). The deleterious surface of present-Mars has long since been rec-
ognized as a reason why habitable conditions, if they exist on Mars today, are likely
to be localized to the deep subsurface (Boston et al. 1992). Today, the largest quan-
tities of water are found in the subsurface (e.g. Holt et al. 2008). The specific inter-
est in lava tubes on Earth is motivated by their presence in volcanic, basaltic environ-
ments analogous to the geological context of lava tubes on Mars (Cushing et al. 2007;
Williams et al. 2010). On Mars, lava tubes provide a natural way to access the deep subsur-
face. As caves can potentially occur in any type of geological environment, understanding
their geology and biota provides a comprehensive understanding of near-surface habitabil-
ity of a diversity of planetary materials that can be applied to understanding extra-terrestrial
subsurface habitability (Abbey et al. 2005). Pitch Lake
Pitch Lake on the Caribbean island of Trinidad and Tobago may be used as an
analogue of the methane lakes of Titan (Schulze-Makuch et al. 2011). The Pitch Lake has
very low water activity, being filled with hot asphalt, and bubbling hydrocarbon gases (such
as methane). It has been shown that active microorganisms survive on its extreme conditions
(Schulze-Makuch et al. 2011). Rio Tinto
Rio Tinto in southern Spain is an analogue for rivers present on early Mars. It
is characterized by acidic headwaters (pH value of 2.3) due to oxidation of sulphide ore de-
posits in this region (Bigham and Nordstrom 2000; Fernández-Remolar et al. 2005), and also
a very high concentration of iron (Amils et al. 2007). Despite all these extreme conditions,
microbiological communities thrive. This makes Rio Tinto a good electrochemical and com-
positional analogue of early Mars, in particular to study the possible role of microorganisms
on the formation of iron oxide and sulphate deposits on the planet (Amils et al. 2007). Salten Skov
Salten Skov is a Martian analogue collected from Jutland (Denmark). It
contains high concentrations of iron oxides, and has been used for its similar bulk phys-
ical and magnetic properties to Mars (Gunnlaugsson et al. 2002; Merrison et al. 2004;
Nørnberg et al. 2004, 2008). 2.1 Planetary Field Analogues Sites Just like JSC Mars-1, Salten Skov has a high microbi-
ological and organic content, and therefore is not the best Mars soil analogue from
the organic point of view (Hansen et al. 2005; Garry et al. 2006; Peeters et al. 2009;
Chan et al. 2012). Utah Desert
The Utah Desert has a mineral composition similar to Mars (Borst et al. 2010), with sedimentary deposits of sands, evaporites, clays and gypsum (Kotler et al. 2011). The microbiological and organic content are very heterogeneous; amino acids range from
non-detectable to several thousand parts-per-billion (ppb), while an extraordinary variety of
putative extremophiles was observed (Ehrenfreund et al. 2011; Direito et al. 2011; Martins
et al. 2011). 3.1 Semi-permanent Field-Testing Bases Semi-permanent field testing campaigns with permanent infrastructure have been estab-
lished to carry out multidisciplinary research for mission and technology development and Z. Martins et al. 54 definition. Although not their core business, scientific campaigns can be carried here out
as well (Ansdell et al. 2011). Two of these sites are centered around underwater activi-
ties, the Aquarius Undersea Research Station at the Florida Keys, USA, a site to study
coral reefs (Todd and Reagan 2004) and the Pavilion Lake Research Project (PLRP) that
aims to explain the origin of freshwater microbiolites (Lim and Brady 2011). The Boulby
International Subsurface Astrobiology Laboratory (BISAL, UK) is the world’s first per-
manent subsurface astrobiology laboratory. It explores instrument testing for robotic and
human planetary missions, as well as deep subsurface evaporate geochemistry and bi-
ology (Cockell et al. 2013; Payler et al. 2017). Most of the sites focus on developing
Mars exploration scenarios, sometimes combined with Moon activities. These include the
Haughton Mars Project (HMP) Research Station at Devon Island, Canada (Lee and Os-
inski 2005, http://marsonearth.org/), the McGill Arctic Research Station (MARS) at Axel
Heiberg Island (Pollard et al. 2009), the Pacific International Space Center for Explo-
ration Systems (PISCES) at Hawaii (Schowengerdt et al. 2007; Duke et al. 2007), and
the Ibn Battuta Centre for exploration and field activities in Morocco, established in 2006
http://www.ibnbattutacentre.org/). 3.2 Long-Term Field-Testing Campaigns Long-term field testing campaigns are used to test for planetary instruments and mis-
sions (Ansdell et al. 2011). Examples include the Arctic Mars Analog Svalbard Expe-
dition (AMASE), the Desert Research and Technology Studies (Desert RATS), and the
NASA Extreme Environments Mission Operations (NEEMO) campaigns. The AMASE is
a campaign focused on science and technology, in which technology is tested in support
of the science, and it took place on Svalbard, Norway (Steele et al. 2007). The Desert
RATS field-testing campaigns support future manned mission scenarios, including technol-
ogy related testing (Ross et al. 2013) (http://www.nasa.gov/exploration/analogs/desertrats/). They have taken place in various locations in the United States, including Mauna Kea
(Hawaii) (ten Kate et al. 2013), and Black Point Lava Flow (Arizona), which were se-
lected based on their physical resemblance to the lunar and Martian surface. The scope of
these campaigns includes testing single space suit configurations and multi-day integrated
mission scenarios (Ross et al. 2013), as well as integrating science into the technology-
driven scenarios and communication between scientists and engineers (Yingst et al. 2015). The NEEMO campaigns are technology and mission oriented, and use the Aquarius Sta-
tion, which provide a convincing analogue for space exploration (Thirsk et al. 2007)
(http://www.nasa.gov/mission_pages/NEEMO/). Long-term field testing campaigns are used to test for planetary instruments and mis-
sions (Ansdell et al. 2011). Examples include the Arctic Mars Analog Svalbard Expe-
dition (AMASE), the Desert Research and Technology Studies (Desert RATS), and the
NASA Extreme Environments Mission Operations (NEEMO) campaigns. The AMASE is
a campaign focused on science and technology, in which technology is tested in support
of the science, and it took place on Svalbard, Norway (Steele et al. 2007). The Desert
RATS field-testing campaigns support future manned mission scenarios, including technol-
ogy related testing (Ross et al. 2013) (http://www.nasa.gov/exploration/analogs/desertrats/). They have taken place in various locations in the United States, including Mauna Kea
(Hawaii) (ten Kate et al. 2013), and Black Point Lava Flow (Arizona), which were se-
lected based on their physical resemblance to the lunar and Martian surface. The scope of
these campaigns includes testing single space suit configurations and multi-day integrated
mission scenarios (Ross et al. 2013), as well as integrating science into the technology-
driven scenarios and communication between scientists and engineers (Yingst et al. 2015). 3.2 Long-Term Field-Testing Campaigns The NEEMO campaigns are technology and mission oriented, and use the Aquarius Sta-
tion, which provide a convincing analogue for space exploration (Thirsk et al. 2007)
(http://www.nasa.gov/mission_pages/NEEMO/). Field research at Mars analogue sites, such as desert environments can provide impor-
tant constraints for instrument calibration and landing site strategies of robotic exploration
missions to Mars that will investigate habitability and life beyond Earth during the next
decade. Terrestrial analogue studies are used to better understand the nature, the process and
the utilization of geological processes and resource utilization on Earth in order to interpret
and validate information from orbiting or surface missions on extra-terrestrial bodies (Fo-
ing et al. 2011). In this analogue context, all the mission aspects are simulated to perform
fieldwork as a test of human factors procedures. The Mars Desert Research Station (MDRS)
in southern Utah (USA) is an analogue site where human space mission simulations are
performed in order to investigate all possible factors that may interact to affect mission suc-
cess, such as human factors and geological in-situ resource exploration (Schlacht et al. 2010;
Direito et al. 2011; Ehrenfreund et al. 2011; Foing et al. 2011, 2013, 2014; Kotler et al. 2011;
Martins et al. 2011; Orzechowska et al. 2011; Thiel et al. 2011). Every two weeks, exchange
crews of six members come to the station to perform a new mission to establish the knowl- Earth as a Tool for Astrobiology—A European Perspective 55 edge of, and to test the equipment necessary for future successful planetary exploration,
also from a human factors perspective. An illustration of the kind of scientific results that
were obtained includes data relevant for habitability and astrobiology. Astrobiology field
research from the MDRS was conducted during the EuroGeoMars 2009 campaign. Euro-
GeoMars 2009 was an example of a Moon-Mars field research campaign dedicated to the
demonstration of astrobiology instruments and a specific methodology of comprehensive
measurements from selected sampling sites (Foing et al. 2011). Special emphasis was given
to sample collection and pre-screening using in-situ portable instruments. A comprehensive
analysis was applied to a set of selected samples from different geological formations includ-
ing Mancos Shale, Morrison, and Dakota Formation as well as a variety of locations (sur-
face, subsurface and cliffs) partly in-situ in the habitat or in a post-analysis cycle. 3.2 Long-Term Field-Testing Campaigns Individ-
ual technical laboratory analysis studies were compiled and correlations were investigated
of environmental parameters, minerals, organic markers and biota. The results were inter-
preted in the context of future missions that target the identification of organic molecules and
biomarkers on Mars. Results showed that the Utah desert has a mineral composition similar
to Mars (Borst et al. 2010), with sedimentary deposits of sands, evaporites, clays and gypsum
(Kotler et al. 2011). The microbiological and organic content are very heterogeneous, with
amino acids ranging from non-detectable to several thousand parts-per-billion (ppb), while
an extraordinary variety of putative extremophiles was observed (Ehrenfreund et al. 2011;
Direito et al. 2011; Martins et al. 2011). Several field campaigns (e.g. EuroGeoMars2009 and Drilling on the Moon and Mars in
Human Exploration/International Lunar Exploration Working Group (DOMMEX/ILEWG)
Euro-MoonMars) have been conducted at the MDRS. Most of the information we have
from the surface of the Moon and Mars comes from satellite observations. Satellite images
of high resolution are very important to achieve a successful selection of landing sites and
planning of traverses on unfamiliar planetary sites. In order to help in the interpretation of
Mars missions’ measurements from orbit (Mars Express, MRO) or from the surface (MSL
Curiosity) at Gale crater, several field research campaigns (e.g. ILEWG EuroMoonMars)
were performed in the extreme conditions of the Utah desert. These provided data analysis
relevant to Mars geology at multiple scales. The goal of ILEWG EuroMoonMars project
(2013) was to conduct field studies to identify environments analogous to those that Cu-
riosity has been studying at Gale crater. Therefore, a comparative study was made between
satellite images with a spatial resolution of 50–60 cm per pixel. This is comparable to the
resolution of MRO HiRise on Mars. Traverses at MDRS similar to geomorphological fea-
tures seen at the Gale crater were planned as possible and implemented using a rover, drone
and Extravehicular Activity (EVA) simulation walks, before taking rocks and soil samples
(Figs. 4 and 5). The usability of a drone for imaging reconnaissance was tested, and expe-
riences and lessons learnt were assessed concerning geological traverse planning based on
high resolution satellite images (Foing et al. 2014). Fig. 4 Map of geological
features and inverted riverbeds
around MDRS, Utah showing
areas explored by traverses and
EVAs, and through samples
(Foing et al. 2014) 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities Planetary and Low Earth Orbit (LEO) space simulation facilities are designed to reproduce
a wide range of conditions of space, as the ones accessible in LEO (i.e. on satellites or the Z. Martins et al. 56 Fig. 4 Map of geological
features and inverted riverbeds
around MDRS, Utah showing
areas explored by traverses and
EVAs, and through samples
(Foing et al. 2014)
Fig. 5 (Left) Concretions in Utah Brushy Basin Member of the Morrison Formation within a cross-bedded
sandstone, compared to (right) similar concretions, cross-bedding and veins apparent in Curiosity camera
color images (Foing et al. 2014) Fig. 5 (Left) Concretions in Utah Brushy Basin Member of the Morrison Formation within a cross-bedded
sandstone, compared to (right) similar concretions, cross-bedding and veins apparent in Curiosity camera
color images (Foing et al. 2014) Fig. 5 (Left) Concretions in Utah Brushy Basin Member of the Morrison Formation within a cross-bedded
sandstone, compared to (right) similar concretions, cross-bedding and veins apparent in Curiosity camera
color images (Foing et al. 2014) International Space Station (ISS)), or of a specific planet or moon, including vacuum or the
relevant atmospheric pressure and composition, UV radiation, and (surface) temperature. Laboratory planetary analogues allow exposure to conditions which cannot be found in ana-
logue field sites. Indeed, they provide the means to investigate the effect of extra-terrestrial
environmental parameter on biological, chemical and material samples in the laboratory,
and the basis for space experiments including the selection of best landing areas for possi-
ble life detection. All planetary and LEO space simulation facilities are technically limited. Nevertheless, according to their individual focus, they provide valuable research opportuni-
ties with the advantage of fast and easy access and capacity for more or bigger experiments
when compared to space research facilities. Simulation facilities at the various locations are usually divided into those used for or-
ganic chemistry experiments only, and those that are also used for biological experiments or Earth as a Tool for Astrobiology—A European Perspective 57 hardware tests. The required simulated environmental parameters of low temperatures, low
pressure and short wavelength UV are of interest for biological and chemical experiments
and hardware tests. In contrast, cleanliness of the facility, in particular with respect to or-
ganic compounds is of great importance to organic chemical experiments and—depending
on its destination—for final flight hardware. 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities Cleanliness with respect to organisms is ab-
solutely necessary for biological experiments, but with the insertion of biology into any
facility, cleanliness of the facility with respect to organic compounds can no longer be in-
sured. Therefore, experiments with biological material as test objects often are not consistent
with the performance of organic chemical experiments in the same facilities. Exceptions are
compartmentalizations and separation of the experiments, as in the EXPOSE ground simu-
lations. For most biological exposure experiments, the required conditions at a sample site
are (i) simulated temperature ranges from at least −25°C up to a maximum of 120°C, as
this is the range where life can exist in a dormant, viable or reproductive and metabol-
ically active state (Price and Sowers 2004; Junge et al. 2006; Kashefiand Lovley 2003;
Schwartzman and Lineweaver 2004), (ii) irradiation with different spectral ranges, in partic-
ular with respect to the short wavelength cut off, (iii) high or low pressure regimes including
vacuum as in LEO, (iv) high or low relative humidity and (v) different atmospheric com-
positions. Damage induction, repair capacities, adaptation and adaptation strategies, hence
the overall influence on cell functions of the applied parameter or combinations, are investi-
gated for different species. Passive exposure experiments performed in these chambers only
allow the comparison of samples before and after the simulation experiments to evaluate the
effect of the applied parameters. In this case, biological samples are prepared from inactive
forms of selected organisms, i.e. spores, cysts, or organisms in hibernation mode, where
metabolism, growth or any other vitality parameters are not detectable with current meth-
ods. After exposure, the revival of the organisms and their metabolic resumption and growth
is analyzed in comparison with the pre-exposure base data to better understand the effect of
the individual applied parameter. Molecular analysis e.g. of DNA damages and repair mech-
anisms increase our knowledge on the underlying reactions. In particular, organisms that are
derived from extreme terrestrial environments are of high interest to better understand their
adaptation strategies. They are selected from terrestrial environments that differ from stan-
dardized lab conditions, i.e. 1013.25 hPa, 293.15 K (20°C) and the terrestrial atmospheric
composition of about 78% N2 and 20% O2, often from the above presented analogues. Wa-
ter availability is the major necessity for active life as we know it from Earth. 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities Wherever
water is available in the simulated extra-terrestrial environment, active instruments that de-
tect metabolic activity or growth during the exposure experiment complete the simulation
facility design. Currently, one of the main astrobiological focuses is the question of extinct or extant
life and corresponding signatures on Mars. The possible transport of viable organisms to
Mars, accidentally by spacecraft and their survival capacities are of interest for Planetary
Protection means. Indeed, the prevention of any contamination of Mars by Earth organisms
is the major topic of Planetary Protection regulations. Therefore, most simulation facilities
have been designed to simulate either LEO space or Mars surface conditions. The aim of
LEO space simulation facilities is to approach as much as possible LEO pressure of down to
10−7 Pa and, in particular the short wavelength solar UV and vacuum UV (VUV) radiation
non-attenuated by an Earth ozone layer. For Mars, simulation temperatures between 198 K
(−75°C) and 293 K (20° C) are applied (de Vera et al. 2010, 2014a, 2014b; Krala et al. 2011;
Schuerger et al. 2013; Martin and Cockell 2015), approaching temperatures observed in
equatorial to mid-latitudes on Mars (McEwen et al. 2011; Head et al. 2003). In parallel, Z. Martins et al. 58 Fig. 6 A. MOMIE (Image credit LISA). B. PASC (Mateo-Martí et al. 2006). C. The Open University Mars
Simulation Facility (http://www3.open.ac.uk/events/201653_35210.jpg). D. MESCH (Jensen et al. 2008). E. PELS (Martin and Cockell 2015). F. PALLAS (ten Kate and Reuver 2015; Beysens and van Loon 2015) Fig. 6 A. MOMIE (Image credit LISA). B. PASC (Mateo-Martí et al. 2006). C. The Open University Mars
Simulation Facility (http://www3.open.ac.uk/events/201653_35210.jpg). D. MESCH (Jensen et al. 2008). E. PELS (Martin and Cockell 2015). F. PALLAS (ten Kate and Reuver 2015; Beysens and van Loon 2015) atmospheric pressure of about 600 to 800 Pa with a Mars-like atmospheric composition of
about 95% CO2, 2.7% N2, and 1% O2 and traces of water is obtained. The need of adequate
radiation spectra approximating the solar UV regimes is mainly achieved using specific
solar UV lamps (Rabbow et al. 2012; Lorek and Koncz 2013; de Vera et al. 2014a, 2014b). A special type of Mars simulation facility are wind tunnels for the simulation of Martian dust
devils and dust storms to investigate correlated effects, in particular on landers and rovers
hardware, but also for a better understanding of their effect on the Martian environment
itself. 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities Several laboratory setups have been devoted to the study of the evolution of potential bi-
ological records (mainly organic matter such amino acids, carboxylic acids, polycyclic aro-
matic hydrocarbons, nucleobases, etc.) under simulated Martian surface conditions (Fig. 6). Initially, these experiments were focused on the impact of UV radiation and oxidation pro-
cesses. Table 1 highlights some key parameters of some laboratory simulations submitting
pure or mixed organic molecules to simulated Martian UV sources (Hintze et al. 2010;
Johnson and Pratt 2010; Oro and Holzer 1979; Poch et al. 2013, 2014, 2015; Schuerger
et al. 2008; Stalport et al. 2008, 2009, 2010; Stoker and Bullock 1997; ten Kate et al. 2005, 2006). Among these parameters, the use of a Xenon arc lamp as the radiation source
has proven to better reproduce the energy and relative abundance of the UV photons (190
to 400 nm) supposed to reach the surface of Mars (Schuerger et al. 2003). Moreover, tem-
perature and pressure should also be representative of the Martian ones for several rea-
sons: First, because temperature can influence the kinetics of the chemical reactions oc-
curring in the simulation reactor (ten Kate et al. 2006), as pressure may also do (Keppler
et al. 2012). Second, because the chemical stability and physical state (solid or gas) of the
products is largely dependent of these parameters. Thus, when possible, analysis should
be made in situ during the simulation at constant environmental conditions in order to re-
trieve information on both the kinetics of transformation and the nature of the products. With some of these setups, the influence of a mineral matrix has also been investigated
such as in clays (montmorillonite, saponite, nontronite) (Poch et al. 2015; dos Santos et al. 2016), in sulfates (gypsum, jarosite) (dos Santos et al. 2016), in minerals containing ferrous Earth as a Tool for Astrobiology—A European Perspective 59 Table 1 Laboratory simulations of the evolution of pure or mixed organic molecules under simulated Mars
surface UV radiation. The data of the MOMIE experiment are described in Poch et al. (2013, 2014 and 2015)
Reference
Sample
phase
Temperature
(°C)
Pressure
(mbar)
Irradiation
source
Oxidant(s)
In situ
analysis
Oro and Holzer
(1979)
Solid
−10 to 25
0.001 (N2)
Mercury
lamp
Dioxygen
gas
No
Stoker and
Bullock (1997)
Solid
R.T. 100
(CO2+)
Xenon lamp
Yes
ten Kate et al. (2006)
Solid
−63 and
R.T. 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities 10−7 and 7
(CO2)
Hydrogen
and
deuterium
lamps
Yes
Schuerger et al. (2008)
Solid
−80, −10
and +20
7.1 (CO2+)
Xenon lamp
No
Stalport et al. (2009)
Solid
−54
0.01 (N2)
Xenon lamp
No
Johnson and
Pratt (2010)
Aqueous
solution
−135 to +40
7 to 15
(CO2+)
Xenon lamp
Iron
sulfates
No
Hintze et al. (2010)
Solid
−10 and
R.T. 6.9 (CO2+)
Xenon lamp
No
Shkrob et al. (2010)
Aqueous
solution
−196
1000 (N2)
Nd:YAG
pulsed laser
Goethite,
hematite,
anatase
Yes
MOMIE setup
Solid
−55
6(N2)
Xenon lamp
Mineral
Yes Table 1 Laboratory simulations of the evolution of pure or mixed organic molecules under simulated Mars
surface UV radiation. The data of the MOMIE experiment are described in Poch et al. (2013, 2014 and 2015) Table 1 Laboratory simulations of the evolution of pure or mixed organic molecules under simulated Mars
surface UV radiation. The data of the MOMIE experiment are described in Poch et al. (2013, 2014 and 2015) iron (augite, enstatite and basaltic lava) (dos Santos et al. 2016), and in the Mars soil ana-
logues JSC-Mars 1 (Garry et al. 2006; Stalport et al. 2010), Salten Skov (Garry et al. 2006;
Peeters et al. 2009), and Atacama (Peeters et al. 2009). Note that the source for JSC Mars-1
is the ash from the Pu’u Nene cinder cone on Hawaii. Analyzed at the Johnson Space
Center, it mimics the composition and physical properties of Mars (Allen et al. 1998a;
Allen et al. 1998b; Perko et al. 2006). However, the very high organic content of JSC Mars-
1 precludes it as a good analogue regarding organic material (Garry et al. 2006). Finally,
some studies have investigated the influence of energetic particles on the evolution of or-
ganics at the surface of Mars (Kminek and Bada 2006; Gerakines and Hudson 2013; Mate
et al. 2015). The results show that this type of radiation can cause the degradation of simple
organic molecules (glycine) on a timescale of hundreds of millions of years. On the other
hand, UV radiation acts on a much shorter timescale (several days to months) (see for ex-
ample Poch et al. 2014). Furthermore, higher-energy radiation can penetrate deeper into the
soil (up to around 2 m) than UV radiation (up to a few microns or millimeters), potentially
leading to inactive bacteria or spores due to accumulating radiation damage (Dartnell et al. 2007). 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities A large number of simulation facilities (>50) worldwide are capable of mimicking gen-
eral planetary surfaces and space conditions to some extent. Only a limited number of the
available simulation facilities in Europe is listed below to show typical examples of the var-
ious approaches to simulating space and planetary environments in the laboratory. Facilities
restricted to abiotic (non-biological) simulations only are indicated. Three sub-sections are
also included to describe specific types of simulation facilities: 4.1.1 biologically relevant
accessories for in situ analysis in simulation facilities; 4.1.2 Mars wind tunnels; and 4.1.3
simulation facilities for organic aerosols in planetary atmospheres. Z. Martins et al. 60 Mars Organic Molecule Irradiation and Evolution (MOMIE), LISA, Paris, France
The MOMIE experimental set-up allows investigators to simulate the in-situ Mars-like UV
irradiation (Fig. 6A; Poch et al. 2013). It uses Fourier transform infrared (FTIR) spec-
troscopy to monitor the sample, at a temperature (2–218 K) and pressure (1–6 mbar) repre-
sentative of the mean conditions at the Martian surface. The setup is composed of a reactor
(Fig. 6A—section A) housing the sample (on an MgF2 window, in green) which is main-
tained at 6 mbar, thanks to gas circulation system (Fig. 6A—section B), and 218 K using a
cryothermostat (Fig. 6A—section C). The irradiation source is a Xenon arc lamp (Fig. 6A—
section D), and a mobile mirror enables to switch between irradiation or analysis phases via
FT-IR spectroscopy (Fig. 6A—section E). A glove compartment (Fig. 6A—section F) filled
with N2 contains the entire device. The work in this chamber has focused on photochemistry
of organic molecules and the effect of the mineral matrix (Stalport et al. 2008; Poch et al. 2013, 2014, 2015) The Planetary Atmospheres Simulation Chamber (PASC), The Centre for Astrobiol-
ogy Research (CAB), Madrid, Spain
The PASC reproduces atmospheric compositions
and surface temperatures for most planetary objects (Fig. 6B; Mateo-Martí et al. 2006). Temperatures can be set from −269 to 52°C, pressures from 5 to 5 × 10−9 mbar. A residual
gas analyzer regulates the required atmospheric composition with ppm precision, while a
combination of a deuterium lamp and a noble-gas discharge lamp provides the required UV
spectrum. PASC has already been used to study the UV-absorbing properties of a Martian
analogue (basaltic dust) as a function of its thickness and mass (Muñoz-Caro et al. 2006),
the stability of saline water under Mars conditions (Zorzano et al. 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities The facility, containing six separate sample vessels, allows for statis-
tical replication of samples. Control of pressure, gas composition, UV irradiation conditions
and temperature allows for the precise replication of aqueous conditions, including sub-
zero brines under Martian atmospheric conditions. A sample acquisition system allows for
the collection of both liquid and solid samples from within the chamber without breaking
the atmospheric conditions, enabling detailed studies of the geochemical evolution and ge-
omicrobiological habitability of past and present extra-terrestrial environments (Martin and
Cockell 2015). Planetary Analogues Laboratory for Light, Atmosphere, and Surface Simulations
(PALLAS), Utrecht University, The Netherlands
PALLAS is a planetary surface simu-
lation facility at Utrecht University (Fig. 6F). Variable parameters include the atmospheric
composition and pressure (10−7–900 mbar), sample temperature (−80 to +60°C) and so-
lar (UV-Visible) spectrum. The UV spectrum is generated with a combination of a Xenon
solar simulator equipped with a water filter to remove residual heat (250–900 nm) and a
deuterium light source (160–400 nm). Gases can be pre-mixed or mixed within the chamber
to obtain a range of atmospheric conditions. Up to ten samples can be placed, either in the
beam spot of the UV source or in the dark (as control) on a table that can be cooled to −80°C
using a cooling bath. A quadrupole mass spectrometer is mounted onto the chamber, via a
differentially pumped sampling volume, to sample and analyze the atmosphere during the
experiments (ten Kate and Reuver 2015). Planetary and Space Simulation Facilities at the DLR, Germany
There are three plan-
etary simulation chambers in the Institute of Planetary Research at DLR Berlin which are op-
erated by the Spectroscopy Laboratory Group and the Astrobiological Laboratories Group. One is a vacuum chamber (40 × 30 × 30 cm) which simulates conditions on Venus and
Mercury. Samples can reach 500°C and beyond, while keeping the rest of the chamber rela-
tively cold (Maturilli et al. 2010). The second chamber is a Mars Simulation Facility (MSF). The MSF laboratory consists of a cold chamber with a cooled volume of 80 × 60 × 50 cm. The effective operational experimental chamber, which is cooled within the cold chamber,
is a cylinder with inner diameters of 20.1 × 32.4 cm. This chamber operates at 6 mbar
CO2 pressure at −75°C. 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities 2009), the survival of
chemolithoautotrophic bacteria and resistance of the lichen Circinaria gyrosa exposed to
simulated Mars conditions (Gomez et al. 2010; Sánchez Iñigo et al. 2012), and the photo-
stability of nucleobases and peptide biomolecules under UV irradiation (Mateo-Martí et al. 2009; Mateo-Martí and Pradier 2013). Mars Simulation Facilities, The Open University (OU), UK
Two chambers comprise
the Mars simulation facilities at the Open University. The large chamber provides pressure
and temperature conditions representative of the surface of Mars (Fig. 6C). This chamber
has the capability to incorporate large-scale regolith experiments. The small Mars cham-
ber has a solar illumination facility designed for instrument qualification and astrobiology
experiments, which allows an automated variation of the environment (e.g. thermal diur-
nal cycling; Patel et al. 2010). These simulation chambers have been used, for example
to study the degradation of microbial fluorescence biosignatures by solar ultraviolet radi-
ation on Mars (Dartnell and Patel 2013), the influence of mineralogy on the preservation
of amino acids under simulated Mars conditions (dos Santos et al. 2016), and the potential
of interlayer regions of silicate sheets as a favorable habitat for endolithic microorganisms
(Kapitulˇcinová et al. 2015). Mars Environmental Simulation Chamber (MESCH), Aarhus University, Denmark
MESCH is a double-walled cylindrical Mars simulation facility comprised of a cryogenic
environmental chamber, an atmospheric gas analyzer, and a xenon/mercury discharge source
to generate UV radiation (Fig. 6D). The double wall functions as a cooling mantle through
circulation of liquid N2. A carousel controlled by an external motor holds up to 10 samples. Exchange of the samples without changing the environment of the chamber is achieved
due to a load-lock system, which has a small pressure-exchange chamber. Data such as
temperature, pressure, and UV exposure time are used in automated feedback mechanisms,
allowing a wide variety of experiments (Jensen et al. 2008). 61 Earth as a Tool for Astrobiology—A European Perspective Planetary Environmental Liquid Simulator (PELS), University of Edinburgh, UK
The PELS chamber at the University of Edinburgh is designed for examining extra-terrestrial
aqueous environmental conditions (Fig. 6F). The front door has a glass view port in the cen-
ter, so samples and manipulations can be easily viewed. For liquid input/output, vacuum
flanges have been modified with bored-through steel bulkheads, through which polyvinyli-
dene fluoride (PVDF) tubing has been attached to give a corrosion-resistant wetted flow
path and vacuum seal. 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities The solar spectra range as it appears on Mars can be simulated by
the use of a Xe-lamp and the applied above mentioned environmental parameters as well
as the humidity can be varied according to the programed daily cycles. It can be used in
non-biological simulation experiments (in different disciplines such as planetary geology,
astrobiology, environmental chemistry and materials science and sensor testing), and for bi-
ologically relevant experiments by using hardware, which performs in situ measurements
during the simulation experiments (de Vera et al. 2010, 2014a, 2014b see Sect. 4.1.1 of the
present manuscript). The third simulation chamber is a micro-version of the second simula-
tion facility, which can be connected to a cryostate and can even simulate the temperature
conditions of the icy moons and deep space (down to 4 K). It is adapted to be placed under
a microscope and Raman spectroscope, allowing measurements under simulated planetary
conditions testing the influence of environmental conditions on the measurement operations. 62 Z. Martins et al. The use of this facility will be of significant support for the approved future ESA BioSigN
exposure experiment in LEO. By using ocean and deep sea material it will simulate and ap-
proach the conditions which space exposed plume ejecta of the icy moons might face to. In
parallel it will investigate the degree of biosignature detection by spectroscopy under these
extreme icy moon-related space conditions. The astrobiology group of the Radiation Biology Department at the DLR Institute of
Aerospace Medicine operates a total of seven modular Planetary and Space Simulation Fa-
cilities (PSI) in Cologne. A full description of the facilities, their operational parameters
and space experiments performed in these facilities was published by Rabbow et al. (2016)
(http://www.dlr.de/spacesim). The facilities differ with respect to size, provide simulated
space vacuum or planetary atmospheres, are temperature controlled and are equipped with
optical windows and a variety of solar simulators for UV-irradiation. The facilities were
repeatedly used for space mission preparation and reference experiments. 9 Space environment simulation is realized by combining vacuum, down to 10−9 Pa with
Ion getter pumps are connected (“Deep Space” facilities PSI 3, 6 and 9), temperature oscil-
lation at sample site and short wavelength UV irradiation as experienced outside the Earth
Ozone layer. This setup complemented space mission experiments, as e.g. 4.1.1 Biologically Relevant Accessories for “in situ” Analysis in Simulation Facilities 4.1.1 Biologically Relevant Accessories for “in situ” Analysis in Simulation Facilities In analogy to the recommendations in “Space as tool for astrobiology” for advanced astrobi-
ological space hardware (Cottin et al. 2017), in situ measurements during ground based sim-
ulation experiments are of prime importance. The activity of tested samples can be measured
in situ by specific sensor hardware applied during the simulation experiments in addition to
the sensors, which are measuring the environmental conditions during the simulation exper-
iments. For example, the degree of photosynthetic activity can be monitored and recorded
during simulation conditions by a planetary and Mars adapted measurement device named
Mini-PAM (Walz 1999; Lorek and Koncz 2013, de Vera et al. 2010, 2014a, 2014b). Methane
production of methanogens can be observed during Mars simulation experiments due to the
use of specific optical hardware like laser spectroscopy and gives indirectly information on
growth rates of methanogens during simulation experiments (Schirmack et al. 2014). Bio-
logical activity during the simulation experiments may be observed by many other ways:
(i) pH-changing of soil where bio-samples are embedded, (ii) camera based observations
of bleaching effects of soil and rocks and (iii) use of isotopes in atmospheric gases and
use of isotope detecting hardware for check of consumption or production of gases due to
biological activity, or optical density variations measured by photometers. 4.1 Planetary and Low Earth Orbit (LEO) Space Simulation Facilities on the Long Du-
ration Exposure Facility (LDEF) from April 7th 1984 to January 11th 1990, in ERA on
EURECA from August 2nd 1992 to June 24th 1993 and several Biopan experiments on
Foton satellites. Material tests were performed in the big sized facilities, which can be used for Life
Science and Human Flight operations and also for Astrobiology tasks. They are connected
to computer controlled temperature regulation, solar simulators and providing space vacuum
or Mars atmosphere with respect to pressure and gas composition. They performed the space
experiment pre-flight test and mission parallel ground experiments for all three long-term
ESA multiuser exposure facilities EXPOSE-E from February 7th 2008 to September 12th
2009, EXPOSE-R from November 26th 2008 to March 9th 2011, and EXPOSE-R2 from
July 23rd 2014 to June 5th 2016. While simulation of space and Mars surface environment was the main focus in the past
and is adapted for anaerobic organisms in preparation of the ESA MEXEM experiment, the
facilities are upgraded now also for the simulation of the icy moon Europa ocean conditions
in preparation of the ESA IceCold active exposure experiment featuring in situ life measure-
ments. Recently, the stationary vacuum facilities were complemented by small transport and
exposure boxes (TRex-Box) and the option for additional ionizing irradiation in an x-ray fa-
cility under defined vacuum or planetary atmospheric conditions. These past and current ground based experiments performed in simulation facilities can-
not substitute space experiments that use space as a tool for astrobiology to investigate the
effect of the unique combination of environmental parameters on life. Simulation of space
parameters and their combination are technically limited. In particular, short UV with wave-
length <200 nm and the complex ionizing radiation field of space can only be approximately
simulated. But in contrast to space, Earth bound facilities used as tools for astrobiological in-
vestigations provide easy and fast access and more experimental space, an important feature
in particular for the preparation of astrobiological space experiments. In order to allow the exposure of a wide variety of scientifically interesting biological
organisms, the complete DLR laboratory housing the simulation facilities is announced
as Genetic laboratory safety level 1 for the use of genetically modified organisms, and as
Biosafety level 2 laboratory according to German law. Earth as a Tool for Astrobiology—A European Perspective 63 4.1.3 Simulation Facilities for Organic Aerosols in Planetary Atmospheres Experimental setups are developed in planetary science to simulate the production of organic
aerosols in planetary atmospheres. These laboratory analogues are often named “tholins” in
the literature since the seminal work by Sagan and Khare in 1979 (Sagan and Khare 1979). The abundance, the prebiotic interest as extra-terrestrial nitrogen-rich organic matter, and
the poor knowledge of their chemical composition make Titan’s atmospheric aerosols one
of the most studied objects through laboratory simulation. However, tholins have also been
produced to investigate the formation and/or evolution of aerosols in other atmospheres such
as the early Earth’s atmosphere (Trainer et al. 2004), the Jovian atmosphere (McDonald et al. 1991; Sagan et al. 1993), and Triton’s atmosphere (McDonald et al. 1994). The main principle of these experiments is to couple an energy source to a gas mix-
ture composed of the main atmospheric components in order to mimic the initiation of
the complex chemical networks taking place in the given atmosphere and leading to the
production of organic aerosols. The energy sources used in the laboratory are proxies
for the solar irradiation and the precipitation of magnetospheric electrons. Those natu-
ral sources are reproduced in the laboratory by UV photon fluxes (Carrasco et al. 2014;
Imanaka and Smith 2010; Trainer et al. 2012; Tran et al. 2008; Trainer et al. 2006)
and/or by plasma discharges (Somogyi et al. 2005; Coll et al. 1999; Horvath et al. 2009;
Imanaka et al. 2004; Sciamma-O’Brien et al. 2010; Hörst and Tolbert 2013). Pressure and
temperature are strongly dependent on the experiments, and can hardly reproduce the whole
range and extreme conditions occurring in real atmospheres. As developed in (Carrasco et al. 2013a, 2013b), wall effects can be encountered at low pressure conditions, when the mean
free path exceeds the typical dimensions of the photo-reactor. Considering these constraints,
it is important to propose and develop various and complementary experiments. The larger
the set of conditions is explored, the better our knowledge on the sensitivity enables to ex-
trapolate the analogues properties to the atmospheric aerosols properties. A complementary
approach consists in targeting an organic growth mechanism by initiating the polymerization
of a specific molecule, minor in the atmospheric composition but expected as a possible pre-
cursor of the aerosol nucleation process. Thus, tholins of interest for Titan were synthesized
by polymerization of HCN (Vuitton et al. 2010) and C4N2 molecules (Couturier-Tamburelli
et al. 2014). 4.1.2 Mars Wind Tunnels To do so, a re-circulating wind tunnel is housed
within an environmental chamber that operates at the atmospheric pressure (6–10 mbar),
composition and temperature of Mars. An axially mounted fan driven by an electric motor
draws gas down the central wind tunnel and returns it in an outer cylindrical cavity. This
design has been chosen to maximize the available open wind tunnel area (cross section)
while maintaining smooth fluid flow given the limited environmental chamber dimensions. It is of importance when utilizing the recirculating system that the area of the return flow is
comparable (if not greater than) to that of the wind tunnel in order to minimize turbulence
and irregularities in the wind stream. Fins are used to minimize rotation effects on the wind
by the fan (Merrison et al. 2002, 2004, 2007, 2008, 2010, 2012). The Aarhus Wind Tunnel Simulator (AWTS), University of Aarhus, Denmark
The
AWTS facility was specifically built to reproduce wind conditions on the Martian surface
and to study the transport of airborne dust. To do so, a re-circulating wind tunnel is housed
within an environmental chamber that operates at the atmospheric pressure (6–10 mbar),
composition and temperature of Mars. An axially mounted fan driven by an electric motor
draws gas down the central wind tunnel and returns it in an outer cylindrical cavity. This
design has been chosen to maximize the available open wind tunnel area (cross section)
while maintaining smooth fluid flow given the limited environmental chamber dimensions. It is of importance when utilizing the recirculating system that the area of the return flow is
comparable (if not greater than) to that of the wind tunnel in order to minimize turbulence
and irregularities in the wind stream. Fins are used to minimize rotation effects on the wind
by the fan (Merrison et al. 2002, 2004, 2007, 2008, 2010, 2012). 4.1.3 Simulation Facilities for Organic Aerosols in Planetary Atmospheres 4.1.2 Mars Wind Tunnels Dust devils and dust storms occur frequently on the Martian surface (Greely et al. 1977). Both slow and fast moving dust interacts with its environment, with abrading effects on
landers and rovers, and also on mineralogy (Merrison et al. 2010). In terrestrial dust storms
the interaction of dust particles leads to the generation of an electric field (e.g. Freier 1960),
a process which on Mars has been related to oxygen enhancement (Atreya et al. 2006)
and methane destruction in the atmosphere (Farrell et al. 2006). To study the effects of the
moving dust, several wind tunnel facilities have been established. A selection of Mars wind
tunnels available on the USA is described below. The Mars Surface Wind Tunnel (MARSWIT), NASA Ames Research Center, USA
MARSWIT, the first Mars wind tunnel was established in the 1960s at NASA Ames Re-
search Center (Greeley et al. 1977, 1980, 2000; Greeley 2002). This pentagon-shaped cham-
ber, a 30 m high concrete tower, can be evacuated to a minimum pressure of 3.8 mbar. Large experimental apparatus can be placed inside the chamber. High-pressure air (up to
9.86 kg/cm2) is introduced into the tunnel to induce the flow of air. At these pressures a
maximum free-stream airspeed of 180 m/s can be achieved at Mars-like pressures of 5 mbar
(500 Pa) (Greeley et al. 1977). The Arizona State University Vortex Generator (ASUVG), USA
As dust devils have
entirely different dynamics than dust flows and storms, ASUVG was built specifically to
simulate dust devils. It consists of three components: the frame, the vortex generator, and
the test table. The vortex generator includes a cylinder with a “bell mouth” to alleviate
boundary effects at the edge of the cylinder, a motor drive, and a fan blade system. The dust
is placed on a test table that can be raised, lowered, or moved laterally to simulate different
motions of a dust devil During the tests, the ambient temperature, relative humidity, wind
speeds, and surface pressures on the test bed beneath the vortex can be measured (Greeley
et al. 2003; Greeley 2002; Neakrase et al. 2006; Neakrase and Greeley 2010). Z. Martins et al. 64 The Aarhus Wind Tunnel Simulator (AWTS), University of Aarhus, Denmark
The
AWTS facility was specifically built to reproduce wind conditions on the Martian surface
and to study the transport of airborne dust. 4.1.3 Simulation Facilities for Organic Aerosols in Planetary Atmospheres Organic growth processes through negative ion chemistry was also investigated
between CN−ions and HC3N molecules (Žabka et al. 2012). The first aim of these laboratory experiments is to address the question of how atmo-
spheric chemistry can sustain the formation of complex organic matter and possibly prebi-
otic buildings blocks in a planetary system (Raulin et al. 2012). This laboratory approach 65 Earth as a Tool for Astrobiology—A European Perspective Fig. 7 The plasma experience PAMPRE (Szopa et al. 2006) can be used to produce Titan’s tholins at various
initial methane concentrations (Mahjoub et al. 2012), as a support to the interpretation of several observations
from the Cassini-Huygens space mission (on the right, copyright NASA) Fig. 7 The plasma experience PAMPRE (Szopa et al. 2006) can be used to produce Titan’s tholins at various
initial methane concentrations (Mahjoub et al. 2012), as a support to the interpretation of several observations
from the Cassini-Huygens space mission (on the right, copyright NASA) focuses on the chemical processes. Firstly, the chemistry is decoupled from the complex
transport occurring in an atmosphere, and submitting aerosols to the large variety of phys-
ical conditions encountered on the global scale. Secondly, the chemical kinetics is accel-
erated to give access to long time-scale processes, which are hardly accessible from direct
observations. Laboratory studies have been used for example to investigate Titan’s aerosol
chemical composition through geological time-scale (Mahjoub et al. 2012), and the low iso-
topic nitrogen fractionation of the Archean organic matter on the early Earth (Kuga et al. 2014). And thirdly, analogues can be analyzed with highly efficient analytical techniques
yet out of reach in space, providing unique information on the chemical composition and
formation processes of the organic aerosols: Nuclear magnetic resonance (NMR) (Derenne
et al. 2012; He and Smith 2014), high resolution mass spectrometry (Gautier et al. 2014;
Pernot et al. 2010; Somogyi et al. 2005) or thermal characterization (He et al. 2014;
Nna-Mvondo et al. 2013). focuses on the chemical processes. Firstly, the chemistry is decoupled from the complex
transport occurring in an atmosphere, and submitting aerosols to the large variety of phys-
ical conditions encountered on the global scale. Secondly, the chemical kinetics is accel-
erated to give access to long time-scale processes, which are hardly accessible from direct
observations. Laboratory studies have been used for example to investigate Titan’s aerosol
chemical composition through geological time-scale (Mahjoub et al. 4.2 Interstellar and Cometary Organic Chemistry Simulation Facilities Numerous laboratory experiments have been developed to study the complex organic chem-
istry occurring in interstellar and cometary ices (Allamandola et al. 1988; Palumbo et al. 2004; Briani et al. 2013; Kobayashi et al. 1995; Moore and Hudson 2005; Meinert et al. 2012; Danger et al. 2013). The principle of such experiments is the following; from ob-
servations of the most abundant species present in the ices in late stages of the molecular
clouds (out of which stars and planetary systems form), together with some comparison with
cometary atmospheres, one can built templates of ices to study their evolution in the labora-
tory. The templates consist in the deposition of a gaseous mixture representative of cosmic
ices molecules in a high vacuum chamber and on a cold (10–80 K) inert window. At this
stage, the main analytical tool is infrared spectroscopy because, historically, this method was
used to understand the composition and behavior of such ices in space (d’Hendecourt et al. 1996). Since cosmic ices can be subjected to various energetic processes (VUV irradiation,
cosmic-rays, and thermal heating), all these processes are straightforward to produce in the
laboratory to study the ice chemistry that progressively forms complex organic molecules
and actual organic matter. Globally these processes are templates of the general evolution
of the ices, from molecular clouds to their incorporation in comets and other minor bodies
in the solar (or any planetary) system. For astrobiological purposes, the philosophy of such
experiments is linked to the possible generation of so-called prebiotic molecules that can be
found in these ices and/or organic residues. Indeed, one can note that when the sample is
warmed up to room temperature for further analysis using more precise and sophisticated
tools, a refractory organic residue always remains on the substrate as the volatiles sublime
(Fig. 8). Mayo Greenberg, who was the first to conceive such an experimental approach
called this residue “Yellow Stuff” (Greenberg 1982) because of its light-yellow color. The
necessity to proceed to such experimental simulations is dictated by the fact that the very
high level of complexity obtained from solid state reactions within the bulk of the ices gen-
erates a very large number of products. Therefore, it would be impossible to predict the issue
of such non-directed Miller-type experiments using an ab-initio model implying thousands
of species and reaction channels. 4.1.3 Simulation Facilities for Organic Aerosols in Planetary Atmospheres 66 Z. Martins et al. One of the difficulties encountered when simulating the synthesis of organic aerosols is to
prevent samples from air contamination when collecting them and bringing them to external
analytical devices (Cable et al. 2012). Without any specific precaution the analogues adsorb
and react on their surface with oxygen, leading to a few percent of oxygen in their elemental
composition (Fleury et al. 2014). This issue becomes a critical interference when studying
the primordial incorporation of oxygen in the aerosols in planetary conditions (Hörst et al. 2012). Some efforts can be made to collect the analogues in oxygen free gloves boxes, but
this caution is not enough. A total oxygen-free chain would be required to transport the
samples from their site of synthesis to their site of analysis. This is to date hardly achievable
and remains a strong limitation to simulate in the laboratory analogues of planetary organic
aerosols with a controlled content of oxygen. 4.1.3 Simulation Facilities for Organic Aerosols in Planetary Atmospheres 2012), and the low iso-
topic nitrogen fractionation of the Archean organic matter on the early Earth (Kuga et al. 2014). And thirdly, analogues can be analyzed with highly efficient analytical techniques
yet out of reach in space, providing unique information on the chemical composition and
formation processes of the organic aerosols: Nuclear magnetic resonance (NMR) (Derenne
et al. 2012; He and Smith 2014), high resolution mass spectrometry (Gautier et al. 2014;
Pernot et al. 2010; Somogyi et al. 2005) or thermal characterization (He et al. 2014;
Nna-Mvondo et al. 2013). The second aim of these laboratory experiments is to support space missions giving rise
to useful collaborations between laboratory, modelling and observation studies. These col-
laborations involve both the preparation of new missions and the exploitation of on-going
and past missions. Planetary atmospheres are complex systems coupling chemical, dynam-
ical and radiative processes. The observation data reflect all this complexity and can be dif-
ficult to interpret. The chemical and physical properties of the laboratory analogues provide
access to essential parameters for de-convoluting atmospheric data measurements. Labo-
ratory analogues are extensively used for example as a support of interpretation for the
Cassini-Huygens space mission (Fig. 7). Optical refractive indices of laboratory analogues
were used to interpret the light-scattering properties by Titan’s aerosols from the Descent im-
ager/spectral radiometer (DISR) instrument (Hadamcik et al. 2009; Hadamcik et al. 2013). Mid- and far-IR absorption properties of Titan’s aerosol analogues helped to identify and
confirm aerosols signatures obtained by the Visible and Infrared Mapping Spectrometer
(VIMS) and the Composite Infrared Spectrograph (CIRS) instruments (Gautier et al. 2012;
Sebree et al. 2014), but also identified some limitations of the laboratory approach through
discrepancies between laboratory analogues and Titan’s aerosols signatures (Gautier et al. 2012; Imanaka et al. 2012; Kim et al. 2011). Mass analysis at high resolution of labora-
tory analogues also provided some leads on the chemical composition of the large species
detected in Titan’s ionosphere by the Cassini Plasma Spectrometer (CAPS) instrument at
low mass resolution (Carrasco et al. 2013a, 2013b). Moreover, laboratory analogues are
used as model organic matter to calibrate and optimize space instrumentation for future
space missions in order to detect and analyze organics in space. Analogues of Titan’s at-
mospheric aerosols have for example been used to improve the analysis of organics in
space by pyrolysis-Gas Chromatography-Mass Spectrometry (pyr-GC-MS) (He et al. 2014;
Morisson et al. 2016). 4.2 Interstellar and Cometary Organic Chemistry Simulation Facilities The diversity of organic compounds synthesized during such laboratory simulations is
remarkable but their identification is not always evident (Colangeli et al. 2004; Cottin et al. 1999; Danger et al. 2013), and depends on the various analytical methods and protocols used
in the procedure. Surprisingly, if templates of abundant interstellar ices are considered, es-
sentially containing H, C, O and N roughly in cosmic abundances, the nature of the complex
molecules tends not to depend too much on the ice composition (Muñoz Caro and Schutte
2003). Similarly, the nature of the energy sources, either VUV photons or fast energetic Earth as a Tool for Astrobiology—A European Perspective 67 particles (including heavy swift high energy ions) leads, at least to the first order to similar
radical-molecule reactions giving very similar organic residues (Muñoz-Caro et al. 2014). This is due to the nature of the solid-state chemistry which is generated by the irradiation
process, and by further warming-up of the sample. This chemistry is basically dictated by
diffusion of reactants and radical-molecule recombination, which appear to be a somewhat
random process. The chemistry considered and simulated in these laboratory experiments
can occur to ice mixtures either on icy coated dust grains in interstellar clouds (potentially
pre-cometary ices), or within the Solar Nebula during the accretion of icy planetesimals, or
in the outer layers of comet ices in the Solar system. The fact that these simulations do lead
to a rather unique class of organic materials that may share some similarities with chondritic
organic matter is a good argument in favor of the validity of this approach, which can be con-
sidered as a systemic one, and ultimately allows for the build-up of an astrophysical scenario
for the origin of the organic matter in the solar system (or any forming planetary system). This organic matter may later be involved (or not) in prebiotic processes that may be at work
on telluric planets in presence of the right environmental conditions, mainly liquid water and
a free energy source (UV-visible photons being the most probable one because of entropy
considerations). Indeed, the full nature of this astrophysical scenario is highly convoluted
and these simulations must be considered as templates that are able to reproduce some of
the organic complexity observed in meteorites, and further on may suggest prebiotic routes
for the emergence of life by the delivery of exogenous organic matter on the early Earth. 4.2 Interstellar and Cometary Organic Chemistry Simulation Facilities One
does not need to fully reproduce all the aspects of the true historical scenario (indeed an im-
possible task), but instead highlight the main points and bottlenecks that such a scenario
may encounter. Direct implication on astrobiology, as far as the habitability of a planet, the
subsequent prebiotic activity in a given environment and the emergence of life in the form of
autocatalytic chemical networks are considered, is central to this type of laboratory studies
and further potential and expected developments. p
p
p
For direct relevance to astrobiology, it must be noted that a great number of amino acids
(such as glycine, alanine, sarcosine, valine, proline, serine, etc.) are reported in residues
obtained after UV irradiation of ice mixtures containing molecules such as of H2O, NH3,
CH3OH, HCN, CO and CO2 (Bernstein et al. 2002; Muñoz-Caro et al. 2002; Meinert et al. 2012). Those are detected using classical GC-MS techniques, sometimes with very high sen-
sitivity and separation properties such as in the case of bi-dimensional GC-MS. In some ex-
periments, contrary to what can be done in the analysis of meteorites, labelling of the carbon
used in the experiments (i.e. using 13C) leads to results in which potential terrestrial contam-
inations issues can be fully discarded. Analytical protocols require usually the dissolution
in water of the soluble organic matter present in the residues, followed by acid hydrolysis
(for the liberation of amino acids often trapped in some form of macromolecular material),
as well as derivatization methods in order to allow the use of the GC technique, which
requires the compounds to be volatile. Although somewhat controversial, non-hydrolysed
samples versus hydrolysed ones do show very similar composition in amino acids, the hy-
drolysed fraction showing usually a larger abundance (Nuevo et al. 2008). Nevertheless, free
amino acids do exist in the residues, as in primitive carbonaceous meteorites (Martins et al. 2015). It is a matter of debate whether hydrolysis of the synthetic organic matter present in
these residues is relevant to what could have occurred on the primitive Earth. It is however
expected that organic matter exogenously delivered to our primitive planet was mixed to
an ocean of liquid water, and that similar chemistry occurred. Many other potentially pre-
biotic molecules are present in these residues. 4.2 Interstellar and Cometary Organic Chemistry Simulation Facilities Sugars were detected in the same suite of
experiments using strictly the same methodology (but not the same extraction and detection
protocol due to the different chemical properties of the searched target molecules). Glyco-
laldehyde and glyceraldehyde, two molecules suspected to be important for the formation Z. Martins et al. 68 Fig. 8 A typical experimental
setup allowing the photolysis of
cometary ice analogues made by
deposition of a gas mixture on a
cold sample carrier and cooled
down to 10 K in a cryostat (the
UV lamp can be replaced in some
setups by an ion or an electron
gun). The ice evolution can be
analyzed in situ by IR
spectroscopy. The room
temperature residue can then be
collected for further analysis such
as GC-MS, High Performance
Liquid Chromatography (HPLC)
and Very High Mass Resolution
Spectrometry (VHRMS). Schematic of the experimental
set-up from Briani et al. 2013
of ribonucleotides in a particularly primitive Earth-like environment (Powner et al. 2009),
were found (de Marcellus et al. 2015). More recently, the detection of the aldopentose ri-
bose together with a full family of structurally related sugar molecules have been reported
in quite high abundances in the same laboratory ices (Meinert et al. 2016). One may also
note that nucleobases are suspected to be present in the residue. Indeed, the detection of
il
d i
l
t
i
hi
d (N
t l 2009 2014) U
t
thi Fig. 8 A typical experimental
setup allowing the photolysis of
cometary ice analogues made by
deposition of a gas mixture on a
cold sample carrier and cooled
down to 10 K in a cryostat (the
UV lamp can be replaced in some
setups by an ion or an electron
gun). The ice evolution can be
analyzed in situ by IR
spectroscopy. The room
temperature residue can then be
collected for further analysis such
as GC-MS, High Performance
Liquid Chromatography (HPLC)
and Very High Mass Resolution
Spectrometry (VHRMS). Schematic of the experimental
set-up from Briani et al. 2013 of ribonucleotides in a particularly primitive Earth-like environment (Powner et al. 2009),
were found (de Marcellus et al. 2015). More recently, the detection of the aldopentose ri-
bose together with a full family of structurally related sugar molecules have been reported
in quite high abundances in the same laboratory ices (Meinert et al. 2016). 4.2 Interstellar and Cometary Organic Chemistry Simulation Facilities One may also
note that nucleobases are suspected to be present in the residue. Indeed, the detection of
uracil, and in only one case cytosine was achieved (Nuevo et al. 2009, 2014). Up to this
moment no other nucleobases have been identified, but a systematic search is underway in
some laboratories. The issue of chirality is also being investigated through laboratory simulations. It has
been reported that an asymmetric (i.e. a chiral electromagnetic field) vacuum UV photolysis
of a racemic mixture of leucine in the solid-state results in the production of an enantiomeric
excess of one of the forms of the amino acid (Meierhenrich et al. 2005). Moreover, enan-
tiomeric excesses can be detected when amino acids are directly synthesized within an achi-
ral ice mixture under circularly polarized light (CPL), with an enantiomeric excess of up to
2.54% (de Marcellus et al. 2011; Modica et al. 2014). In this case, the evolution of the ice
under VUV light does produce chiral precursors of organic compounds. If the VUV light is
generated as circularly polarized light using a synchrotron light source such as the SOLEIL
(Source Optimisée de Lumière d’Énergie Intermédiaire du Laboratoire pour l’utilisation du
rayonnement électromagnétique) in France, on a line dedicated to the production of chi-
ral photons, enantiomeric excesses can be generated and observed within the same organic
residues formed in the warming-up process. Remarkably Modica et al. (2014) found that five
different amino acids present the same (L- or D-) excesses in the experiment, depending on
the helicity of the CPL used. When no CPL is used (e.g. in the linear polarization irradiation
regime), a racemic sample is obtained. 4.3 Instrument Testing Facilities Before deployment on a mission, instruments need to reach a certain technical readiness
level (TRL). This TRL can be reached by testing the instrument both in the field (as de-
scribed earlier) and in the lab under realistic planetary conditions. Several chambers have
been developed specifically for instrument testing. Earth as a Tool for Astrobiology—A European Perspective 69 Chemistry and Camera (ChemCam) Environmental Chamber
The ChemCam Envi-
ronmental Chamber was developed at the University of Toulouse, France to test the first
Laser Induced Breakdown Spectroscopy instrument under simulated Martian environment
(pressure and composition). The instrument was part of ChemCam on the Curiosity Rover
(Cousins and Crawford 2011). In each experiment, five samples are placed in the cham-
ber (kept at room temperature) and the ChemCam instrument is installed 3 meters from the
sample (Cremers and Radziemski 2006). Sample Analysis at Mars (SAM) Environmental Chamber
The SAM environmental
chamber was developed at NASA Goddard Space Flight Center, USA to mimic the ther-
mal conditions in a rover on the surface of Mars, allowing simultaneous instrument suite
qualification The SAM chamber consists of six independent thermal zones, in which the
temperature can be cycled between −40°C and +50°C, and the pressure varied from 10−6
to 1000 mbar (Mahaffy et al. 2012). 5 Conclusions and Recommendations Over the past few decades, the role of the Earth as a tool for Astrobiology has been empha-
sized by establishing a wide range of planetary analogue field sites and simulation facilities
in the laboratory. But these existing infrastructures and logistics are still unknown to the
broader community and often could complement each other. The first activities, such as the
EU supported EUROPLANET or CAFE, have just started to coordinate systematic interac-
tions and common projects combing planetary field site research with planetary simulation
experiments. The goal is to achieve as many investigations as possible on the origins of
life and habitability of Mars and other planets or moons by experiments in the field com-
bined with planetary simulations in the lab. But further work and synergistic cooperation
is needed. A number of different national Polar Expeditions, Desert research projects and
Deep sea missions are often organized field campaigns with the topic of “climate change” or
“environmental issues”, and offer in parallel to researchers of other disciplines opportunities
to participate. These are opportunities to cooperate and to share also results, which might
be of significant relevance either for astrobiology and Earth sciences researchers. To further
enhance the utilization of the Earth and Earth-based systems as a tool for Astrobiology with
emphasis on synergistic cooperation we propose the following recommendations to ESA. 5.1 Analogue Studies, Field Work and Laboratory Simulations • Obtain more calls for opportunities for field test campaigns in environments that are sim-
ilar to solar system bodies. To facilitate these field campaigns, a central point of contact
(potentially hosted at ESA) could be established to help organize campaigns, to keep an
oversight of the conditions on the field sites, and to work together with the local respon-
sible parties. One of its responsibilities would be to oversee the remote monitoring of the
field sites, through satellite data or ground stations for field sensors. • Establish an EU or ESA supported international survey of all expeditions performing sci-
entific work in planetary field analogue sites, which could provide logistics and infrastruc-
ture, enabling an interdisciplinary scientific cooperation. This can minimize the costs, and
also allow astrobiologists to learn methods, operations and strategies from other scientific
communities. In this way scientists share their own knowledge and results for general and
common use. Z. Martins et al. 70 • Improvement of infrastructures and resources to undertaking astrobiological research is
necessary. This means that the combination of field work and planetary simulation in-
vestigations is not sufficiently developed at present. For example, support is needed for
the scientific community involved in the study of biosignatures and life detection, as well
as for future space missions dedicated to the search for life on other planets and moons. A systematic approach with tests on instruments under simulated planetary and space
conditions as well as tests on the operation capacity in the field is needed. A systematic approach with tests on instruments under simulated planetary and spa
conditions as well as tests on the operation capacity in the field is needed. • Support the use of simulation facilities, which is currently covered in the ESA-member
states by national or international cooperation agreements. In addition, the planetary field
analogue projects should be supported by ESA like it is accomplished by NASA in the
United States. • A European Astrobiology Platform (or Institute) should be implemented to optimize
the scientific return by using a coordinated infrastructure and funding system. This is
in agreement with recent recommendations by the AstRoMap European Astrobiology
Roadmap, which was supported by the European Commission Seventh Framework Pro-
gramme (Horneck et al. 2016) • Data obtained on astrobiological research should be shared with the scientific community
through an open access database. Coordinated funding must insure the ability to establish
this database. 5.1 Analogue Studies, Field Work and Laboratory Simulations • Data obtained on astrobiological research should be shared with the scientific community
through an open access database. Coordinated funding must insure the ability to establish
this database. • Develop a coordinated EU or ESA program of opportunities for public education and
outreach, including training for scientists, and information to the public about the state of
the art in Astrobiology. 5.2 ESA Field Campaign Facilities General Curation Facility
It would be beneficial for the community to develop a com-
mon sampling protocol, and a tool for packing and transporting the samples in oxygen-free
conditions, to be able to share the analogues within the community without altering them in
the transport. In the same way, the PFA and ISAR should be expanded in order to contribute
meaningfully with the proposed European/ESA curation facility (n.b. not country specific)
to host planetary field analogue samples. In fact, the ISAR is being transferred to the ESA
facility under construction at Harwell in the UK for this purpose. There should be a inter-
action with the European curation of astromaterials received by a sample return mission
from future space exploration programs (e.g. the requirements for such facility are being de-
fined by the EC Horizon 2020 project EURO-CARES, http://www.euro-cares.eu/), in order
to have a parallel between planetary field analogue samples and actual collected samples
from the sample return missions as foreseen within the Solar System exploration program
to asteroids, Mars, the Moon, and comets. Whether or not both sets of samples should be
placed at the European Sample Curation Facility (ESCF) is still to be discussed. Analytical Facilities
An ESA supported User Analytical Facility Network should be es-
tablished to facilitate uniformity in analyses and therefore allowing the different campaigns
to be compared to each other. This User Analytical Facility Network could be comprised of
instrumentation at associated institutions as well as instrumentation at ESA centers avail-
able for this purpose. The aim of this network would be to enable all participating scientists
to make use of the most sophisticated instrumentation around. The network should include
instrumentation for ultra-trace sample analysis, such as High-Resolution MS facilities (ToF-
SIMS, NanoSIMS, LA-ICPMS), Microscopy facilities (CLSM, AFM, SEM, TEM, FIB),
Spectroscopy facilities (Infrared, Raman, Fluorescence, NMR) and Analytical chemistry fa-
cilities (LC-ToF-MS, GC-MS). 71 Earth as a Tool for Astrobiology—A European Perspective ESA Archiving Facilities
An archiving facility should be built, consisting of two parts. A
Ground Reference Sample Storage, where samples collected on the field testing campaigns
will be stored for future reference, and an Electronic Database, where simulated planetary
environment parameters and environmental field parameters are logged, field campaign data
and laboratory data stored, and all these can be interconnected with existing databases. Acknowledgements
This work has been supported by ESA, through a Topical Team grant from 2011
to 2016. 5.2 ESA Field Campaign Facilities We are grateful to Oliver Angerer, Jason Hatton and René Demets from ESA for their help and
support. UPEC and LISA provided important administrative help (Mireille Paul, Sabrina Hammache, Valérie
Simard, Nicolas Vautrin). Zita Martins is funded by a Royal Society University Research Fellowship (grant
UF080820 and grant UF130324). Nathalie Carrasco acknowledges the financial support of the European
Research Council (ERC Starting Grant PRIMCHEM, grant agreement no. 636829). Pascale Ehrenfreund
acknowledges support from the NASA Astrobiology Institute. Louis d’Hendecourt acknowledges support
from the French Space Agency (CNES) Exobiology Group. Silvano Onofri acknowledges the Italian Space
Agency (ASI) for financial support. Inge Loes ten Kate acknowledges the Netherlands Space Office (NSO),
the Netherlands Organization for Scientific Research (NWO) and Raheleh Motamedi for fruitful discussions. Jean-Pierre de Vera acknowledges the Helmholtz-Gemeinschaft for financial support through the Helmholtz-
Alliance Planetary Evolution and Life. Frances Westall acknowledge support from the French Space Agency
(CNES), the Agence Nationale de la Recherche (ANR), the Centre national de la recherche scientifique
(CNRS-MI-2014), the MASE project (supported by European Community’s Seventh Framework Programme
(FP7/2007-2013) under Grant Agreement no. 607297), and the H2020 EUROCARES programme. Open Access
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Activities Mauna Kea 2012 field test. Acta Astronaut. 113, 120–131 (2015)
Ž J. Žabka, C. Romanzin, C. Alcaraz, M. Polášek, Anion chemistry on Titan: a possible route to large N-bearing
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'We Can Also Do Stupid': The EU's Response to America First Protectionism
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RSCAS 2019/52
Robert Schuman Centre for Advanced Studies
Global Governance Programme-349 “We can also do stupid”: Simon J. Evenett “We can also do stupid”:
The EU’s response to America First protectionism Simon J. Evenett EUI Working Paper RSCAS 2019/52 This text may be downloaded only for personal research purposes. Additional reproduction for other
purposes, whether in hard copies or electronically, requires the consent of the author(s), editor(s). If cited or quoted, reference should be made to the full name of the author(s), editor(s), the title, the
working paper, or other series, the year and the publisher. ISSN 1028-3625 © Simon J. Evenett, 2019
Printed in Italy, July 2019
European University Institute
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www.eui.eu
cadmus.eui.eu Robert Schuman Centre for Advanced Studies The Robert Schuman Centre for Advanced Studies, created in 1992 and currently directed by
Professor Brigid Laffan, aims to develop inter-disciplinary and comparative research on the
major issues facing the process of European integration, European societies and Europe’s place
in 21st century global politics. The Centre is home to a large post-doctoral programme and hosts major research programmes,
projects and data sets, in addition to a range of working groups and ad hoc initiatives. The
research agenda is organised around a set of core themes and is continuously evolving,
reflecting the changing agenda of European integration, the expanding membership of the
European Union, developments in Europe’s neighbourhood and the wider world. For more information: http://eui.eu/rscas The EUI and the RSCAS are not responsible for the opinion expressed by the author(s). Abstract The reaction of the European Commission to a series of provocative protectionist acts by the Trump
Administration is characterised and then evaluated according to six criteria. The traditional “playbook”
for dealing with high-profile US protectionism is found wanting, calling further into question the
apparent logic of retaliation. Indeed, the European Commission’s current approach appears to be more
of a tactic to buy time than an effective strategy to alter U.S. policy. Implications for the next European
Commission are also drawn. The Global Governance Programme The Global Governance Programme is one of the flagship programmes of the Robert Schuman Centre. It is a community of outstanding professors and scholars, that produces high quality research and
engages with the world of practice through policy dialogue. Established and early-career scholars work
on issues of global governance within and beyond academia, focusing on four broad and
interdisciplinary areas: Global Economics, Europe in the World, Cultural Pluralism and Global
Citizenship. The Programme also aims to contribute to the fostering of present and future generations of policy and
decision makers through its executive training programme: the Academy of Global Governance, where
theory and ‘real world’ experience meet and where leading academics, top-level officials, heads of
international organisations and senior executives discuss on topical issues relating to global governance. For more information: http://globalgovernanceprogramme.eui.eu The European University Institute and the Robert Schuman Centre are not responsible for the opinions
expressed by the author(s). *
Part of a warning by European Commission President Juncker delivered on 3 March 2018, one week before President
Trump issued a Proclamation imposing tariffs on imported steel and aluminium. The full quote is: "So now we will also
impose import tariffs. This is basically a stupid process, the fact that we have to do this. But we have to do it. We will now
impose tariffs on motorcycles, Harley Davidson, on blue jeans, Levis, on Bourbon. We can also do stupid. We also have
to be this stupid.” Source: http://www.euronews.com/2018/03/03/juncker-responds-to-trump-s-trade-tariffs-we-can-also-
do-stupid- Keywords America First, protectionism, European Union, commercial policy 2
Social media interventions aside, on my review of the press conducted for this paper, of all the US officials quoted the
Secretary of Commerce has made the most negative comments about EU commercial policy. For example, in a 17 May
2018 article in Inside US Trade, Secretary Ross is reported as saying: “The United States is one of the least-protectionist
major countries, and we have deficits to show for it. China and Europe are highly protectionist and their positive trade
balances with us reflect it…[China and Europe] eloquently espouse free-trade rhetoric, but—in practice—are far more
protectionist than the United States. Our trade policy’s main objective is to make their real-world behavior match their free-
trade speeches” (Inside U.S. Trade 2018a). ** I thank, without implicating, Per Altenberg, Bernard Hoekman, Sébastien Jean, Patrick Low, Piotr Lukaszuk, and Edwin
Vermulst for comments on earlier versions of this paper. An initial version of the argument in this paper was published in
December 2018 in the Japan Institute of International Affairs journal “Kokusai Mondai.” The project to which this paper
contributes has received funding from the European Union's Horizon 2020 research and innovation program under grant
agreement No 770680. 1
See, for example, President Trump’s speech on trade policy in Monessen, Pennsylvania, on 28 June 2016, available at
http://time.com/4386335/donald-trump-trade-speech-transcript/ 1. Introduction** Having campaigned on a platform of America First it was inevitable that questions would arise whether,
once installed in the White House, President Trump was going to follow through with this
confrontational approach to international trade relations.1 After all, albeit with a different style, Barack
Obama had been critical of trade deals signed by his predecessors but, upon assuming office, adopted a
less critical tone to America’s trading partners. In retrospect, the emphasis in 2017 on getting his Cabinet
nominated and confirmed, attempting unsuccessfully to repeal Obama Care, and enacting wide-ranging
tax reforms, left some observers with the impression that, like his predecessors, President Trump’s
protectionist instincts were no more than a campaigning ploy. In fact, the Trump administration used 2017 to plant the seeds of the protectionism that was to come
in 2018 by initiating a number of investigations and reviews. The bitter fruit that resulted has since been
causing indigestion in capitals around the world, in particular in governments that have traditionally
supported a rules-based multilateral trading system. Coming on top of a World Trade Organization
(WTO) weakened by the failure to complete the Doha Round of multilateral trade negotiations, fears
that a meaningful pillar of the post-war economic order is under serious threat arose and arguably
remain. In terms of US tariff hikes implemented and potentially in the works, China is arguably a bigger
target of American ire than the European Union. Still, President Trump and his officials have bitterly
criticised the goods trade imbalance between the EU and the United States and the 10% import tariff on
imported cars into the EU, as well as characterising the EU as having much more protectionist policies. To such critics, the EU’s fine words about multilateralism are just that—rhetoric belied by experience
on the ground.2 The EU has both a direct, and an indirect systemic, stake in current US trade policy. There is no shortage of advice to European policymakers as to how best to react to America First
protectionism. Jean, Martin, and Sapir (2018) recommend a response that blends “firm and credible”
retaliation with the offer of multilateral or plurilateral talks on macroeconomic imbalances, enhanced 1 Simon J. Evenett Simon J. Evenett WTO dispute settlement, stronger rules on subsidies and intellectual property rights, as well as new rules
on state-owned enterprises, environmental protection and global warming, and tax evasion and
optimisation. 1. Introduction** For Pisani Ferry (2018) the current transatlantic tensions, which relate to defence as well
as to trade policy, mark a pivotal moment for the EU. Having thrived under the umbrella of post-war
US institutions, the EU must define its “strategic stance vis-à-vis a more distant and possibly hostile US,
and vis-à-vis rising powers that have no reason to be kind to it. It must stand for its values.” Demertzis
(2018) advocates the EU match US threats and tariffs, demonstrating to the Americans the lose-lose
nature of protectionism. In contrast, Beattie (2018a) argued “[m]atching Mr Trump tariff for tariff will
at some point become counterproductive. If the US no longer wishes to anchor the world economy, it
may be better for other governments to get on and supplant it rather than using trade restrictions to try
to force it back to its former role.” Later he has poured cold water on the prospects of the European
Commission concluding the trade talks called for by Presidents Juncker and Trump in July 2018 on
terms acceptable to the European Union (Beattie 2018b). Dadush and Wolff (2019) see a broader “trade
crisis,” of which America First policies are one (admittedly important) component. They advance a
“Plan A” whose goal is to preserve the multilateral trading system. In their view this would involve the
EU revisiting the “red lines” in sensitive areas, such as agriculture (a step they argue would facilitate
bilateral talks with the United States as well), and macroeconomic reforms in EU member states running
large current account surpluses. Should Plan A fail and the multilateral trading system fragment into
blocs then a Plan B would be needed, in which separate bilateral negotiations with China and the United
States would be envisaged. The purpose of this paper is to characterise and evaluate how the European Commission, acting on
behalf of the European Union, has reacted to the provocative trade policy actions taken by the Trump
Administration during 2018 and 2019. These matters are of interest, not only for what they reveal about
how the EU attempts to defend its commercial interests when under pressure, but also because at this
time of writing further American protectionism cannot be ruled out. The world trading system—still put
on the pedestal as an example of well-functioning global governance by many scholars3—may look very
different a year from now. 3
In my view far too many scholars have let their normative preference for the current rules-based trading system blind them
to the system’s evident and growing deficiencies (in the negotiating function, monitoring and deliberation functions, and
increasingly the dispute settlement function.) 1. Introduction** That alone says something about the robustness of the current arrangements
and, by implication, about the need to reform them. This paper starts by characterising the four pillars of the America First trade policy of the Trump
Administration. As that policy has been implemented, many of the United States trading partners,
including the European Union, have responded using the classic “playbook” for dealing with American
trade discrimination. Section three reprises that playbook, its apparent logic and, more importantly, the
weaknesses in that logic. As the playbook had evidently failed by mid-2018, a closer look at the actions
taken by the European Commission is warranted and can be found in section four. These inform the
assessment of the European Commission’s approach presented in section five. Concluding remarks
follow. Inevitably, such an assessment is preliminary as the matters under investigation are unfolding. Still, enough evidence is available to draw some tentative conclusions about the European response to
one of the biggest stress tests that the modern world trading system has seen since its creation. Robert Schuman Centre for Advanced Studies Working Papers 2. The four pillars of the America First trade policy of the United States In addition to annual trade policy agenda documents (released in March of each year), the Trump
Administration issued a National Security Strategy in December 2017 that had a substantial trade policy
component. While some may be tempted to divine the “real” strategy underlying the Trump
Administration’s America First trade policy from these documents, on the principle that “actions speak 3
In my view far too many scholars have let their normative preference for the current rules-based trading system blind them
to the system’s evident and growing deficiencies (in the negotiating function, monitoring and deliberation functions, and
increasingly the dispute settlement function.) Robert Schuman Centre for Advanced Studies Working Papers Robert Schuman Centre for Advanced Studies Working Papers 2 “We can also do stupid”: The EU’s response to America First protectionism louder than words,” another approach is to focus on the trade policy steps taken since President Trump
was inaugurated in January 2017. Those steps can be assembled into four groups—each constituting a different pillar of the America
First strategy, see Table 1. Reflecting the different types of trade policy, there is a multilateral pillar, a
preferential trade pillar, a bilateral pillar specifically focusing on trade relations with China, and a
unilateral pillar. In addition to the usual drumroll of trade remedy cases, the latter pillar includes
unilateral acts based on investigations into whether different types of imports constitute a national
security threat to the United States, the latter being it seems a favoured tool of the Trump
Administration.4 In the multilateral pillar, the Trump Administration has continued the Obama Administration practice
of criticising the operation of the World Trade Organization’s (WTO) Dispute Settlement Understanding
and vetoing the appointment of new members of the Appellate Body.5 Again, like the Obama
Administration, current US officials have raised concerns at the WTO about the extent and manner of
Chinese state intervention. Clear differences with its predecessor can, however, be found in the Trump Administration’s policy
towards regional trade agreements (RTAs). In addition to ordering a review of all existing RTAs, the
Trump administration sought amendments to both the US-Korea RTA and the North American Free
Trade Agreement. The United States has withdrawn from one mega-regional trade agreement and ceased
negotiating another. 4
In another piece of evidence of the contemporary legacy of the trade policy of the Reagan Administration, a study by the
Congressional Research Service noted that the last time a US president undertook a Section 232 investigation into the threat
posed by imports was in 1986. At that time, the Commerce Department found that imports of metal cutting and metal-
forming machine tools threatened to impair the national security of the United States (Fefer et al. 2018). 5
See, for example, the trenchant critique on page 6 of USTR (2019a) as well as remarks on pages 25-27.
6
See USTR (2019a) page 23. 7
For an overview of the trade barriers imposed during the Sino-US trade war see chapter 3 of Evenett and Fritz (2
8 9
President Trump postponed making a decision to impose tariffs on imported cars and car parts on 17 May 2019. USTR has
been given until 13 November 2019 to negotiate an arrangement for these goods with relevant trading partners, including
the European Union. 8
Secretary Ross, in an interview on CNBC on 24 May 2018, stated “Economic security is military security. And without
economic security, you cannot have military security.” As reported in Financial Times (2018a). 7
For an overview of the trade barriers imposed during the Sino-US trade war see chapter 3 of Evenett and Fritz (2019).
8
Secretary Ross, in an interview on CNBC on 24 May 2018, stated “Economic security is military security. And without
economic security, you cannot have military security.” As reported in Financial Times (2018a).
9
President Trump postponed making a decision to impose tariffs on imported cars and car parts on 17 May 2019. USTR has
been given until 13 November 2019 to negotiate an arrangement for these goods with relevant trading partners, including
the European Union. 2. The four pillars of the America First trade policy of the United States The Trump Administration has also been critical of G20 initiatives, in particular as
they relate to reducing excess capacity in the steel sector.6 The Trump Administration also successfully
opposed to the renewal of the G20 pledge on protectionism, which was dropped from the communiqué
of the G20 Leaders’ Summit in December 2018. Trump Administration officials reserve particular criticism for China. This is not the place to state
and evaluate each of those criticisms, many of which were also articulated by Obama Administration
officials. What matters was the change in approach, with the current US Administration prepared to
invoke wide-ranging tariffs on Chinese exports unless fundamental changes in Chinese industrial and
other policies are undertaken. During 2018 and again in May 2019, President Trump has threatened to
put tariffs on all of China’s exports to the United States, covering some half a trillion dollars of trade. However, as of July 2019, $278 billion of American imports from China are subject to tariffs.7 The final pillar—the unilateral pillar—is frequently framed by US officials as relating to the threat
posed by international trade to the national security of the United States.8 The Trump Administration
has to date launched four investigations under Section 232 of the Trade Expansion Act of 1962. By far
the most significant in terms of potential trade affected, which at this time of writing has not yet resulted
in tariffs being imposed9, relates to the apparent threat that imports of cars and car parts pose to US
national security. The European Union exported, for example, €37 billion of cars and car parts to the 8
Secretary Ross, in an interview on CNBC on 24 May 2018, stated “Economic security is military security. And without
economic security, you cannot have military security.” As reported in Financial Times (2018a). 9
President Trump postponed making a decision to impose tariffs on imported cars and car parts on 17 May 2019. USTR has
been given until 13 November 2019 to negotiate an arrangement for these goods with relevant trading partners, including
the European Union. European University Institute European University Institute 3 Simon J. Evenett United States in 2018 (Eurostat 2019). Still, the tensions inflamed by the imposition of U.S. tariffs on
national security grounds on steel and aluminium imports was substantial.10 The commercial interests of the European Union are implicated in each of these four pillars. 3. Reprising the “playbook” for American protectionism When the United States previously resorted to blatant protectionism, such as when President George W. Bush announced the imposition of higher tariffs on imported steel in the form of safeguard measures on
5 March 2002, many of America’s trading partners have followed what might be termed a playbook. Former European Commissioner for Trade, Mr. Karel De Gucht, explained the essence of this approach
as follows: “You retaliate on sensitive products. The approach is always to achieve the maximum result
with the minimum effort. You retaliate to hurt somebody.”11 Recognising that imposing tariffs on imports from the United States harms European buyers, the goal
then is to select products for retaliation that have the greatest impact on US political decision-making at
the least possible cost to European interests. With respect to the latter, this shifts the calculus in favour
of retaliating on products for which ready alternatives to US suppliers exist. With respect to the former, however, the key point is that retaliation is driven by the desire to alter
the political economy of trade policymaking in the country that has undertaken protectionism, with the
ultimate goal of rescinding that protectionism as soon as possible. It is for this reason that, in the context
of protectionism imposed by the United States, high-profile exports from states represented by senior
members of the United States Congress are frequently targeted. High-profile exports from electorally
significant states may also be chosen.12 This is in addition to making direct representations to the most
senior members of the committees overseeing trade policy in the United States Congress. The list of American exports targeted for retaliation by the European Commission (after US tariffs
on steel and aluminium came into effect for European exporters on 1 June 2018) appear to have followed
this playbook. Orange juice from Florida, a state of some electoral importance, was targeted. As was
bourbon whiskey from Kentucky, which is represented in the United States Senate by the majority
leader, Mr. Mitch McConnell. Cranberries and Harley-Davidson motorcycles, which are made in
Wisconsin (amongst other locations), were targeted too. A congressional district in Wisconsin was
represented at the time by the then-speaker of the US House of Representatives, Mr. Paul Ryan. It is not clear that following the playbook has been successful. 10 In a speech at Georgetown University in Washington, D.C. on 7 March 2019 Commissioner Malmström made clear:
“Europe was seriously offended by the imposition of steel and aluminium tariffs last summer under section 232. We do not
consider that our exports are a security threat to the United States. We are friends and allies, not a threat. And now we are
carefully watching the president’s decision on car and car parts” (Malmström 2019). 2. The four pillars of the America First trade policy of the United States Even
the Chinese pillar affects European interests as European firms have invested in both China and the
United States and may be adversely affected by tariffs imposed on imports into either nation. Disruption
to supply chains and to the associated corporate strategies built on them are another legitimate concern. Moreover, US actions threaten the principles and the operation of the system of world trade rules which
European officials have stated repeatedly that they value. Given there is much at stake, how then have
European officials traditionally reacted to high-profile acts of US trade discrimination? 12 Bear in mind the vagaries of the US Electoral College and the tendency of some states to be “swing” states.
13 See Financial Times (2018c) and Wall Street Journal (2018). 12 Bear in mind the vagaries of the US Electoral College and the tendency of some states to be “swing” states. 11 As quoted in Financial Times (2018b). Bear in mind the vagaries of the US Electoral College and the tendency of some states to be “swing” states.
See Financial Times (2018c) and Wall Street Journal (2018) 13 See Financial Times (2018c) and Wall Street Journal (2018). 13 See Financial Times (2018c) and Wall Street Journal (2018). 11 As quoted in Financial Times (2018b). 3. Reprising the “playbook” for American protectionism Press reports point to criticism from
Congressional officials of Trump Administration’s trade policy13, which is a necessary condition for the
playbook to work, and some contend that the July 2018 meeting between Presidents Trump and Juncker
went better as a result. However, as of July 2019, US steel and aluminium tariffs were not reversed nor Robert Schuman Centre for Advanced Studies Working Papers 4 “We can also do stupid”: The EU’s response to America First protectionism was the European Union permanently exempted from them. Consequently, it makes sense to revisit the
logic underlying the playbook. Recall the goal of surgical retaliation is to alter the “political economy” of trade policy decision-
making in the United States. For the playbook to be successful, enough affected US exporters have to
persuade US policymakers to remove the offending protectionism so that their access to the EU market
is restored. Several things can go wrong here—as the saying goes there is many a slip between cup and
lip. First, US protectionism may be part of a larger package of policy changes that benefit the US
exporters affected by EU retaliation. Recall that the US Congress passed, at the prompting of the Trump
Administration, a generous package of tax cuts last year. Consequently, the US exporters that are
targeted by the EU may feel that, on net, they have gained from the Trump Administration’s policies. Moreover, Congressional representatives may rebuff US exporter concerns, arguing that overall they
are better off with the package of policies, of which protectionism is only one part.14 Second, if the US exporter believes the European retaliation is going to last “too long” or get out of
hand then, if sales to the EU market are critical, one response is to set up or expand production in the
EU. While the EU may benefit from such “tariff jumping” foreign direct investment, for the playbook
to work, however, the affected US exporters have to remain in the United States and fight the Trump
Administration’s protectionism. This is not just a theoretical conjecture. Recall that, after Harley-
Davidson was targeted for retaliation by the European Commission, it announced that it would supply
much more of the EU market from production facilities in Europe. Third, a US firm exporting a product targeted by the EU for retaliation can choose how it supplies
the EU market. 14 This point, among others, was made in 26 May edition of The Economist, whose cover was devoted to relations between
President Trump and the US business community (Economist 2018). 4. The European Commission’s reaction to America First protectionism In characterising the European Commission’s approach to Trump Administration trade policy, it may
be tempting to discuss its reaction to actions taken under each of the four pillars of the America First
policy. Ultimately that is the approach taken here, however it is important to acknowledge the potential
linkages across the pillars. For example, the European Commission joining forces with the United States
(and Japan) to advance the level playing field agenda (which ostensibly has Chinese policies in its sights)
may be difficult to sustain support for if the United States is concurrently imposing tariffs on EU exports. Moreover, trying to find common ground with the United States on improvements to the WTO’s
Dispute Settlement Understanding cannot be made easier if both parties are bringing cases against each
other to Geneva and alleging rule violations by the other. Lastly, negotiating away trade barriers with
the Trump Administration, perhaps as part of a broader package to normalise trade relations, may seem
counterintuitive given it was the same US administration that walked away from the Transatlantic Trade
and Investment Partnership negotiations. Such complications will be difficult to ignore, will
undoubtedly be raised by some in intra-EU deliberations, and ought to influence how the EU approach
to handling transatlantic trade tensions is evaluated in the round. In describing the EU approach, it helps to have the key decisions taken in Beijing, Brussels, and
Washington, DC in mind.15 It is now apparent that 2017 was the quiet before the storm. Inevitably, it
takes time for senior US officials to be vetted, nominated, and confirmed by the US Senate. Moreover,
the Trump Administration pursued non-trade policy priorities in 2017, such as the unsuccessful repeal
of President Obama’s health care legislation and the successful enactment of a large tax reform bill in
December 2017. Still, the groundwork for the tariffs imposed in 2018 and 2019 was laid in 2017, with investigations
launched in April and August 2017.16 Since no high-profile trade barriers were erected by the US in
2017, it was possible for some to claim then that President Trump had pulled his (trade-related) punches. A more plausible explanation is that investigations take time and that for the Trump Administration in
2017 obtaining a landmark legislative achievement was a bigger priority. Simon J. Evenett Simon J. Evenett Having written this, one should acknowledge that EU retaliation against American protectionism
may also serve a domestic political purpose, that is, of not being seen to be pushed around by a foreign
power. The merits of such virtue signalling can be questioned, not least because alternatives to retaliation
do not appear to be given much consideration during policy deliberation. Indeed, as will become clear,
a major trading partner of the United States did turn its cheek when hit by U.S. tariffs on imported steel
and aluminium in 2018 suggesting that this political “logic” of retaliation is far from apparent to
everyone. 15 In this regard the timeline presented (and frequently updated) in Bown and Kolb (2019) is valuable.
16 Other investigations were launched in May 2018 (into whether cars and car parts imports represented a threat to the national
security of the United States) and in July 2018 (into whether uranium imports represented a threat to national security as
well.) 3. Reprising the “playbook” for American protectionism For example, a US firm may have production facilities in the United States and in
Mexico. Such a firm could switch production for the EU market from the United States to a Mexican
plant so as to avoid paying the duties in the EU. Here switching production substitutes for expending
political capital lobbying elected US representatives. Fourth, the relative size of switching costs for exporters matter. If a US firm finds it more expensive
to supply the EU market after retaliation then, if the cost of switching exports to a new overseas
destination are low, then it may be preferable to do that than lobbying US elected representatives. Fifth, to the extent that the playbook seeks to influence US administration decisions then it does so
indirectly by influencing the views of US elected representatives. If the US president of the day feels
that he or she can ignore the critical Congressmen and women and Senators, then the playbook is
unlikely to work. In this regard, it is worth recalling two further structural factors: comparatively
speaking the US economy is less dependent on exports than most (implying that fewer elected
representatives will see defending exports as central to their constituents’ well-being) and that the
support or otherwise that elected representatives give a US president depends on factors other than trade
policy (and those other factors may dominate in the calculus of any elected Congressman or woman or
Senator.) In sum, there are plenty of reasons why following the traditional playbook won’t result in a reversal
of high-profile acts of US protectionism. Simply put, the playbook is contingent on too many factors
falling into place for it to be reliable. Economists have traditionally been nervous about supporting
retaliatory measures on welfare grounds and the concerns raised above ought to add to that disquiet. The
uncertain gains from following the playbook have to be weighed up against the certain losses to
European buyer welfare from raising tariffs on US exports. European University Institute 5 15 In this regard the timeline presented (and frequently updated) in Bown and Kolb (2019) is valuable. 17 The EU does conduct a High-level Economic and Trade Dialogue with China. According to the European Commission,
the seventh meeting of that Dialogue, which took place on 25 June 2018, involved an agreement “to set up a working group
to concretely co-operate on reform to help the WTO meet new challenges and to further develop rules in key areas relevant
for the global level playing field, such as industrial subsidies” (EC 2018a). Chinese reports of this meeting make no mention
of the global level playing field or subsidies. WTO reform With respect to the WTO, the European Commission developed a range of proposals that were leaked
in July 2018 (EC 2018b).18 These proposals were organised around three themes: new and upgraded
rules especially as they relate to subsidies, state-linked firms, and services as well modalities for
negotiations; notification and monitoring obligations; and WTO dispute settlement. The European
Commission tabled proposals to modernise the WTO in September 2018 (EC 2018c) and to reform the
Appellate Body in November 2018 (WTO 2018). In this regard it is worth noting that on more than one
occasion President Trump has stated that he might withdraw the United States from the WTO unless it
is reformed.19 4. The European Commission’s reaction to America First protectionism During the first year of the Trump Administration its officials took the opportunity to articulate a
number of themes that were to recur, namely, the perceived link between economic prosperity and
national security; the alleged link between foreign trade distortions and associated US bilateral trade
deficits; the deficiencies of the World Trade Organization’s agreements and procedures and their
adverse impact on the interests and legitimate objectives of the United States; and the apparent
incompatibility between the Chinese economic development model and extant multilateral trade rules. However lacking in merit these claims and linkages are, no one can plausibly claim surprise in hearing
them for the first time in 2018. In sum, arguments were articulated and means established
(investigations) in 2017, laying the groundwork for the trade tensions that were to come to pass in 2018. Robert Schuman Centre for Advanced Studies Working Papers 6 “We can also do stupid”: The EU’s response to America First protectionism Trade relations between the European Union and the United States entered a new, more adversarial
phase following the imposition of tariffs by the Trump Administration from 2018. Across-the-board
safeguard duties were imposed on imported solar panels and washing machines on 22 January 2018. More importantly, on 1 March 2018 the United States announced that additional tariff of 25% on all
imported steel would be imposed along with a 10% tariff on imported aluminium, although on 22 March
2018 imports from the European Union were temporarily exempted. That temporary exemption was
further extended on 30 April 2018 but ended on 1 June 2018. On 22 June 2018 the European Union
retaliated by imposing tariffs on $3.2 billion of American exports. Looking across the four pillars, differences in the degree to which the European Commission’s
reaction has evolved can be discerned. The de facto abandonment of the Transatlantic Trade and
Investment Partnership negotiations by the Trump Administration did not require much of a reaction
from the European Commission, except expressions of disappointment. 19 On the matter of WTO reform it is should also be noted that the European Union is a member of the Ottawa Group, led by
Canada, that is seeking to identify proposals that a broad base of governments can support. The level playing field agenda With respect to the pillars relating to China and to multilateral fora, the European Commission has
sought to engage with the United States, engaging in dialogue and developing proposals. In the case of
China, this engagement also included Japan and led to the elaboration of a series of scoping papers
published in May 2018. A statement released by the three parties on 23 May 2019, following a meeting
in Paris, referred to “progress made in discussions on text-based work on increasing transparency,
identifying harmful subsidies that merit stricter treatment and ensuring that appropriate benchmarks can
be used” and included an instruction to officials to finalise such text (EC 2019a). As yet, it is not known
if any trilateral proposals have been put to the Chinese government17 or what the process is for taking
these proposals forward. 18 For an evaluation of these proposals see Evenett (2018). 18 For an evaluation of these proposals see Evenett (2018). Reaction to unilateral US acts The unilateral pillar, especially as it relates to the national security-related investigations undertaken by
the United States, is the area where the European Commission’s position has evolved the most over
time. Initially, the Trump Administration temporarily exempted EU steel and aluminium exports from
duties, which the European Commission sought to make permanent. Failing that, from March 2018, the European University Institute 7 Simon J. Evenett Simon J. Evenett Commission made it clear it would bring a WTO case against the United States, impose retaliatory duties
on US imports in line with the Commission’s reading of WTO rules, and initiate a safeguard action on
steel imports to ensure that shipments no longer sold in US markets were not redirected towards Europe. Coupled with this three-prong strategy was the insistence, initially at least, that the EU would not
negotiate with the United States on this matter until the threat of tariffs were withdrawn. In a number of
press reports it has been suggested that US Administration officials saw the threat of tariffs as a means
of gaining negotiating leverage and that trading partners were expected to offer or to accept curbs on
steel and aluminium exports to the US, or otherwise lift trade barriers facing US exports. If this account
of the US approach is correct, then the preconditions were in place for a dialogue of the deaf across the
Atlantic. Once the expectation arose that the EU exemption from steel and aluminium tariffs would not be
extended beyond 1 June 2018, conflicting signals were sent by the governments of France and Germany
to the United States in late April and early May 2018. While EU leaders were able to agree at a meeting
in Sofia that discussions (rather than negotiations) with the United States on the bilateral economic
relationship could begin only after the threat of US tariffs was dropped, the appearance of divisions
among the member states, while hardly a new phenomenon, could not have enhanced the position of the
European Commission in its discussions with American counterparts. The announcement towards the end of May 2018 that the United States would investigate whether
cars and car parts threaten to impair its national security received did not go unnoticed, in particular in
Germany which is a major exporter of these products. Reaction to unilateral US acts The willingness of officials in Berlin to
countenance a deal with the United States apparently without the withdrawal of the threat of steel- and
aluminium-related tariffs likely influenced the events that followed. The ultimate imposition on 1 June 2018 of tariffs on EU steel and aluminium exports to the US
served as a reminder that the threats to EU commercial interests were no longer hypothetical. Soon after
the EU imposed tariffs on an amount of US exports equivalent to 44% of the value of EU exports harmed
by the new US tariffs on steel and aluminium.20 Moreover, it was reported that, in a submission to the
US Department of Commerce, the European Commission warned that global retaliation to any US tariffs
on cars and car parts could affect up to $294 billion of US exports (Financial Times 2018d). The intended
audience for this submission may have included other affected US trading partners and not only
decision-makers in Washington, DC. 20 The balance, 56%, would be hit by EU tariffs should the Commission’s WTO case against the United States prevail or after
three years, whichever comes sooner.
21 The text of this Joint Statement can be accessed at https://eeas.europa.eu/delegations/united-states-america/48861/joint-us-
eu-statement-following-president-junckers-visit-white-house_en. 21 The text of this Joint Statement can be accessed at https://eeas.europa.eu/delegations/united-states-america/4886
eu-statement-following-president-junckers-visit-white-house_en. 20 The balance, 56%, would be hit by EU tariffs should the Commission’s WTO case against the United States p
three years, whichever comes sooner. The Juncker-Trump meeting in July 2018 and reaction A shift in the EU’s approach to handling trade tensions with the United States became apparent after
Presidents Juncker and Trump met in Washington, DC on 25 July 2018. Before the meeting it was
reported that, consistent with its view that any deals must benefit all participants, the EU would offer to
negotiate a plurilateral deal to lower trade barriers by the world’s major car exporters. In addition, a
review of EU barriers to US exports was to be offered as well as joint approaches to Chinese trade
practices (New York Times 2018). It appears the meeting of 25 July 2018 did not go according to plan. Suggestions to negotiate a
plurilateral deal among car exporters were not embraced, presumably by the American side. Instead,
according to a joint statement issued after the meeting21, four steps were agreed. First, it was agreed “to
work together toward zero tariffs, zero non-tariff barriers, and zero subsidies on non-auto industrial
goods. We will also work to reduce barriers and increase trade in services, chemicals, pharmaceuticals, Robert Schuman Centre for Advanced Studies Working Papers 8 “We can also do stupid”: The EU’s response to America First protectionism medical products, as well as soybeans.” Notice the phrase “work together” can have a different meaning
than “negotiate.” Perhaps the more important point is that the scope of this barrier-reduction exercise
goes beyond (and seems to exclude) cars and inevitably invited comparisons to the lapsed Transatlantic
Trade and Investment Partnership negotiations. Second, greater cooperation on energy policy was envisaged with specific reference made to EU
interest in importing more liquid natural gas from the United States. Third, a dialogue on regulatory
standards and their effects on trade would be launched. Fourth, the EU and the US agreed to work with
like-minded trading partners to reform the WTO and to address third party trade distortions (more code
for Chinese trading practices.) Lastly, the following cryptic phrase was included in the statement “While we are working on this,
we will not go against the spirit of this agreement, unless either party terminates the negotiations.” This
has been interpreted principally by European observers22 to mean that the US will not impose new tariffs
on EU exports while discussions are ongoing. Subsequently, this meeting was seen as declaring a truce in transatlantic trade tensions. The Juncker-Trump meeting in July 2018 and reaction An Executive
Working Group, jointly chaired by the European Commissioner for Trade and the USTR, was
established to take this initiative forward. Reaction within the EU to this deal was, however, mixed. German and Dutch government representatives welcomed the deal. Meanwhile, the French government
sought clarifications about its contents, especially as it relates to sensitive agricultural trade policies and
environmental and health standards. The criticism was made both in Paris and by a leading member of
the European Parliament that the European Commission was unable to secure the elimination of the US
tariffs on steel and aluminium first (Financial Times 2018e). Others pointed out that full-fledged trade negotiations would require a separate mandate from the EU
member states and that the EU was not going to engage in managed trade of soybeans and liquid natural
gas. Yet it transpires that the European Union has imported much more soybeans from the United States
since the 25 July 2018 meeting. In a memorandum to EU Member States representatives on 30 January
2019, European Commission officials noted “There has been a significant increase in US exports since the start of the 2018/19 marketing year
on 1 July 2018. Over the first 31 weeks of the marketing year, up to 27 January 2019, the EU
imported 6.1 million tonnes of soybeans from the US, representing an increase of 114% compared
to the same period in 2017-18. Given this strong trade performance, the US presently has a 77%
share of all EU imports of soybeans (compared to 39% in the same period in 2017-8)” (EC 2019b). How this feat was pulled off and which trading partners lost market share was not reported. Furthermore,
the European Commission initiated a process by which the use of U.S. soybeans to produce biofuels in
the European Union will be permitted. How this feat was pulled off and which trading partners lost market share was not reported. Furthermore,
the European Commission initiated a process by which the use of U.S. soybeans to produce biofuels in
the European Union will be permitted. After the initial reaction to the meeting of the two Presidents, attention switched to which items
would be included in the subsequent negotiating agenda. Representatives of both parties diverged from
the Presidents’ joint statement in important respects. Commissioner Malmström suggested including
cars in the negotiations to eliminate tariffs on industrial goods. The Juncker-Trump meeting in July 2018 and reaction American public officials suggested
including agricultural trade reform in the talks. 22 See, for example, https://www.politico.eu/article/donald-trump-jean-claude-juncker-announces-trade-negotiations-with-
eu/. or example, https://www.politico.eu/article/donald-trump-jean-claude-juncker-announces-trade-negotiations-with- Negotiating mandates negotiating objectives were published
but before the European Council approved the Commission’s negotiating mandate.) The European
Commission’s report argued: “It should be kept in mind that the two Presidents agreed in this joint agenda to focus on a limited
range of areas where results could be realistically achieved quickly and without entering areas of
significant sensitivity for either side. This is why for example the joint agenda does not include
agriculture—which is a sensitivity for the EU side—or public procurement—which is a sensitivity
for the US. In this sense, cooperation under the joint agenda should not be compared with the wide-
ranging and comprehensive scope of a typical modern EU trade agreement…” (EC 2019b). “It should be kept in mind that the two Presidents agreed in this joint agenda to focus on a limited
range of areas where results could be realistically achieved quickly and without entering areas of
significant sensitivity for either side. This is why for example the joint agenda does not include
agriculture—which is a sensitivity for the EU side—or public procurement—which is a sensitivity
for the US. In this sense, cooperation under the joint agenda should not be compared with the wide-
ranging and comprehensive scope of a typical modern EU trade agreement…” (EC 2019b). This statement begs the question as to what the specifically the joint agenda is?23 One way to reconcile
this statement from the European Commission with the published negotiating objectives of the United
States is that the latter document is for domestic consumption and that there is an understanding that the
agenda is narrower and arguably less ambitious. Such a reconciliation beggars belief. European Commission officials have also made clear that the “removal of restrictions on exports of
steel and aluminium is a precondition for the conclusion of negotiations” (EC 2019b) and that they
would refuse to negotiate further with the United States should it impose tariffs on imported cars and
car parts on national security grounds.24 When asked to give an assessment of the status of EU-U.S. talks
in June 2019, Commissioner Malmström made specific reference to progress on trade in soybeans and
liquefied natural gas. She conceded that officials were still at the exploration stage on conformity
assessment and that negotiations on an agreement to free up trade in industrial goods had not started yet. 23 As a result of a legal requirement in the United States relating to securing Trade Promotion Authority for an upcoming
trade negotiation, the United States and the European Commission did not engage in a “scoping exercise” before publication
of the United States’ negotiating objectives. 24 Austrian Economy Minister Margarete Schramböck was reported in November 2018 saying “Agricultural products are not
part of that mandate so they are not the object of the negotiation. We expect the Americans to move because the Europeans
have already moved. We have shown that the quota for non-hormone treated beef and veal—there’s been a lot of movement
on that.” The implication is that a country-specific import quota had been established for the United States, or was planned.
Whether the United States is prepared to forgo all of its negotiating objectives on agriculture for this quota remains to be
seen. At the time this statement was made Austria held the presidency of the European Union. See Inside U.S. Trade
(2018b). Negotiating mandates The United States has made clear its objectives for the subsequent talks with the European Union. In its
annual strategy statement of 2019, USTR states “Despite this significant trade volume, U.S. exporters
in key sectors have been challenged by multiple tariff and non-tariff barriers for decades, leading to European University Institute 9 Simon J. Evenett chronic U.S. trade imbalances with the EU” (USTR 2019a, page 29). USTR published detailed
objectives for the negotiation of a trade agreement with the European Union on 11 January 2019. These
objectives are far-reaching covering 24 areas of public policy and give little sense of prioritisation
(USTR 2019b). chronic U.S. trade imbalances with the EU” (USTR 2019a, page 29). USTR published detailed
objectives for the negotiation of a trade agreement with the European Union on 11 January 2019. These
objectives are far-reaching covering 24 areas of public policy and give little sense of prioritisation
(USTR 2019b). On agricultural trade reform, the United States is seeking “comprehensive market access for U.S. agricultural goods in the EU by reducing or eliminating tariffs” as well as reforms to European sanitary
and phytosanitary rules. The United States is also seeking access to EU government procurement
markets while seeking to exclude from the negotiations its own Buy America policies. Another U.S. negotiating objective is to preserve exceptions on national security grounds (USTR 2019b). It was only until 9 April 2019 that the European Council gave the European Commission a mandate
to negotiate with the United States on the elimination of tariffs on industrial goods. Negotiations on
agricultural products were specifically excluded by the Council. Moreover, the Council made specific
reference to “sensitivities” that it felt should be taken into account during negotiations (EC 2019c). Another decision by the European Council allowed for negotiations over conformity assessment. In contrast to the U.S. document on negotiating objectives, the corresponding EU documents are
sparse, to say the least. The impression that both parties to this negotiation are not aligned on its agenda
is hard to dispel. That impression is reinforced by statements in the report to EU member state
representatives mentioned earlier (that was finalised after the U.S. Negotiating mandates Tellingly she observed “And here there is a discrepancy on mandates” especially as it relates to 23 As a result of a legal requirement in the United States relating to securing Trade Promotion Authority for an upcoming
trade negotiation, the United States and the European Commission did not engage in a “scoping exercise” before publication
of the United States’ negotiating objectives. 24 Austrian Economy Minister Margarete Schramböck was reported in November 2018 saying “Agricultural products are not
part of that mandate so they are not the object of the negotiation. We expect the Americans to move because the Europeans
have already moved. We have shown that the quota for non-hormone treated beef and veal—there’s been a lot of movement
on that.” The implication is that a country-specific import quota had been established for the United States, or was planned. Whether the United States is prepared to forgo all of its negotiating objectives on agriculture for this quota remains to be
seen. At the time this statement was made Austria held the presidency of the European Union. See Inside U.S. Trade
(2018b). 24 Austrian Economy Minister Margarete Schramböck was reported in November 2018 saying “Agricultural products are not
part of that mandate so they are not the object of the negotiation. We expect the Americans to move because the Europeans
have already moved. We have shown that the quota for non-hormone treated beef and veal—there’s been a lot of movement
on that.” The implication is that a country-specific import quota had been established for the United States, or was planned. Whether the United States is prepared to forgo all of its negotiating objectives on agriculture for this quota remains to be
seen. At the time this statement was made Austria held the presidency of the European Union. See Inside U.S. Trade
(2018b). 24 Austrian Economy Minister Margarete Schramböck was reported in November 2018 saying “Agricultural products are not
part of that mandate so they are not the object of the negotiation. We expect the Americans to move because the Europeans
have already moved. We have shown that the quota for non-hormone treated beef and veal—there’s been a lot of movement
on that.” The implication is that a country-specific import quota had been established for the United States, or was planned. 26 In a statement Commissioner Malmström put the RTA with MERCOSUR in the following context: “Over the past few
years the EU has consolidated its position as the global leader in open and sustainable trade. Agreements with 15 countries
have entered into force since 2014, notably with Canada and Japan. This agreement adds four more countries to our
impressive roster of trade allies” (EC 2019d). 25 Senator Grassley also observed “As long as Lighthizer has put out a statement and a basis for negotiation that disagrees
with Europe because it [has] agriculture in it, then I think it’s a moot question until Europe comes around” (Inside US
Trade 2019b). Greater impetus to the negotiation of selected regional trade agreements (RTAs) In addition to taking the steps above the European Commission has placed greater emphasis on
completing and starting new RTA negotiations with trading partners other than the United States. The
logic appears to be to demonstrate that, while others may turn inward, the European Union wants to
open up further, at least on a reciprocal basis. On 22 May 2018 the European Council agreed to start
separate RTA negotiations with Australia and New Zealand. On 17 July 2018 the European Union and
Japan signed a strategic partnership agreement, which came into force on 1 February 2019. A “political
agreement” with the four members of the MERCOSUR group was reached on 28 June 2019, in the
margins of the G20 Leaders’ Summit in Osaka, Japan.26 The EU signed a free trade agreement and an
investment protection agreement with Vietnam on 30 June 2019. Existing RTAs with Chile and Mexico
are being “modernised.” 27 In a revealing remark on 9 June 2019 Commissioner Malmström said that “we haven’t really started those talks” (about a
possible resolution to threat of U.S. tariffs on cars and car parts) (Inside U.S. Trade 2019a). Negotiating mandates Whether the United States is prepared to forgo all of its negotiating objectives on agriculture for this quota remains to be
seen. At the time this statement was made Austria held the presidency of the European Union. See Inside U.S. Trade
(2018b). Robert Schuman Centre for Advanced Studies Working Papers 10 “We can also do stupid”: The EU’s response to America First protectionism agricultural trade and government procurement. She noted “there is no way EU member states will
engage on agriculture” (Inside U.S. Trade 2019a). Pressure for the inclusion of agricultural trade matters in the negotiation has come from influential
members of Congress. Senator Grassley, chairman of the Senate Finance Committee which oversees
trade matters, has consistently argued for the inclusion of agriculture in the negotiating agenda. He has
argued that doing so creates more room for beneficial trade-offs. He has also put the matter more
negatively and is reported as stating in April 2019 “I don’t think you can go ahead unless there’s an
agreement on what you’re going to debate. So, the talks won’t proceed” (Inside U.S. Trade 2019b).25
Senator Grassley was also opposed to the suggestion, made by the United States Trade Representative,
that the negotiation proceed in stages. Other developments in 2019 Two other developments have further complicated EU trade relations with the United States during
2019. The first is the long-standing sore that relates to subsidies to wide-bodied aircraft producers,
Boeing and Airbus. Following a WTO ruling in its favour, USTR proposed on 12 April 2019 that
additional import tariffs up to 100% would be imposed on imports from the EU worth $21 billion (USTR
2019c). Moreover, on 1 July 2019 USTR proposed adding 89 more products to this retaliatory list,
bringing the total value of EU exports at risk up to $25 billion. Imposition of additional tariffs on this
scale will not go unnoticed by European Union member states.27 The second development was the decision by the USTR, upon the direction of President Trump, to
start an investigation under Section 301 of the Trade Act of 1974 of the newly-enacted French Digital
Services Tax. This announcement was made on 10 July 2019. The associated Federal Register notice
notes “The tax applies only to companies with annual revenues from the covered services of at least
€750 million globally and €25 million in France” and that “Available evidence, including statements by
French officials, suggest that France expects the tax to target certain large, U.S.-based tech giants”
(USTR 2019d). This particular investigation would determine whether the French measure constitutes
de facto discrimination against American companies, whether the retroactive application of the tax was 25 Senator Grassley also observed “As long as Lighthizer has put out a statement and a basis for negotiation that disagrees
with Europe because it [has] agriculture in it, then I think it’s a moot question until Europe comes around” (Inside US
Trade 2019b). 27 In a revealing remark on 9 June 2019 Commissioner Malmström said that “we haven’t really started those talks” (about a
possible resolution to threat of U.S. tariffs on cars and car parts) (Inside U.S. Trade 2019a). 11 European University Institute 11 Simon J. Evenett fair, and whether the new French tax diverged “from norms reflected in the U.S. tax system and the
international tax system in several respects.” The investigation may take up to a year, opening in 2020
another possible front in transatlantic trade tensions. fair, and whether the new French tax diverged “from norms reflected in the U.S. 28 There again, given the Appellate Body deadlock, resolution of the US section 232 measures through the WTO within 20
months appears unlikely, if not impossible. I thank Edwin Vermulst for making this point to me. Other developments in 2019 tax system and the
international tax system in several respects.” The investigation may take up to a year, opening in 2020
another possible front in transatlantic trade tensions. 5. Assessing the EU’s approach to managing trade tensions with the United States The following questions can be used to evaluate the European Commission’s approach: The following questions can be used to evaluate the European Commission’s approach: 1. Did the approach result in the removal of America First-related US tariffs or the removal of the
threat of the imposition of such tariffs? 1. Did the approach result in the removal of America First-related US tariffs or the removal of the
threat of the imposition of such tariffs? 2. Has the approach resulted in other outcomes (such as reforms to the WTO or changes in
government policy) that are likely to reduce trade tensions globally? 2. Has the approach resulted in other outcomes (such as reforms to the WTO or changes in
government policy) that are likely to reduce trade tensions globally? 3. Was the approach consistent over time? 3. Was the approach consistent over time? 4. Was the approach coherent or well founded in logic or evidence? 5. Was the approach consistent with the multilateral trade principles the EU seeks to uphold? 6. Was the approach optimal? Were there no preferable alternatives? The first two questions relate to outcomes. The third through fifth questions relate to the choice of means
rather than results. The sixth question relates to alternative means. Each is evaluated in turn. The first two questions relate to outcomes. The third through fifth questions relate to the choice of means
rather than results. The sixth question relates to alternative means. Each is evaluated in turn. Removal of tariffs and the threat of new tariffs As of this writing, the European Commission’s approach has not resulted in the removal of the tariffs
imposed by the US on imported steel and aluminium on national security grounds. Moreover, until the
United States confirms to the contrary, the threat of tariffs on imported cars remains, notwithstanding
the talks initiated following the meeting of Presidents Juncker and Trump in July 2018. While this points
to a rather negative evaluation of the European Commission’s approach, it should be noted that it may
be too soon to tell. After all, when President Bush imposed tariffs on imported steel in March 2002,
these were followed by threats of retaliation by the European Union. When the United States lost the
associated WTO dispute, the EU threatened retaliation again and the Bush Administration withdrew the
tariffs soon after in December 2003. Those steel tariffs which were supposed to last for three years ended
up being removed after only 20 months.28 Even if one is prepared to grant the point that it is too soon to come to a judgement, the disagreements
between Brussels and Washington, DC over the scope of the negotiating mandate for liberalising
industrial goods trade casts doubt on developing a path that results eventually in the steel and aluminium
tariffs being removed. Of course, one could try to argue that in the absence of the European
Commission’s approach then matters would have been worse, but what evidence is there to support that
conjecture? By setting up a bilateral negotiating initiative with counterparts in the Trump
Administration, perhaps one could credit the European Commission for stalling higher U.S. tariffs on
imported cars and any retaliation that would bring forth from the EU. Maybe the best that can be said
for the European Commission’s approach is that it is a holding operation. 29 Again, one might fall back on the argument that it is too soon to judge the European Commission’s approach. Given the
likelihood that by the end of 2019 the WTO’s Appellate Body will have collapsed, then this creates a deadline by which to
judge the steps taken by any WTO member that is seeking to preserve the current system of dispute settlement. Time is not
on the side of the “too soon to say” defence. 30 Put differently, to the extent that WTO rules on immediate retaliation against safeguard measures limit the size of US
exports affected, then this further weakens the effectiveness of the EU playbook. In turn this begs the question whether the
EU can simultaneously support the multilateral trading system and deploy an effective playbook that induces changes in
American trade policy? Systemic payoff With respect to any systemic payoff in terms of lower trade tensions, it is hard to point to any benefits
that have followed from the European Commission’s approach. As of this writing, there are no reforms
to WTO rules or procedures to credit to the European Commission. Nor is it evident that the European 12 Robert Schuman Centre for Advanced Studies Working Papers “We can also do stupid”: The EU’s response to America First protectionism Commission joining forces with Japan and the United States has resulted in first-order changes in
Chinese government behaviour. Moreover, it is not apparent that the United States has moderated it
criticisms of China as a result of either cooperating with the EU and Japan. Likewise, any moderation
of US criticism of the WTO’s dispute settlement procedures on account of adoption of the EU’s WTO
reform proposals. In sum, the best one can say about the European Commission’s approach here is that
the steps it took in 2018 to reduce systemic trade tensions have laid the foundation for future
improvements.29 Commission joining forces with Japan and the United States has resulted in first-order changes in
Chinese government behaviour. Moreover, it is not apparent that the United States has moderated it
criticisms of China as a result of either cooperating with the EU and Japan. Likewise, any moderation
of US criticism of the WTO’s dispute settlement procedures on account of adoption of the EU’s WTO
reform proposals. In sum, the best one can say about the European Commission’s approach here is that
the steps it took in 2018 to reduce systemic trade tensions have laid the foundation for future
improvements.29 Coherence With respect to logical coherence and compelling supporting evidence, concerns have already been
raised earlier in this paper about the effectiveness of imposing retaliatory tariffs as a way to induce the
Trump Administration to reverse the imposition of tariffs on imported steel and aluminium. Reinforcing
the doubts about the effectiveness of the standard EU playbook is the limited scale of the US exports
subject immediately to EU retaliatory tariffs. If the playbook was followed with conviction then, unless
there is a lack of suitable targets (which itself would be revealing), WTO rules notwithstanding, surely
as many US exports should be hit as soon as possible?30 This is not an argument for mass retaliation. Rather it is to question the value of token retaliation and to encourage reflection on what factors are
holding back the scale of EU retaliation.31 Consistency over time With respect to consistency over time, the European Commission’s approach fails this standard. The
initial EU approach sought to deny the United States negotiating leverage by arguing that Brussels would
not negotiate under the threat of tariff increases. Since the perceived American leverage depended on
their tariff increase, demanding that the United States abandon the latter before talks began was not
going to succeed. Plus, as Secretary Ross pointed out, if China could negotiate under the shadow of
threatened tariff increases then why can’t the European Union? Worse, as the likelihood that steel and
aluminium tariffs would be imposed on EU exports grew, combined with the additional threat of tariffs
on car and car part exports, it is not surprising that some Member States felt that there was enough at
stake to merit a different approach and made direct overtures to Washington, DC. That the European
Union has not presented a united front is hardly lost on decision-makers in the US capital and may well
encourage the latter to hold out even longer for concessions from the European Union in return for
abandoning any tariffs on cars and car parts. Given their extensive experience, perhaps American
policymakers have a playbook for dealing with the European Union on trade policy matters? 31 After all, some of the factors limiting EU retaliation may make a lot of sense. 31 After all, some of the factors limiting EU retaliation may make a lot of sense. Optimality With respect to the optimality of the European Commission’s approach, one way to test this is to ask
“compared to what alternative?” Here it is worth contrasting the approach taken by the Government of
Japan with that of the European Commission. On a per capita basis, both Japan and the European Union
exported approximately the same amounts of steel and aluminium to the United States in 2017. Both are
large exporters of cars and car parts to the United States. In short, both jurisdictions have significant
stakes in terms of potential trade flows affected, after normalising for their difference in size. Both too
have a stake in the successful operation of the multilateral trading system and both are on record
expressing concerns about Chinese government practices and associated economic development model. Japan’s response to America First trade policy has four aspects to it. First, as noted earlier, Japan and
the EU joined forces with the United States to develop proposals for a level commercial playing field. Second, while Japan has yet to file a WTO dispute settlement case against the United States concerning
the latter’s tariffs on steel and aluminium, news reports in June 2018 cite Japanese government officials
stating they want to cooperate with the EU on the case it has brought against these tariffs.32 Third, Japan
also notified the WTO on 18 May 2018 of its right to suspend equivalent concessions in respect of
exports from the United States. Crucially, however, to date Japan has not chosen to impose retaliatory
tariffs on the United States. For sure, the United States has not exempted Japan from these tariffs despite
lobbying by Prime Minister Abe, actions and outcomes that in many respects parallel that of the
European Union. However, by not retaliating, Japan has avoided the associated welfare losses and
avoided the accusation that it has broken WTO rules in order to defend them. Fourth, Japan and the United States have initiated negotiations towards a regional trade agreement. The Trump Administration is pursuing this negotiation under its Trade Promotion Authority. Pursuant
to that, negotiating objectives were published in December 2018 and confirmed the traditional U.S. practice of seeking negotiations on a wide range of matters implicating goods trade, services trade,
digital trade, investment, intellectual property, and state-owned enterprises. 32 On 8 June 2018 Japan requested to join the consultations between the European Union and the United States over the
former’s WTO dispute settlement case concerning the steel and aluminium tariffs imposed by the Trump Administration.
To date, Japan has not brought its own WTO dispute settlement case against the United States on this matter. Adherence to multilateral trade principles With respect to adherence to multilateral trade principles, the justification for the European
Commission’s retaliation against the US imposition of tariffs on imported steel and aluminium was that
the latter act constituted de facto a safeguard action. This, of course, is contested by the United States,
which argues that the latter act follows from an investigation of the implications for national security of 29 Again, one might fall back on the argument that it is too soon to judge the European Commission’s approach. Given the
likelihood that by the end of 2019 the WTO’s Appellate Body will have collapsed, then this creates a deadline by which to
judge the steps taken by any WTO member that is seeking to preserve the current system of dispute settlement. Time is not
on the side of the “too soon to say” defence. 30 Put differently, to the extent that WTO rules on immediate retaliation against safeguard measures limit the size of US
exports affected, then this further weakens the effectiveness of the EU playbook. In turn this begs the question whether the
EU can simultaneously support the multilateral trading system and deploy an effective playbook that induces changes in
American trade policy? 13 European University Institute 13 Simon J. Evenett the said imports. For those persuaded by this particular American argument the European Union’s
retaliatory tariffs violate WTO rules, leaving the EU open to the charge that it is, paradoxically, breaking
its WTO obligations so as to defend the rules-based world trading system. Whatever the legal merits of
the EU’s case here, the optics are unfortunate and should surely have shifted the decision-making
calculus further away from retaliating in the first place. Another concern is that the European Union’s increased imports of US soybeans since July 2018, the
proposed new rule to allow US soybeans to be converted into biofuel in the EU, and the apparent
granting of a country-specific quota for American beef, might be regarded as rewards for the United
States for violating accepted multilateral trade principles. Moreover, the vast shifts in soybeans sourcing
to the United States smacks of the kind of managed trade that the European Commission publicly
eschews. Robert Schuman Centre for Advanced Studies Working Papers Robert Schuman Centre for Advanced Studies Working Papers 6. Concluding remarks and implications for the next European Commission President Juncker reacted to the threat of US steel and aluminium tariffs in March 2018 by stating that
“We can also do stupid.” So how stupid was the European Commission’s response to the flagrant US
protectionism of 2018? To be sure, the years 2018 and 2019 have been an extraordinary time for senior
trade policymakers. Still, outbreaks of US protectionism are not new (even if the scale of recent
interventions are unusually large), the United States’ political system is relatively more transparent than
many other nations, and by initiating investigations in 2017 the Trump Administration gave notice that
it might act aggressively in 2018. Arguably, the European Commission’s response has fallen short. Perhaps the best evidence to
support this comes from the Commission itself. Specifically, there was a marked shift in approach in
July 2018 when the European Commission conceded that it would negotiate with the United States under
the threat of new tariffs (on car and car parts) and with steel and aluminium tariffs still in place. If the
strategy of the United States government was to gain negotiating leverage by raising tariffs or
threatening to do so, then it has worked in so far as discussions are envisaged concerning reducing
barriers to Amerian exports. Of course, whether those discussions amount to anything is another matter
(and reasons for doubt were presented in this paper.) The contrast between Japan’s response to date to America First protectionism and that of the
European Union is instructive. After failing to win exemptions for Japanese steel, the government in
Tokyo decided against erecting trade barriers against the United States or against other steel producers. In so doing, the harm to Japanese buyers from higher trade barriers has been avoided. European
importers have not been so fortunate. Indeed, there is a whiff of opportunism in the July 2018 decision
by the European Commission to impose a preliminary safeguard on imported steel. Fears of trade
deflection following US protectionism could become the favoured argument of European interests
seeking to erect more barriers to imports. Should the United States follow through and impose 25%
tariffs on all imports from China then such fears will be fanned in Europe. This paper also examined the logic of retaliation to foreign protectionism. 33 2018/1013.
34 According to the World Steel Association, in 2017 Japanese steel plants produced 104.7 million tons of crude steel (World
Steel Association 2018). The largest EU producer of steel, Germany, produced 43.4 million tons of crude steel in 2017. Optimality However, the United States
Trade Representatives indicated that it may pursue the negotiation with the Japan in stages rather than
have a comprehensive negotiation (Congressional Research Service 2019). Another difference in approach between Japan and the European Commission is that on 17 July 2018
the latter imposed a preliminary safeguard measure of 25% duties on imported steel justified, in part, by
concerns about trade deflection. The additional 25% steel tariffs applied in the United States may make
it commercially unattractive for some steel producers to sell there and those shipments may be diverted
to EU markets, depressing prices there and threatening steel producers in the European Union. A careful Robert Schuman Centre for Advanced Studies Working Papers 14 “We can also do stupid”: The EU’s response to America First protectionism reading of the relevant Commission Implementing Regulation33 reveals that much of the evidence
mustered applies to the era before the Section 232-related tariffs were imposed by the United States in
2018. Only steel imports from members of the European Economic Area were exempted. While the
harm to European steel buyers was arguably limited by the adoption of a tariff rate quota, rather than an
across-the-board tariff on all affected steel imports, this action contributes to the “multilateralization”
of the problem created by the original US tariff increase. Despite being a major steel producer34, to date
Japan has not chosen to follow suit thus avoiding further welfare losses associated with restricting
imports. In sum, a comparison between Japan’s and the European Commission’s approach is instructive. Both
nations failed to win exemptions from US steel and aluminium tariffs. Japan, however, has shown
greater restraint in terms of erecting tariff barriers in response to America First protectionism, whether
in direct retaliation or on account of potential trade deflection. Whether such Japanese restraint endures,
especially if the United States imposes tariffs on imported cars and car parts, remains to be seen. At the
moment, however, on several grounds Japan’s approach has the edge over the response of the European
Union. 34 According to the World Steel Association, in 2017 Japanese steel plants produced 104.7 million tons of crude steel (World
Steel Association 2018). The largest EU producer of steel, Germany, produced 43.4 million tons of crude steel in 2017. 6. Concluding remarks and implications for the next European Commission When the contemporary
realities of US politics and the full range of options available to firms are taken into account, the logic
of the traditional EU playbook for dealing with high-profile acts of US protectionism was found wanting. European University Institute 15 Simon J. Evenett Simon J. Evenett It is important to remember that the purpose of retaliation is to alter the political economy of trade policy
making in the nation that erected the offending trade barrier. Circumstances change and so European
trade policymakers should be cautious about assuming that a playbook that may have “worked” in the
past continues to do so. It is important to remember that the purpose of retaliation is to alter the political economy of trade policy
making in the nation that erected the offending trade barrier. Circumstances change and so European
trade policymakers should be cautious about assuming that a playbook that may have “worked” in the
past continues to do so. Looking forward, what has the Juncker Commission bequeathed to its successor? Rather than induce
a reversal of U.S. tariff increases on imported European steel and aluminium, the best that can be said
for the approach taken during the Juncker Commission is that it stalled for at least one year the
imposition of American tariff increases on cars and car parts. In doing so, pressures from European
Union member states to retaliate further against American protectionism have been contained, at least
for now. But how long can this approach last? That trade officials in Brussels and Washington, DC are so far
apart in defining the negotiating agenda for bilateral trade talks, and that so little progress has been made
since Presidents Juncker and Trump met, calls into question the durability of the current approach of the
European Commission. Getting a bilateral negotiation up and running may have been an effective short-
run tactic to stall pressures for further protectionism on both sides of the Atlantic, but to date little in
terms of strategy to reverse American tariff increases or, more generally, to alter American trade policy
can be divined from the approach of the Juncker Commission. When developments in 2019 are taken into account, further doubts about the length of time that the
European Commission can hold the line arise. 6. Concluding remarks and implications for the next European Commission The determination of the Trump Administration to impose
duties up to 100% on $25 billion of EU exports on account of the Airbus-Boeing dispute will raise
hackles among affected European interests and among the EU member states. Moreover, USTR’s
investigation into the new French digital services tax sets up a potential clash for 2020. Plus the U.S. presidential election cycle which will soon begin in earnest is not known for its enlightening effect on
American trade policymaking. The potential upside of the nascent US-EU negotiation on industrial goods and the other elements of
the work programme following the Juncker-Trump meeting of July 2018 are unlikely to be enough to
contain the trade tensions raised by these developments, let alone any decision by the Trump
Administration to impose tariffs on cars and car parts. Consequently, the new European Commission
should expect to find itself revising its approach to America First trade policy—the fragile construct
arranged by the Juncker Commission is likely to buckle under pressure. That revision should give serious consideration to which objectives can be realistically accomplished
by the European Commission given the political dynamics on both sides of the Atlantic. Evidently, the
standard “playbook” won’t do. But the deeper question is whether, even with its heft, the European
Union can induce first-order changes in the trade policy of the United States? And, should the United
States continue to turn inward, it does not follow that the European Union should follow, consciously
or unconsciously by repeated attempts to retaliate against American trade discrimination. There will, of
course, be self-serving advocates of retaliation by the European Union but those advocates should be
forced to spell out the logic and evidence linking their proposed trade restrictions to more enlightened
trade policy in the United States. But that is not all. There is still the risk that the United States will impose high tariffs on all imports
from China. Should that come to pass, then fears that some of the half a trillion dollars of affected
Chinese exports will be deflected into EU markets will be articulated. Were that to provide certain
European interests with a pretext for widespread application of safeguards on European imports, then
transatlantic trade tensions morph into a far more serious conflict between the European Union and
many of its trading partners. Alas, there is scope for European Union trade policy to become a lot more
stupid. Robert Schuman Centre for Advanced Studies Working Papers 7. References Beattie (2018a). Alan Beattie. “How best to respond to Trump’s trade attacks? Just do nothing.”
Financial Times. 16 July 2018. Beattie (2018b). Alan Beattie. “The dangers for Brussels of talking trade with Trump,” Financial Times. 4 September 2018. Bown and Kolb (2019). Chad P. Bown and Melina Kolb. “Trump’s Trade War Timeline: An Up-to-
Date Guide.” Trade & Investment Policy Watch Blog. June 15. Congressional Research Service (2019). Congressional Research Service. “U.S.-Japan Trade
Agreement Negotiations.” In Focus. 5 June 2019. Dadush, Uri, and Guntram Wolff (2019). “The European Union’s response to the trade crisis,” Policy
Contribution Issue no. 5, Bruegel. March. Demertzis (2018). Maria Demertzis. “The EU should not sing to Trump’s tune on trade.” Blog Post. Bruegel. 17 May 2018. Brussels. EC (2018a). European Commission. “EU and China discuss economic and trade relations at the 7th
High-level Economic and Trade Dialogue.” 25 June 2018. EC (2018b). European Commission. WTO-EU’s proposals on WTO modernisation. 5 July 2018. Brussels. EC (2018c). European Commission. WTO modernisation: Introduction to future EU proposals. 18
September. EC (2019a). European Commission, Japan, and the United States. Joint Statement of the Trilateral
Meeting of the Trade Ministers of the United States, European Union, and Japan. 23 May. Paris. EC (2019b). European Commission. “EU-US Relations: Interim Report on the work of the Executive
Working Group.” Note for the TPC/INTA. 30 January 2019. EC (2019c). Council of the European Union. COUNCIL DECISION authorising the opening of
negotiations with the United States of America for an agreement on the elimination of tariffs for
industrial goods. 6052/19. 9 April 2019. EC (2019d). European Commission. “EU and Mercosur reach agreement on trade,” Press Release. 28
June 2019. Economist (2018). The Economist. The Affair: Why corporate America loves Donald Trump. 26 May
2018 edition. Eurostat (2019). Eurostat. “International trade in cars.” April. Eurostat (2019). Eurostat. “International trade in cars.” April. Evenett (2018). Simon J. Evenett. “Triage? Assessing the EU’s Modernisation Proposals for the WTO.”
Intereconomics: Review of Economic Policy. Evenett and Fritz (2019). Evenett, Simon J., and Johannes Fritz. Jaw Jaw not War War: Prioritising
WTO Reform Options. 24th Global Trade Alert report. Fefer et. al. (2018). Rachel F. Fefer, Vivian C. Jones, Keigh E. Hammond, Brandon J. Murrill, Michaela
D. Platzer, and Brock Williams. “Section 232 Investigations: Overview and Issues for Congress.”
Congressional Research Service. 21 August 2018. Washington, D.C. Financial Times (2018a). Financial Times. 6. Concluding remarks and implications for the next European Commission Robert Schuman Centre for Advanced Studies Working Papers 16 “We can also do stupid”: The EU’s response to America First protectionism 7. References “Trump stirs global anger with car tariff threat.” 25 May
2018. Financial Times (2018b). Financial Times. “EU proves reluctant playmate in Trump’s game of chicken
on trade.” 25 June 2018. European University Institute 17 Simon J. Evenett Financial Times (2018c). Financial Times. “US to announce $12bn in farm aid to ease tariff impact.”
24 July 2018. Financial Times (2018d). Financial Times. “EU warns of $300bn hit to US over car import tariffs.” 1
July 2018. Financial Times (2018e). Financial Times. “Germany welcomes ‘breakthrough’ US-EU trade accord.”
26 July 2018. Inside U.S. Trade (2018a). Inside U.S. Trade. “Ross says WTO rules prevent 'reciprocal' trade with EU,
China.” 17 May 2018. Inside U.S. Trade (2018b). Inside U.S. Trade. “EU hopes to have negotiating mandate for U.S. talks by
early 2019.” 16 November 2018. Inside U.S. Trade (2019a). Inside U.S. Trade. “EU trade chief: Auto talks with U.S. have yet to begin;
e-commerce goal may be ‘optimistic’.” 11 June 2019. Inside U.S. Trade (2019b). Inside U.S. Trade. “Grassley: Without ag in U.S.-EU talks, valuable ‘trade-
offs’ left out.” 19 April 2019. Jean, Martin, and Sapir (2018). Sébastien Jean, Philippe Martin, and André Sapir. “International trade
under attack: what strategy for Europe.” Policy Contribution No. 12. Bruegel. August 2018. Brussels. Malmström (2019). Cecilia Malmström. “The Next Transatlantic Project.” John D. Greenwald
Memorial Lecture, Georgetown University. 7 March. ew York Times (2018). “E.U. to Offer Trade Proposals in Bid to Ease Tensions.” 24 July 2018. Pisani Ferry (2018). Jean Pisani Ferry. “Is Europe America’s Friend or Foe?” Opinion. Bruegel. 30 July
2018. Brussels. USTR (2019a). United States Trade Representative. 2019 Trade Policy Agenda and 2018 Annual Report
of the President of the United States on the Trade Agreements Program. Washington, DC. USTR (2019b). United States Trade Representative. United States-European Union Negotiations:
Summary of Specific Negotiating Objectives. Washington, DC. January. USTR (2019c). United States Trade Representative. Notice of Hearing and Request for Public
Comments: Enforcement of U.S. WTO Rights in Large Civil Aircraft Dispute. 5 July 2019. USTR (2019d). United States Trade Representative. Initiation of a Section 301 Investigation of France’s
Digital Services Tax. 10 July 2019. Wall Street Journal (2018). Wall Street Journal. “U.S., Europe Call a Truce on Trade—Trump, Juncker
agree to hold off on further tariffs as they work to take down barriers.” 26 July 2018. World Steel Association (2018). World Steel Association. World Steel in Figures 2018. WTO (2018). 7. References Communication from the European Union, China, Canada, India, Norway, New Zealand,
Switzerland, Australia, Republic of Korea, Iceland, Singapore and Mexico. WT/GC/W/752. 26
November. 18 Robert Schuman Centre for Advanced Studies Working Papers 18 “We can also do stupid”: The EU’s response to America First protectionism Table 1: The four pillars of America First trade policy and the European Union’s principal response
lar
Multilateral
Regional trade agreements
Bilateral/China
Unilateral/”National security”
atters of interest
the United
ates or relevant
a
WTO reform, in particular of
the Dispute Settlement
Understanding
Withdrawal from Trans-Pacific
Partnership; the Trans-Atlantic Trade
and Investment Partnership
negotiations put on ice. Section 301 investigation of
Chinese policies towards
intellectual property and related
matters
Section 232 investigation of steel and
aluminium imports
G20 Global Forum on Steel
Excess Capacity
Renegotiation of the North American
Free Trade Agreement
Section 232 investigation of
automobiles and automotive parts
G20 trade work programme,
including protectionist
pledge (up for renewal in
December 2018)
Renegotiation of the US-Korea Free
Trade Agreement
Section 232 investigation of uranium
imports
ncipal European
ion response
Propose reforms to the WTO
specifically:
New rules on subsidies
and state-related
businesses.
Strengthened
notification and
monitoring provisions.
Two-step process to
reform the WTO dispute
settlement
understanding. Develop a trilateral approach
with Japan and the United States
that involves information
exchange about third parties’
policies and development of
new multilateral trade rules to
level the commercial playing
field.
With respect to steel and
aluminium tariffs, having failed
to obtain an exemption, a WTO
case was brought, safeguard
duties placed to limit trade
deflection, and phased-in
retaliatory tariffs adopted.
A joint US-EU initiative
launched on 25 July 2018 with,
among others, the goal of
removing trade barriers on
industrial goods other than cars. As part of this accord neither
side is supposed to levy new
tariffs on the other, with possible
implications for the ongoing
Section 232 investigation into
car imports.
Public threats of retaliatory
tariffs on 18-20 billion euros of
US exports to the EU if the US
imports tariffs on EU cars. Table 1: The four pillars of America First trade policy and the European Union’ illars of America First trade policy and the European Union’s principal response European University Institute European University Institute 19 Simon J. Evenett Professor of International Trade and Economic Development, University of St. Gallen
Academic Director, University of St. Gallen MBA programme. HSG-MBA Blumenbergplatz 9
9000 St Gallen
Switzerland Email: simon.evenett@unisg.ch
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A Mathematical Model of the Olfactory Bulb for the Selective Adaptation Mechanism in the Rodent Olfactory System
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A Mathematical Model of the Olfactory Bulb
for the Selective Adaptation Mechanism in the
Rodent Olfactory System Zu Soh1*, Shinya Nishikawa2, Yuichi Kurita1, Noboru Takiguchi3, Toshio Tsuji1* 1 Department of System Cybernetics, Institute of Engineering, Hiroshima University, Higashi-Hiroshima,
Japan, 2 Department of System Cybernetics, Graduate School of Engineering, Hiroshima University,
Higashi-Hiroshima, Japan, 3 Division of Material Sciences, Graduate School of Natural Science and
Technology, Kanazawa University, Kanazawa, Japan * sozu@bsys.hiroshima-u.ac.jp (ZS); tsuji@bsys.hiroshima-u.ac.jp (TT) a1111 Abstract (
)
j
pone.0165230
Editor: Johannes Reisert, Monell Chemical Senses
Center, UNITED STATES
Received: May 17, 2016
Accepted: October 7, 2016
Published: December 19, 2016
Copyright: © 2016 Soh 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: © 2016 Soh 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: The data was
uploaded to Dryad. DOI is doi:10.5061/dryad. h504m. RESEARCH ARTICLE Abstract To predict the odor quality of an odorant mixture, the interaction between odorants must be
taken into account. Previously, an experiment in which mice discriminated between odorant
mixtures identified a selective adaptation mechanism in the olfactory system. This paper
proposes an olfactory model for odorant mixtures that can account for selective adaptation
in terms of neural activity. The proposed model uses the spatial activity pattern of the mitral
layer obtained from model simulations to predict the perceptual similarity between odors. Measured glomerular activity patterns are used as input to the model. The neural interaction
between mitral cells and granular cells is then simulated, and a dissimilarity index between
odors is defined using the activity patterns of the mitral layer. An odor set composed of three
odorants is used to test the ability of the model. Simulations are performed based on the
odor discrimination experiment on mice. As a result, we observe that part of the neural activ-
ity in the glomerular layer is enhanced in the mitral layer, whereas another part is sup-
pressed. We find that the dissimilarity index strongly correlates with the odor discrimination
rate of mice: r = 0.88 (p = 0.019). We conclude that our model has the ability to predict the
perceptual similarity of odorant mixtures. In addition, the model also accounts for selective
adaptation via the odor discrimination rate, and the enhancement and inhibition in the mitral
layer may be related to this selective adaptation. OPEN ACCESS
Citation: Soh Z, Nishikawa S, Kurita Y, Takiguchi N,
Tsuji T (2016) A Mathematical Model of the
Olfactory Bulb for the Selective Adaptation
Mechanism in the Rodent Olfactory System. PLoS
ONE 11(12): e0165230. doi:10.1371/journal. pone.0165230 OPEN ACCESS
Citation: Soh Z, Nishikawa S, Kurita Y, Takiguchi N,
Tsuji T (2016) A Mathematical Model of the
Olfactory Bulb for the Selective Adaptation
Mechanism in the Rodent Olfactory System. PLoS
ONE 11(12): e0165230. doi:10.1371/journal. pone.0165230
Editor: Johannes Reisert, Monell Chemical Senses
Center, UNITED STATES
Received: May 17, 2016
Accepted: October 7, 2016
Published: December 19, 2016
Copyright: © 2016 Soh 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. This selective adaptation function may be consistent with measure-
ment results given by Giraudet et al. [18], which suggest that, in most cases, the response of the
mitral cells to a binary mixture is dominated by one of the components. Although selective adaptation in the olfactory system has been observed, its application to
the prediction of perceptual similarity has not been studied. This paper thus proposes a mathe-
matical model based on previous olfactory models [19, 20] that attempted to simulate the
selective adaptation toward odorant components. We use the proposed model to generate neu-
ral activity patterns in the olfactory bulb, including the glomeruli, mitral cells, and granular
cells, and compare the activity patterns of mitral cells evoked by different odorant mixtures. The similarity between odorant mixtures is identified by examining the correlation between
activity patterns, and the results are compared to the perceptual similarity sensed by mice. The
results show that the proposed model is capable of predicting the discrimination rate of mice
with a strong correlation (r > 0.8, q < 0.05). This suggests that the model is capable of predict-
ing perceptual similarity considering the selective adaptation mechanism. The remainder of this paper is organized as follows. Section 2 explains the structure of the
proposed model and our parameter adjustment algorithm, as well as the simulation procedure. Section 3 presents the simulation results and related discussion, and Section 4 states our con-
clusions. An odor discrimination experiment conducted on mice is described in S1 Appendix. S1 Appendix also lists and explains the symbols used in the model. Introduction Predicting the quality of an odor composed of multiple odorant components is a challenging
problem. Even if the smell of each odorant component was known, the resultant quality of an
odorant mixture may differ from the linear addition of the respective odorant qualities [1]. An interesting approach to predicting odorant quality is that proposed by Haddad et al.,
in which an odorant quality space was derived from about 1400 kinds of odorant descriptor
using principal analysis [2]. Their group also applied the developed method to predict the
pleasantness of an odorant [3]. Our group proposed a model that can predict glomerular Predicting the quality of an odor composed of multiple odorant components is a challenging
problem. Even if the smell of each odorant component was known, the resultant quality of an
odorant mixture may differ from the linear addition of the respective odorant qualities [1]. Funding: This work was supported by Japan
Society for the Promotion of Science KAKENHI
Grant Number 26730144 (ZS) and 25540125 (TT)
(URL: https://www.jsps.go.jp/). The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript. An interesting approach to predicting odorant quality is that proposed by Haddad et al.,
in which an odorant quality space was derived from about 1400 kinds of odorant descriptor
using principal analysis [2]. Their group also applied the developed method to predict the
pleasantness of an odorant [3]. Our group proposed a model that can predict glomerular PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 1 / 16 Olfactory Model for Selective Adaptation Mechanism activity patterns from the chemical structure of an odorant [4], and found that the similarity
indices between the activity patterns of rats correlates with the perceptual similarity of humans
[5]. Hence, the prediction of the quality of a single odorant is gradually becoming a reality,
although more accurate prediction is desirable. Recently, odor quality predictions for odorant
mixtures have been studied. Snitz et al. used odorant descriptors to form a chemical space and
proposed a distance measure between the odorant mixtures that enables the perceptual simi-
larity to be predicted [6]. Haddad et al. proposed a method that can convert artificial sensor
responses into the pleasantness of both odorants and odorant mixtures [7]. Competing Interests: The authors have declared
that no competing interests exist. In addition to the techniques described above, this paper focuses on the brain expression of
odors evoked on the olfactory bulb, including the odor maps on the glomerular layer [8–13],
to predict the perceptual similarity of odorant mixtures. Previous studies [14, 15] have revealed
the close relationship between neural activity and odor quality, and neural interaction in the
olfactory system can change the characteristics of odor perception. For example, Youngentob
et al. reported that the glomerular activity patterns can predict the perceptual similarity of
odorants [14], and the same holds for odorant mixtures according to Grossman et al. [15]. In
addition, through odor discrimination experiments on mice, we have found that selective
adaptation contributes significantly to perceptual similarity [16]. That is, after associating the
reward with a mixture of three odorants, the mice adapted to certain components in the mix-
ture, leading to trouble distinguishing between the full mixture and certain subsets of these
odorants. The same phenomenon was observed in an experiment on humans, who used selec-
tive adaptation to improve the reliability of identification of odor components in a mixture. This is because humans cannot identify the respective odor characteristics of more than three
odorant components [17]. This selective adaptation function may be consistent with measure-
ment results given by Giraudet et al. [18], which suggest that, in most cases, the response of the
mitral cells to a binary mixture is dominated by one of the components. This is because humans cannot identify the respective odor characteristics of more than three
odorant components [17]. Materials and Methods
Olfactory model The proposed model is composed of a glomerular layer and a mitral cell/granular cell layer, as
shown in Fig 1. The model takes measured glomerular activity patterns as input, and outputs a
dissimilarity index between odors. The parameters and symbols used in the model are summa-
rized in Tables A-E in S1 Appendix. 2 / 16 PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 Olfactory Model for Selective Adaptation Mechanism Fig 1. Schematic diagram of the proposed olfactory model. The model consists of a glomerular layer, mitral and granular layer, and a dissimilarity
evaluation module. The model takes the glomerular activity patterns of odorants composing an odor as input, and considers respiration cycles to simulate
the glomerular response to odorant mixture. The neural activity in mitral and granular cells is simulated based on the models proposed in a previous study
[20, 21]. The dissimilarity evaluation module defines a dissimilarity index E and compares the activity patterns evoked in the mitral layer by different input
odorant mixtures. Fig 1. Schematic diagram of the proposed olfactory model. The model consists of a glomerular layer, mitral and granular layer, and a dissimilarity
evaluation module. The model takes the glomerular activity patterns of odorants composing an odor as input, and considers respiration cycles to simulate
the glomerular response to odorant mixture. The neural activity in mitral and granular cells is simulated based on the models proposed in a previous study
[20, 21]. The dissimilarity evaluation module defines a dissimilarity index E and compares the activity patterns evoked in the mitral layer by different input
odorant mixtures. doi:10.1371/journal.pone.0165230.g001 Glomerular Layer. The glomerular layer, which is composed of 1805 glomerular units,
takes each measured glomerular activity pattern of the odorant components as input. The
number of glomerular units is consistent with the actual number of glomeruli distributed on
the olfactory bulbs of mice [21]. It then generates the glomerular activity evoked by an odorant
mixture taking the respiratory cycle into account. The input of the i-th glomerular unit ci(nq)
generated by odorant np is determined by the following procedure (cf. Fig 2), where q is the
index of the odorant component. 1. Obtain a whole olfactory bulb surface image of the glomerular activity pattern correspond-
ing to odorant nq from a web database (http://gara.bio.uci.edu/). 1. Obtain a whole olfactory bulb surface image of the glomerular activity pattern correspond-
ing to odorant nq from a web database (http://gara.bio.uci.edu/). PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 Materials and Methods
Olfactory model The original image of the glomerular activity pattern (left) is composed of 357 × 197 pixels, and the grayscale of each pixel
corresponds to the activity strength. The original image is divided into 1805 lattices, approximately equal to the actual number of glomeruli distributed
on the olfactory bulb. The average activity strength is calculated for each pixel and converted into a vector representing an activity pattern. doi:10.1371/journal.pone.0165230.g002 doi:10.1371/journal.pone.0165230.g002 and was employed in the olfactory model proposed by Li et al. [19] (cf. Fig 1: Breath Function): Isniff;iðtÞ ¼
PiðogÞðt tinhaleÞ þ Isniff;iðtinhaleÞ;
if 0 ≦t ≦tinhale;
Isniff;iðtexhaleÞexp ðt texhaleÞ
texhale
;
if tinhale < t ≦texhale;
ð1Þ
8
>
<
>
: ð1Þ where tinhale = 220 [ms], texhale = 400 [ms], τexhale = 33 [ms] are the inhalation duration, where tinhale = 220 [ms], texhale = 400 [ms], τexhale = 33 [ms] are the inhalation duration,
exhalation duration, and time constant of exhalation, respectively [19]. In addition,
P ¼ ½P1ðogÞ; . . . ; PkðogÞ; . . . ; P1805ðogÞ 2 R18051 denotes the maximum output of a glomer-
ular unit to odor oγ, and is calculated by Eq (2). This equation was derived under the
assumption that a glomerular activity pattern of an odorant mixture is the linear combina-
tion of its odorant components [10]. Note that this is a simplified formulation, because
Grossman et al. [15] reported that a simple linear addition is not always applicable. PiðogÞ ¼ 1
k
Xk
q¼1 f ciðnqÞ
;
f ciðnqÞ
¼
1;
if ciðnpÞ ≧y
0;
if ciðnpÞ < y ;
ð2Þ
( ð2Þ where k is the number of odorant components in an input odor, nq is the odorant compo-
nent in odorant mixture oγ, and θ is a threshold variable. The threshold function f() was
applied to enhance the contrast, because the 2-deoxyglucose (2-DG) method can blur the
spatial response. The output Pi(oγ) of the i-th glomerular unit is sent to the mitral unit. Mitral and Granular layer. The mitral and granular layer was derived from the Li–Hop-
field model [19] and the Erdi model [20]. PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 Materials and Methods
Olfactory model 2. Based on the procedure used in our previous study [5], the image file of the activity pattern
is divided into 1805 lattices corresponding to the glomerular units. First, each pixel of the
activity pattern image is converted into a z-score corresponding to its activation level based
on the color bar given by the database. A lattice filter is then adapted to divide the activity
pattern into 1805 lattices. 3. Calculate the average z-score of each lattice and determine the input ci(nq). The above procedure provides the input vector CðnqÞ ¼ ½c1ðnqÞ; . . . ; ckðnqÞ; . . . ; c1805ðnqÞ 2
R18051 for odorant component nq. The output Isiniff,i(t) of the i-th glomerular unit at time t is given by the following equations,
which are based on a respiratory function that originated from measurement data of a rabbit The output Isiniff,i(t) of the i-th glomerular unit at time t is given by the following equations,
which are based on a respiratory function that originated from measurement data of a rabbit PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 3 / 16 Olfactory Model for Selective Adaptation Mechanism Fig 2. Method of generating the model input from measured glomerular activity patterns provided by a web database (http://gara.bio.uci. edu/) (adapted from [5]). The original image of the glomerular activity pattern (left) is composed of 357 × 197 pixels, and the grayscale of each pixel
corresponds to the activity strength. The original image is divided into 1805 lattices, approximately equal to the actual number of glomeruli distributed
on the olfactory bulb. The average activity strength is calculated for each pixel and converted into a vector representing an activity pattern. d i 10 13 1/j
l
016 230 002 Fig 2. Method of generating the model input from measured glomerular activity patterns provided by a web database (http://gara.bio.uci. edu/) (adapted from [5]). The original image of the glomerular activity pattern (left) is composed of 357 × 197 pixels, and the grayscale of each pixel
corresponds to the activity strength. The original image is divided into 1805 lattices, approximately equal to the actual number of glomeruli distributed
on the olfactory bulb. The average activity strength is calculated for each pixel and converted into a vector representing an activity pattern. doi:10.1371/journal.pone.0165230.g002 Fig 2. Method of generating the model input from measured glomerular activity patterns provided by a web database (http://gara.bio.uci. edu/) (adapted from [5]). Materials and Methods
Olfactory model These models, which can simulate the neural dynam-
ics caused by the interaction between mitral and granular cells, can be written as follows: Ii ¼ Isniff;i þ Ibackground
ð3Þ ð3Þ _X ¼ HGY 1
tx
X þ c
XN
j¼1 LGX þ I
_Y ¼ WGX 1
ty
Y þ Ic
8
>
>
>
<
>
>
>
:
ð4Þ ð4Þ PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016
4 / 16 4 / 16 Olfactory Model for Selective Adaptation Mechanism Where X ¼ ½x1; . . . ; xk; . . . ; x1805 2 R18051 and Y ¼ ½y1; . . . ; yk; . . . ; y1805 2 R18051 are the
internal states of the mitral and granular cells, respectively, I ¼ ½I1; . . . ; Ik; . . . ; I1805 2 R18051
is the input from the glomeruli, Ibackground is the background noise, Ic is the excitatory input
from the olfactory cortex, and τx = τy = 7 [ms] are time constants [20]. H; L; G 2 R18051805 are
synapse connection matrices, where H represents the connection from granular cells to mitral
cells, L represents that between mitral cells, and G represents that from mitral cells to granular
cells. In addition, GY ¼ ½gyðy1Þ; . . . ; gyðykÞ; . . . ; gyðy1805Þ 2 R18051 corresponds to the mem-
brane potentials of mitral and granular cells, respectively. Based on the Li–Hopfield model, the
membrane potentials are calculated by the following equations [19]: gxðxiÞ ¼
S0
x þ S0
xtanh xi z
S0
x
;
if x < z;
S0
x ¼ 0:14
S0
x þ Sxtanh xi z
Sx
;
if x ≧z;
Sx ¼ 1:4
;
ð5Þ
8
>
>
>
<
>
>
>
: ð5Þ gyðyiÞ ¼
S0
y þ S0
ytanh yi z
S0
y
! ;
if y < z;
S0
y ¼ 0:29
S0
y þ Sytanh yi z
Sy
! ;
if y ≧z;
Sx ¼ 2:9
;
ð6Þ
8
>
>
>
>
>
<
>
>
>
>
>
: ð6Þ where the threshold z is set to 1.0 based on the Erdi model [20]. As several tens of mitral cells
typically receive excitatory input from the same glomerulus [22], they can be considered to
form a column [23]. PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 Materials and Methods
Olfactory model Thus, a mitral unit represents a mitral cell column, and each mitral unit is
connected to one glomerular unit. In the same manner, the granular units represent groups of
granular cells. Thus, in the proposed model, the mitral units and granular units share the same
spatial distribution as the glomerular units. To determine the synapse connection matrices, we consider each type of neuron to form a
layer on a spherical surface in the olfactory bulb. First, a unit is placed on a two-dimensional
α-β coordination, as shown in Fig 3. Each mitral unit is then connected to the other mitral
units within a distance of zm units based on the actual connection structure of mitral cells [18]. In the same manner, each granular unit is connected to the mitral units within a distance of zg
units. If this distance exceeds the extent of the two-dimensional surface, the connection is
folded back to the opposite end, considering the spherical surface of the olfactory bulb shown
on the right of Fig 3. The gray shadow in Fig 3 is an example of the connection range from the
i-th unit located at (αi, βi). To achieve such a connection, the following equations are used to
determine the synapse connection matrices: ð7Þ daði;jÞ ¼ minððminðaA;i ai; aiÞ minðaA;i aj; ajÞÞ
2; ðai ajÞ
2Þ
ð7Þ dbði;jÞ ¼ minððminðbB;i bi; biÞ þ minðbB;i bj; bjÞÞ
2; ðbi bjÞ
2Þ
ð8Þ dbði;jÞ ¼ minððminðbB;i bi; biÞ þ minðbB;i bj; bjÞÞ
2; ðbi bjÞ
2Þ
ð8Þ ð8Þ dði;jÞ ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
d2
aði;jÞ þ d2
bði;jÞ
q
ð9Þ dði;jÞ ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
d2
aði;jÞ þ d2
bði;jÞ
q ð9Þ PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016
5 / 16 5 / 16 Olfactory Model for Selective Adaptation Mechanism Fig 3. Configuration of synapse connections. The figure represents the connection range of a mitral or a granular unit. A unit at the center of the
grey circle is connected to all units within a range of the circle. If the connection range exceeds the limit, it is folded back to the other end, considering
the bulbous shape of the olfactory bulb, using Eqs (7)–(12). Fig 3. Configuration of synapse connections. The figure represents the connection range of a mitral or a granular unit. PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 where dα(i,j) and dβ(i,j) denote the distance between two units along the α-axis and β-axis,
respectively. H0 ¼ ½H0ð1;1Þ; . . . ; H0ðj;kÞ; . . . ; H0ð1805;1805Þ 2 R18051805 is the synapse connection
matrix from granular units to mitral units, Materials and Methods
Olfactory model A unit at the center of the
grey circle is connected to all units within a range of the circle. If the connection range exceeds the limit, it is folded back to the other end, considering
the bulbous shape of the olfactory bulb, using Eqs (7)–(12). doi:10.1371/journal.pone.0165230.g003 doi:10.1371/journal.pone.0165230.g003 H0ði;jÞ ¼
Ri;j
zm
; if 0 < dði;jÞ < zm
0; if zm < dði;jÞ
ð10Þ
8
>
<
>
:
W0ði;jÞ ¼
Rw;i
zg
; if 0 < dði;jÞ < zg
0; if zg < dði;jÞ
ð11Þ
8
>
<
>
:
L0ði;jÞ ¼
Rl;i;
if 0 < dði;jÞ < zg
0; if zg < dði;jÞ
ð12Þ
( H0ði;jÞ ¼
Ri;j
zm
; if 0 < dði;jÞ < zm
0; if zm < dði;jÞ
ð10Þ
8
>
<
>
: ð10Þ ð11Þ ð12Þ where dα(i,j) and dβ(i,j) denote the distance between two units along the α-axis and β-axis,
respectively. H0 ¼ ½H0ð1;1Þ; . . . ; H0ðj;kÞ; . . . ; H0ð1805;1805Þ 2 R18051805 is the synapse connection
matrix from granular units to mitral units, where dα(i,j) and dβ(i,j) denote the distance between two units along the α-axis and β-axis,
respectively. H0 ¼ ½H0ð1;1Þ; . . . ; H0ðj;kÞ; . . . ; H0ð1805;1805Þ 2 R18051805 is the synapse connection
matrix from granular units to mitral units, PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 6 / 16 Olfactory Model for Selective Adaptation Mechanism W0 ¼ ½W0ð1;1Þ; . . . ; W0ðj;kÞ; . . . ; W0ð1805;1805Þ 2 R18051805 is that from mitral units to granular
units, and L0 ¼ ½L0ð1;1Þ; . . . ; L0ðj;kÞ; . . . ; L0ð1805;1805Þ 2 R18051805 is that between mitral units. Note that, in Eqs (10) and (11), Ri,j, Rw,i, Rl,i are random numbers selected from the normal
distribution N(1.0,0.05), and each connection parameter is divided by the connection range
parameters zm or zg so that the input strength to a unit is independent of the connection range
parameters. Dissimilarity Evaluation Part. The mitral cells transfer neural activity to the olfactory
cortex, and higher brain functions judge the odor quality based on individual experiences
[23]. In the proposed model, the dissimilarity evaluation simulates this role by calculating a
dissimilarity index based on the Pearson correlation between the spatial patterns of the
mitral layer evoked by different odors. Materials and Methods
Olfactory model The action potential of each mitral unit responding
to an odor oγ(nq) composed of odorants nq is averaged over the respiration duration
(texhale = 400 [ms]) using the following equation to give the spatial activity pattern of the
mitral layer Sog ¼ ½S1;og; . . . ; Sk;og; . . . ; S1805;og 2 R18051. Si;og ¼
Z texhale
0
xiðtÞ
maxðxiÞ dt
ð13Þ ð13Þ The dissimilarity index between arbitrary odors o1 and o2 is then calculated by the follow-
ing equation, which reflects the ease of discrimination, that is, the discrimination rate
obtained from the mice experiment. The dissimilarity index between arbitrary odors o1 and o2 is then calculated by the follow-
ing equation, which reflects the ease of discrimination, that is, the discrimination rate
obtained from the mice experiment. E ¼ 1 Pn
i¼1ðSi;1 Si;1ÞðSi;2 Si;2Þ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 ðSi;1 Si;1Þ
2
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 ðSi;2 Si;2Þ
2
q
ð14Þ ð14Þ Therefore, the dissimilarity index E can be compared to the observed discrimination rate
to test whether the model can predict the perceptual similarity exhibited by mice, from
which the selective adaptation mechanism in the olfactory system was identified. PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 STEP 1: Simulation of conditioning training STEP 1: Simulation of conditioning training Based on the mice experiment [16], the rewarded odor ([IA, EB, Ci], cf. Table F in S1
Appendix) was input to the proposed model. Assuming that selective adaptation is caused
by the interaction between mitral cells and granular cells, the proposed model used the fol-
lowing Hebbian rule [20, 24] to learn the synapse connection matrix H: _H ijðtÞ ¼ Z1H2
ij þ Z2gxðxiðtÞÞgyðyjðtÞÞ
ð15Þ ð15Þ _H ijðtÞ ¼ Z1H2
ij þ Z2gxðxiðtÞÞgyðyjðtÞÞ where the learning rates were set to η1 = 10−5 and η2 = 10−3 [19], and the adjustment was termi-
nated when the connection parameters converged to constant values. Here, the activity pattern
of the mitral layer when rewarded odor [IA, EB, Ci] was input after parameter learning is
denoted as S1 ¼ ½S1;1; . . . ; Sk;1; . . . ; S1805;1 2 R18051. STEP 2: Odor discrimination simulation Based on the mice experiments, five discrimination target odors (except the rewarded
odor [IA, EB, Ci], cf. Table F in S1 Appendix) were input to the model with adjusted
synapse connection matrix H, and each activity pattern obtained was represented by
S2 ¼ ½S1;2; . . . ; Sk;2; . . . ; S1805;2 2 R18051. Activity patterns S1 evoked by the rewarded
odor and S2 evoked by the discrimination target were substituted into Eq (14), and the dis-
similarity index E was calculated. As the dissimilarity index corresponds to the discrimina-
tion rate obtained from the experiments on mice, we computed the Pearson correlation
between the dissimilarity indices and the discrimination rate. Parameter analysis. To test the dependency of the prediction accuracies on the connec-
tion structure in the mitral and granular layers, various connection ranges zm and zg were iter-
atively searched. Based on a previous study [25, 26], we assumed that granular cells have
a wider range of influence than the mitral cells, and searched for parameters in the range
3 ≦zm ≦7, 13 ≦zg ≦17. We then determined the combination of zm and zg that yielded the
highest correlation between the dissimilarity index E and discrimination rate of rats. We also
investigated the changes in the activity patterns of the mitral layer and synapse connection
parameters in H along with the Hebbian learning. Olfactory Model for Selective Adaptation Mechanism generate different connection matrices allow us to test the robustness of prediction ability
against different configurations of initial values. Prediction of perceptual similarity. Simulation procedure In our simulations, the synapse connection matrix H was adjusted using a Hebbian learning
rule [20, 24] based on the experimental procedure applied to mice [16], and then the dissimi-
larity index obtained from the model was compared to the experimentally observed discrimi-
nation rate. We consider the proposed model to correctly account for selective adaptation if it
can predict the perceptual similarity of mice. This section describes the simulation procedure,
including a parameter adjustment and comparison method between the simulations and
experiments. Configurations. The majority of the parameters included in the proposed model were
determined based on the Li–Hopfield model [19] and the Erdi model [20], as shown in Tables
C and D in S1 Appendix. The newly introduced parameters in the proposed model are the
threshold θ that determines the activity pattern of the glomerular layer, and zm, zg that deter-
mine the synapse connection range of mitral units and granular units, respectively. The thresh-
old parameter was set to θ = 0.60 to enhance the strongly activated part corresponding to a z-
score greater than 2 measured by the 2-DG method. The synapse connection range parameters
were set to zm = 4 and zg = 15 under the assumption that granular cells have a wider influential
range than the mitral cells [25]. In addition, each component of the synapse connection matri-
ces H, L, W was determined by Eqs (10)–(12), and 20 sets of initial connection matrices were
generated to perform the following simulations. The random numbers Ri,j, Rw,i, Rl,i used to 7 / 16 PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 Results and Discussion
Results (a) Output from the glomerular layer for odorant input [IA, Ci, EB]. The figure shows the steps taken to generate output for the glomerular layer. The activity strength is represented in grayscale, where whiter pixels
correspond to higher activity. The uppermost row shows the glomerular activity pattern for the odorant composing odor [IA, EB, Ci] obtained from
Johnson et al. [27–31]. The middle row shows the binarized activity patterns given by adapting Eq (2) after dividing the pixels into 1805 lattices and
generalizing the activity strength into the range [0, 1]. The third row shows the output from the glomerular layer, and the bottom row shows that from the
mitral layer generated by the procedure described in Section 2. (b) Comparison between discrimination rates of mice obtained from experiments and
dissimilarity obtained from simulations (ζm = 4, ζg = 15). The figure compares the dissimilarity index E obtained from the simulation and discrimination
rate for each odor. The orange bars denote simulation results and the blue bars represent the experimental results. The error bars added to the
experimental results address the standard deviation in 10 mice, and the error bars added to the simulation results correspond to the standard deviation Fig 4. Prediction of perceptual similarity for odors composed of IA, EB, and Ci. (a) Output from the glomerular layer for odorant input [IA, Ci, EB]. The figure shows the steps taken to generate output for the glomerular layer. The activity strength is represented in grayscale, where whiter pixels
correspond to higher activity. The uppermost row shows the glomerular activity pattern for the odorant composing odor [IA, EB, Ci] obtained from
Johnson et al. [27–31]. The middle row shows the binarized activity patterns given by adapting Eq (2) after dividing the pixels into 1805 lattices and
generalizing the activity strength into the range [0, 1]. The third row shows the output from the glomerular layer, and the bottom row shows that from the
mitral layer generated by the procedure described in Section 2. (b) Comparison between discrimination rates of mice obtained from experiments and
dissimilarity obtained from simulations (ζm = 4, ζg = 15). The figure compares the dissimilarity index E obtained from the simulation and discrimination
rate for each odor. The orange bars denote simulation results and the blue bars represent the experimental results. Results and Discussion
Results Fig 4 shows the simulation results for an odor set composed of odorants Isoamyl acetate (IA),
Etyhl butyrate (EB), and Citral (Ci). Fig 4(a) shows the transformation from glomerular activ-
ity patterns of IA, EB, and Ci into the input of the glomerular layer based on the procedure
described in Section 2. Fig 4(b) compares the dissimilarity index E obtained from the simula-
tion along with the discrimination rate of mice. Fig 4(c) shows a scatter plot of the dissimilarity
index E and discrimination rate. Fig 4(b) and 4(c) confirm a strong correlation (r = 0.88,
p = 0.019) between the discrimination rate of mice and the dissimilarity index E. = 0.019) between the discrimination rate of mice and the dissimilarity index E. To evaluate the prediction ability of perceptual similarity, multiple testing was performed
using Bonferroni’s method on the discrimination rate of mice between odor pairs. The results PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 8 / 16 Olfactory Model for Selective Adaptation Mechanism Fig 4. Prediction of perceptual similarity for odors composed of IA, EB, and Ci. (a) Output from the glomerular layer for odorant input [IA, Ci, EB]. The figure shows the steps taken to generate output for the glomerular layer. The activity strength is represented in grayscale, where whiter pixels
correspond to higher activity. The uppermost row shows the glomerular activity pattern for the odorant composing odor [IA, EB, Ci] obtained from
Johnson et al. [27–31]. The middle row shows the binarized activity patterns given by adapting Eq (2) after dividing the pixels into 1805 lattices and
generalizing the activity strength into the range [0, 1]. The third row shows the output from the glomerular layer, and the bottom row shows that from the
mitral layer generated by the procedure described in Section 2. (b) Comparison between discrimination rates of mice obtained from experiments and
dissimilarity obtained from simulations (ζm = 4, ζg = 15). The figure compares the dissimilarity index E obtained from the simulation and discrimination
rate for each odor. The orange bars denote simulation results and the blue bars represent the experimental results. The error bars added to the
experimental results address the standard deviation in 10 mice, and the error bars added to the simulation results correspond to the standard deviation Fig 4. Prediction of perceptual similarity for odors composed of IA, EB, and Ci. Olfactory Model for Selective Adaptation Mechanism of 20 sets of synapse connection parameters. Orange and blue lines above the bars denote a significant difference of p<0.01 between odor pairs. Orange lines represent multiple comparison results from the simulation, and blue lines represent that of experiments on mice. (c) Scatter plot between
discrimination rates of mice obtained from experiments and dissimilarity index E obtained from simulations. The figure shows a scatter plot between
the dissimilarity index and discrimination rate. The error bars correspond to those in (b). doi:10.1371/journal.pone.0165230.g004 doi:10.1371/journal.pone.0165230.g004 are shown in Fig 4(b), where blue lines above pairs of bars denote a significant difference
(p<0.01) between the corresponding odor pairs, and in the second column of Table 1, where
double asterisks represent a significant difference. Multiple testing was also adopted for the
dissimilarity index E between odor pairs. The results are shown in Fig 4(b) and the third col-
umn of Table 1. These results show that the dissimilarity index E of [IA, EB] is significantly
lower (p<0.01: green background in Table 1) than that for the other five odors ([IA Ci], [EB
Ci], [IA], [EB], [Ci]), the dissimilarity index of [Ci] is significantly higher (p<0.01: yellow
background in Table 1) than that of the other odors ([IA EB], [IA Ci], [EB Ci], [IA], [EB]), and
there is little significant difference among the dissimilarity indices of the four odors [IA, Ci],
[EB, Ci], [IA], and [EB] (white background in Table 1). This result is generally consistent with
the discrimination rates of mice, as shown in the second column of Table 1. In the above simulations, the Hebbian learning rule [20, 24] was used to learn the synapse
connection matrix H. Fig 5(a) shows the parameters in H with respect to time, and Fig 5(b)
shows the activity pattern of the mitral layer with respect to the respiratory cycle. Fig 5(a) con-
firms the convergence of the synapse connection parameters within five respiratory cycles. Fig
5(b) demonstrates that part of the input activity pattern has been enhanced, while the remain-
der is inhibited as the Hebbian learning evolves. The parameters zm and zg, determine the number of neighbor units connected to each
mitral and granular unit, and correspond to the axonal length extended from the mitral cells
and granular cells in the actual olfactory bulb, respectively. Results and Discussion
Results The error bars added to the
experimental results address the standard deviation in 10 mice, and the error bars added to the simulation results correspond to the standard deviation PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 9 / 16 doi:10.1371/journal.pone.0165230.t001 between the dissimilarity index E and discrimination rate of mice [16] are shown in Fig 6,
from which the best parameter combination was determined to be zm = 4 and zg = 15. between the dissimilarity index E and discrimination rate of mice [16] are shown in Fig 6,
from which the best parameter combination was determined to be zm = 4 and zg = 15. To test the dependency on the con-
nection structure, the parameters zm and zg, were iteratively varied over the range 3–7 and 13–
17, respectively (giving a total of 25 parameter combinations). The resultant correlations Table 1. Difference between discrimination rates (Experiment) of odor pairs and that between dissimi-
larity index (Simulation). Odor pair
Experiment
Simulation
[IA, EB]-[IA, Ci]
**
**
[IA, EB]-[EB, Ci]
**
**
[IA, EB]-[IA]
**
**
[IA, EB]-[EB]
**
**
[IA, EB]-[Ci]
**
**
[IA, Ci]-[EB, Ci]
-
-
[IA, Ci]-[IA]
-
-
[IA, Ci]-[EB]
-
**
[EB, Ci]-[IA]
-
-
[EB, Ci]-[EB]
-
-
[IA]-[EB]
-
-
[Ci]-[IA]
-
**
[Ci]-[EB]
**
**
[Ci]- [IA, Ci]
**
**
[Ci]- [EB, Ci]
**
**
**: p<0.01
doi:10.1371/journal.pone.0165230.t001 Table 1. Difference between discrimination rates (Experiment) of odor pairs and that between dissimi-
larity index (Simulation). PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 10 / 16 Olfactory Model for Selective Adaptation Mechanism Fig 5. Results of Hebbian learning. (a) Convergence of the synapse connection strength of H. (b) Changes of activity patterns in
mitral layer. The deep red parts are the most activated and blue parts are least activated. doi:10.1371/journal.pone.0165230.g005 Fig 5. Results of Hebbian learning. (a) Convergence of the synapse connection strength of H. (b) Changes of activity patterns in
mitral layer. The deep red parts are the most activated and blue parts are least activated. doi:10 1371/journal pone 0165230 g005 Fig 5. Results of Hebbian learning. (a) Convergence of the synapse connection strength of H. (b) Changes of activity patterns in
mitral layer. The deep red parts are the most activated and blue parts are least activated. doi:10.1371/journal.pone.0165230.g005 doi:10.1371/journal.pone.0165230.g005 doi:10.1371/journal.pone.0165230.g005 PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 Discussion Fig 4(a) shows that the output of the glomerular layer for the odor [IA, EB, Ci] is a linear addi-
tion of the activity patterns of each odorant component. This representation is consistent with
the measurement data provided by Belluscio and Katz [11]. The output of the mitral layer sup-
pressed part of the activity in the glomerular layer, which may correspond to selective adapta-
tion. Indeed, Fig 4(c) confirms that there is a strong correlation between the dissimilarity
index E and the discrimination rate of mice (r = 0.88, p = 0.019). The mice find it difficult to
distinguish between the full mixture odor [IA, EB, Ci] and the subset mixture odor [IA, EB]. These results can be interpreted as the mice learning to selectively adapt and attend to certain
glomeruli, which happen to be very similar for the full mixture as well as the subsets that mice
have difficulty identifying. Fig 4 also show that accurate perceptual similarity of odors [IA, Ci], [EB, Ci], [IA Ci], [IA],
and [EB] to odor [IA EB Ci] are difficult to predict, and the correlation largely depends on [IA, 11 / 16 PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 Olfactory Model for Selective Adaptation Mechanism Fig 6. Parameter set ζm, ζg and prediction accuracy. Pearson correlations between
and discrimination rate of the mice obtained from experiments are plotted with different
doi:10.1371/journal.pone.0165230.g006
Olfactory Model for Selective A Fig 6. Parameter set ζm, ζg and prediction accuracy. Pearson correlations between dissimilarity index E
and discrimination rate of the mice obtained from experiments are plotted with different ζg and ζm. Fig 6. Parameter set ζm, ζg and prediction accuracy. Pearson correlations between dissimilarity index E
and discrimination rate of the mice obtained from experiments are plotted with different ζg and ζm. doi:10.1371/journal.pone.0165230.g006 PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 12 / 16 Olfactory Model for Selective Adaptation Mechanism EB] and [Ci]. However, Table 1 demonstrates that the model can predict three levels of odor
similarity: odor [IA, EB] is significantly similar to [IA, EB, Ci], odor [Ci] is significantly dis-
similar to [IA, EB, Ci], and odors [IA, Ci], [EB, Ci], [IA], and [EB] are moderately similar to
[IA, EB, Ci]. In this regard, the results of multiple testing presented in Table 1 indicate the
robustness of the prediction ability of the model against random initial values of connection
matrices H, W, and L. Discussion The proposed model can therefore be considered to have the ability to
predict the perceptual similarity between odors. In other words, as selective adaptation was
observed in the odor discrimination rate of mice and the model can predict this discrimination
rate, we can conclude that the model is sufficiently accurate to account for selective
adaptation. Fig 5(a) shows that the synapse connection parameters converged after Hebbian learning,
and the learning step can be terminated after five respiratory cycles. Fig 5(b) shows that part of
the activity pattern input from the glomerular layer to the mitral layer was enhanced, whereas
the other part was suppressed. This simulation result may correspond to the measurement
data reported by Giraudet et al. [19], which indicated that one of the binary mixture compo-
nents generally dominates the activity pattern on the mitral layer. Therefore, the measurement
data of Giraudet et al. [19] may be a neural representation of selective adaptation in the olfac-
tory system. Further investigation into selective adaptation should therefore measure the activ-
ity of mitral cells. In addition, as the selective adaptation emerged from Hebbian learning, our
approach could be used to account for the difference in perceptual characteristics caused by
odor experiences. From the results of the parameter search described in Fig 6, the connection range parame-
ters of mitral cells and granular cells were determined to be zm = 4 and zg = 15, which yields
the highest correlation with the odor discrimination rates of mice [16]. The anatomy of the
olfactory bulb, however, is not consistent with this straightforward result, because the den-
drites extending from the granular cells are shorter than those from the mitral cells [32]. How-
ever, as the number of granular cells is approximately 100 times that of mitral cells [26], we
assumed that the granular cells have a wider influential range than the mitral cells, and
searched within the range of 3 ≦zm ≦7, 13 ≦zg ≦17. While this assumption was derived
from a previous olfactory model proposed by Linster et al. [25], it is also possible that the corti-
cal feedback onto granular cells gives a broader view and suppresses the response of mitral
cells in an odor-selective manner. It has long been known that the olfactory cortex sends feed-
back to the olfactory bulb [33]. The role of this feedback was analyzed by Boyd et al. PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 Conclusion We have proposed an olfactory model based on previous models [19, 20], as well as some bio-
logical facts revealed in previous studies. The proposed model was used to simulate an odor
discrimination experiment performed by Takiguchi et al. [16], and predicted the perceptual
similarity observed in mice. Our results indicate that the proposed model is able to account for
selective adaptation in the olfactory system, as observed in the odor discrimination rate of
mice. We plan to investigate whether the proposed model is also applicable to humans by clari-
fying their selective adaptation characteristics, and then improving the proposed model to pre-
dict humans’ perceptual characteristics. Discussion [34], who
found that pyramidal cells in the olfactory cortex send odor-selective excitatory feedback to
glomerular cells in the olfactory bulb, which gates the response of the mitral cells. The proposed model and the above discussion are based on the assumption that the sup-
pression of certain activities in the olfactory bulb is the cause of selective adaptation. If this is
correct, there are at least two more mechanisms that could be involved in selective adaptation. 1. Intraglomerular inhibition through periglomerular cells Interglomerular inhibition can decorrelate similar sensory inputs [35]. Selective adaptation
may be a side effect of such a decorrelation. 2. Inhibitory feedback from pyramidal cells in the olfactory cortex to the mitral cells via glo-
merular cells 2. Inhibitory feedback from pyramidal cells in the olfactory cortex to the mitral cells via glo
merular cells Selective adaptation should involve a learning process and be modulated according to the
given task, because successive odor discrimination experiments on mice produced substan-
tially improved discrimination rates for odor [IA EB] against odor [IA EB Ci] [16]. Such task-
oriented learning can only be directed by higher brain functions. In addition, the cortical 13 / 16 PLOS ONE | DOI:10.1371/journal.pone.0165230
December 19, 2016 Olfactory Model for Selective Adaptation Mechanism inhibitory feedback to the olfactory bulb helps amplify the odor-evoked inhibition [34], which
is an important aspect of selective adaptation. Therefore, cortical feedback is quite possibly
involved in selective adaptation and its modulation. In addition to the above scenarios, Linster et al. proposed the following mechanism:
3. Synaptic modulation in the olfactory cortex Selective adaptation may also be implemented in the olfactory cortex. Combined analysis of
computer models and behavior experiments on mice suggests that synaptic adaptation and
synaptic potentiation in the olfactory cortex can cause odor-specific habituation [36], which is
similar to selective adaptation. (Please note that this study did not discuss odorant mixtures or
measure activity patterns.) Although our simulation results suggest that selective adaptation forms solely in the olfac-
tory bulb, the above possibility should be considered in future to clarify the relationship
between selective adaptation and internal representation in the olfactory bulb and olfactory
cortex. To achieve this goal, further neural activity data are required from corresponding
behavior experiments. Supporting Information S1 Appendix. Fig A. Odor discrimination experiment using Y-maze. Fig B. Results of odor
discrimination experiment. Table A. Glomerular layer. Table B. Respiration function. Table C. Mitral layer and granular layer. Table D. Parameters related to synapse connection matrices. Table E. Dissimilarity evaluation part. Table F. Experimental procedure. (DOCX) Data curation: SN. Formal analysis: ZS SN. Formal analysis: ZS SN. Funding acquisition: ZS TT. Funding acquisition: ZS TT. Funding acquisition: ZS TT. Investigation: NT. Investigation: NT. Methodology: ZS. Conceptualization: TT. Conceptualization: TT. Data curation: SN. PLOS ONE | DOI:10.1371/journal.pone.0165230
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English
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Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics
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Journal of nanobiotechnology
| 2,017
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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. Elasto‑inertial microfluidics for bacteria
separation from whole blood for sepsis
diagnostics d Asim Faridi, Harisha Ramachandraiah, Indradumna Banerjee, Sahar Ardabili, Sergey Zelenin
Russom* Muhammad Asim Faridi, Harisha Ramachandraiah, Indradumna Banerjee, Sahar Ardabili, Sergey Zelenin
and Aman Russom* Abstract Background: Bloodstream infections (BSI) remain a major challenge with high mortality rate, with an incidence that
is increasing worldwide. Early treatment with appropriate therapy can reduce BSI-related morbidity and mortality. However, despite recent progress in molecular based assays, complex sample preparation steps have become critical
roadblock for a greater expansion of molecular assays. Here, we report a size based, label-free, bacteria separation
from whole blood using elasto-inertial microfluidics. Results: In elasto-inertial microfluidics, the viscoelastic flow enables size based migration of blood cells into a non-
Newtonian solution, while smaller bacteria remain in the streamline of the blood sample entrance and can be sepa-
rated. We first optimized the flow conditions using particles, and show continuous separation of 5 μm particles from
2 μm at a yield of 95% for 5 µm particle and 93% for 2 µm particles at respective outlets. Next, bacteria were continu-
ously separated at an efficiency of 76% from undiluted whole blood sample. Conclusion: We demonstrate separation of bacteria from undiluted while blood using elasto-inertial microfluidics. The label-free, passive bacteria preparation method has a great potential for downstream phenotypic and molecular
analysis of bacteria. Keywords: Micro particle separation, Elasto-inertial microfluidics, Sepsis, Sample preparation death in Germany [1]. In the case of septic shock, stud-
ies have shown that patient mortality will increase by
7.6% for each hour the antibiotic therapy is delayed [2],
and, if the initial antibiotic therapy is inappropriate, the
survival rate decreases from 52 to 10.3% [3]. Therefore,
in infectious diseases and sepsis diagnosis the foremost
important step is to find the suitable treatment and iden-
tification of the bacteria to prevent evolution of resistant
bacteria. Currently, blood-culturing method is the gold
standard for identification of microorganism. However,
the automated method still requires 24–72 h to get the
results [4]. This long turnaround time, especially for the
identification of antimicrobial resistance, is driving the
development of molecular diagnostics, often based on
polymerase chain reaction (PCR) and are used to detect
pathogens either from blood culture bottles [5–7] or
directly from blood [8–13]. While the total time for Journal of Nanobiotechnology Journal of Nanobiotechnology Faridi et al. J Nanobiotechnol (2017) 15:3
DOI 10.1186/s12951-016-0235-4 *Correspondence: aman.russom@scilifelab.se
Division of Proteomics & Nano‑biotechnology, School of Biotechnology,
Royal Institute of Technology KTH, SciLifeLab Tomtebodavägen 23,
17165 Solna, Sweden Background Despite progress in medical science, including the
development of effective therapies, infectious diseases
continue to cause millions of deaths worldwide, and
pathogens in food, animals, water, and plants cause dam-
age and production losses running into billions of dollars. To date, pathogens are typically detected only after they
have already caused massive damage. Improved diag-
nostic methods for infectious pathogens are, therefore,
urgently needed. For example, sepsis—an acute inflam-
matory response of immune-compromised patients to
certain pathogens—is the third most common cause of *Correspondence: aman.russom@scilifelab.se
Division of Proteomics & Nano‑biotechnology, School of Biotechnology,
Royal Institute of Technology KTH, SciLifeLab Tomtebodavägen 23,
17165 Solna, Sweden Faridi et al. J Nanobiotechnol (2017) 15:3 Page 2 of 9 diagnosis has been shortened significantly, the imple-
mentation of these molecular methods in clinics has
been severely hampered by their lack of sensitivity in
comparison with, for instance, blood culturing and the
need for complex, multi-step sample preparation. Some
of the factors affecting the quality in nucleic acid-based
methods are PCR inhibitors, abundant interfering human
DNA, the risk of carryover when processing several sam-
ples, inadequate lyses, and pathogens enclosed within or
adhering to human cells. Despite improvements, sample
preparation remains the bottleneck for the further devel-
opment and implementation of molecular diagnostics
in clinical settings. Hence, molecular diagnostics would
benefit from a rapid, integrated sample-preparation assay
method to isolate and enrich bacteria from complex sam-
ple matrices such as blood.l separation using inertial microfluidics. In addition, the
fact that bacteria are smaller than blood cells and will end
up in the “unfocused” stream, and in essence everywhere
in the channel cross-section makes it difficult to achieve
proper separation. Very recent, Hou et al. [22] used dean
flow fractionation to address this by introducing a sheath
flow at the inlet to pinch blood sample and size based
migration of the blood cells towards the inner wall while
the bacteria are lagging behind and could be extracted at
an efficiency of 70% [23]. In this work, we address this by
employing elasto-inertial microfluidics instead to differ-
entially migrate larger blood cells away from smaller bac-
teria in flow through straight channels. l
Using elasto-inertial microfluidics, it is possible to
migrate particles across streamlines and focus into a
single stream in three-dimensional channel depending
upon their size [24–30]. Background A number of investigations have
recently focused on optimizing different conditions like
concentration of non-Newtonian fluids (elastic forces)
and flow rates (inertial forces) [31]. The inertial and elas-
tic forces have been used in combination to separate
smaller and larger particles from each other [32] and for
separation of blood cell components [33]. Elasto-inertial
microfluidics was utilized by Liu et al. [34] to separate
bacteria (E. coli) from red blood cells without the use of
sheath flow. However, the channel dimension used is not
applicable for other blood cells as the smallest dimension
of the cross-section was 10 μm, which would easily clog
for applications using whole blood. Microfluidics has the potential of eliminating the short-
comings associated with complex sample preparation. Microfluidics provide a higher surface to volume ratio,
a faster rate of mass and heat transfer, and the ability to
handle very small volumes of reagents in microchannels
very precisely. Moreover, microfluidics open up the pos-
sibility for automated platforms with integrated microflu-
idic cartridges thereby reducing the risk of contamination
[9]. Therefore, a recent interest of microfluidic techniques
has been towards the separation of microorganisms from
blood. Different approaches to separate pathogens from
blood using affinity separation [13–15], size [16, 17] or
electrokinetic properties [18] have been demonstrated. These methods typically exploit the difference between
cell properties, such as the size, shape, density, deforma-
bility, electric/magnetic susceptibility, and hydrodynamic
properties. Among these parameters, size is an excellent
label-free biomarker for bacteria separation from blood.l In this paper, using elasto-inertial microfluidics, we
separate bacteria from undiluted whole blood by selec-
tively migrating blood cells away from the walls towards
the centerline of the channel while bacteria are remained
in the streamline they enter and separated. We first inves-
tigate the elastic and inertial forces theoretically using
simulations and experimentally using different sized par-
ticles and viscoelastic solutions. We optimized the flow
conditions to continuously separate large particles (5 μm)
from small particles (2 μm). Following, we applied the
optimal flow conditions to continuously separate bacteria
from undiluted whole blood. p
Very recent, inertial microfluidics has been described
as a high-throughput, simple method for precise manip-
ulation particles based on size [19]. Recently, Wu et al. Background [20], separated bacteria from diluted red blood cells
using ‘‘soft” inertial microfluidics that utilized deflection
of larger cells in an asymmetrical sheath flow around a
curvature while the smaller cells are kept on or near the
original flow streamline. While the yield was about 62%,
they obtained an impressive high purity of 99.7%. Simi-
larly, Mach et al. [21] used a straight channel to separate
bacteria from red blood sample using massively parallel
channels. Here, size-dependent inertial lift forces were
used to focus larger red blood cells as a method of cell
separation and the authors achieved 80% removal of bac-
teria from diluted red blood cells after two passes of the
single channel system.f Results and discussion Elasto‑inertial based particle focusing and separationlf Elasto‑inertial based particle focusing and separationlf Elasto inertial based particle focusing and separation
Elasto-inertial microfluidics harnesses a synergetic effect
of viscoelastic forces and inertial forces to focus parti-
cles based on size (Fig. 1). In pressure-driven viscoelas-
tic flows, the first and second normal stress differences
lead to particle migration across the streamlines and
occupy multiple equilibrium positions including four at
the corners and one at the center in flows through rec-
tangular channels [32]. By moderately increasing the flow
rate so that the fluid inertia becomes non-negligible, it is While promising, the narrow size difference between
microorganisms (typically 1–3 µm) and blood cells
(3–15 µm) has shown to be very difficult for bacteria Faridi et al. J Nanobiotechnol (2017) 15:3 Page 3 of 9 Fig. 1 Schematic illustration of elasto-inertial microfluidics based particle migration and separation in flow through straight channel. A mixture of
small (red) and large (blue) particles entered the channel along the sidewalls and non-Newtonian solution is used as sheath at the center. The net
result of elastic (Fe) and inertial forces (shear-induced lift force, FLS, and wall-induced lift force, FLW) affect the large particles such that they laterally
migrate to three dimensional single stream in the middle of channel while the small particles keep flowing along the wall and can be separated Fig. 1 Schematic illustration of elasto-inertial microfluidics based particle migration and separation in flow through straight channel. A mixture of
small (red) and large (blue) particles entered the channel along the sidewalls and non-Newtonian solution is used as sheath at the center. The net
result of elastic (Fe) and inertial forces (shear-induced lift force, FLS, and wall-induced lift force, FLW) affect the large particles such that they laterally
migrate to three dimensional single stream in the middle of channel while the small particles keep flowing along the wall and can be separated possible to reduce the focusing positions to a single one
at the center of the channel (Fig. 1).f Fig. 2 Comsol simulation showing first normal stress difference
distribution of non-Newtonian fluid in flow through straight rectan-
gular microchannel. The flow rate is varied from 6 µl/h (Re = 0.017) to
390 µl/h (Re = 1.082) at Weissenberg number (Wi): 0.2, 3.5, 5.2 and 7
respectively To quantitatively evaluate the effect of lift forces (FL)
and elastic forces (Fe), it is possible to employ two dimen-
sionless numbers: Reynolds number (Re) and Weissen-
berg number (Wi). Elasto‑inertial based particle focusing and separationlf 2 and 5 µm
particle suspension is introduced at inlet (highlighted as yellow square). At the outlets (highlighted as red square), the 2 µm follow the flow of stream
and collected at the side outlets, while the 5 µm particles migrated away from the streamline and focused at the centerline and collected through
the center outlet. b Focusing profile of 5 µm particles with respect to particle Reynolds number Rp and channel length. The red, yellow and green
bars are corresponding to no lateral displacement, semi displacement and complete displacement into the center channel position respectively. c
Lateral position displacement of 5 µm particles at Rp = 0.008 as the function of length. d Separation of 2 and 5 µm particles at different outlets. The
yield of the 5 μm particles collected at the middle outlet was 95%, and 93% for the 2 μm particles in the side outlet Fig. 3 Elasto-inertial particle focusing and separation. a Particle focusing and separation in flow through straight microfluidic channel. 2 and 5 µm
particle suspension is introduced at inlet (highlighted as yellow square). At the outlets (highlighted as red square), the 2 µm follow the flow of stream
and collected at the side outlets, while the 5 µm particles migrated away from the streamline and focused at the centerline and collected through
the center outlet. b Focusing profile of 5 µm particles with respect to particle Reynolds number Rp and channel length. The red, yellow and green
bars are corresponding to no lateral displacement, semi displacement and complete displacement into the center channel position respectively. c
Lateral position displacement of 5 µm particles at Rp = 0.008 as the function of length. d Separation of 2 and 5 µm particles at different outlets. The
yield of the 5 μm particles collected at the middle outlet was 95%, and 93% for the 2 μm particles in the side outlet Fig. 3 Elasto-inertial particle focusing and separation. a Particle focusing and separation in flow through straight microfluidic channel. 2 and 5 µm
particle suspension is introduced at inlet (highlighted as yellow square). At the outlets (highlighted as red square), the 2 µm follow the flow of stream
and collected at the side outlets, while the 5 µm particles migrated away from the streamline and focused at the centerline and collected through
the center outlet. Elasto‑inertial based particle focusing and separationlf The channel aspect
ration 1:1 [50 µm × 65 µm (width × height)] resulted
in particle focusing at the centerline based on size and
was chosen for further investigation (Fig. 3a). Specifi-
cally, solution containing the particles (2 and 5 µm) is
branched into two streams, one on either side of the
entry stream of the non-Newtonian fluid of PEO intro-
duced as a sheath at the center channel inlet. The par-
ticles entering the channel at the walls migrate towards
the centerline based on size, such that 5 μm particles
are fully focused at the center of the channel while the
smaller 2 μm particles remain close to the walls and
can easily be separated (Fig. 3a). For more systematic
analysis of the effect of flow rate on the particle migra-
tion, the solution containing 5 µm particles was intro-
duced at a constant flow rate of 30 µl/h while the PEO
solution, starting at 300 µl/h was increased at interval of 60 µl/h to cover Re values from 0.25 to 2.1 (correspond-
ing Rp 0.002–0.015 for 5 µm particles). Figure 3b shows
the effects of flow are on focusing length for 5 µm par-
ticles. A minimum of 15 mm channel length is required
to fully migrate 5 µm particles under the given PEO
concentration of 500 ppm. As expected, at low flow rate
(Rp < 0.006) no focusing is observed presumably due to
insufficient elastic forces developed. Furthermore, it is
noticeable that a minimum of 25 mm channel length is
required to obtain particle focusing when the flow rate
is increased (Rp > 0.007). The focusing channel length
is further decreased to 15 mm with increased flow rate
(Rp > 0.012). As the flow rate is increased further, parti-
cle defocusing is observed (see Additional file 2: Figure
S2). Particle defocusing is observed at Rp > 0.016, mainly
due to shear thinning effect. Furthermore, the effect of
non-Newtonian fluid concentration on particle focusing
was investigated. While the minimum required chan-
nel length was similar (15 mm), as the PEO concentra-
tion (viscosity) is increased the focusing is achieved at
relatively lower Rp values (see Additional file 3: Figure
S3). The 2 μm particles remained unfocused under all Fig. 3 Elasto-inertial particle focusing and separation. a Particle focusing and separation in flow through straight microfluidic channel. Elasto‑inertial based particle focusing and separationlf The channel Reynolds number (Re) is
defined to describe the magnitude ratio of inertial force
to viscous force, while Wi describes the magnitude ratio
of elastic force to viscous force (Additional file 1: Figure
S1). Particle’s Reynolds number (Rp) is another impor-
tant dimensionless number that accounts for particle size
[Rp = Re (a/Dh)2]. Using COMSOL multiphysics, the First Normal Dif-
ference distribution (N1) in non-Newtonian fluid was
modelled from very low flow i.e. Re = 0.02 (creeping
flow condition) up to Re = 1.08 (elasto-inertial combined
regime). As can be seen in Fig. 2, at lower Re and when
Wi is close to zero, the fluid is not perturbed notably, due
to insufficient elasto and inertial forces and the first nor-
mal stress difference is not distributed to induce Fe that
could affect particle position. Increasing the flow rate
increases the shear stress on the non-Newtonian solu-
tion, and under stress a non linear viscosity decreasing
behavior (shear thinning) will give rise to the regions of
higher and lower first normal stresses, distributed such
that five equilibrium position for particles are formed
[32, 35] most evident at Wi = 7 in Fig. 2. From the simu-
lation, around Wi = 3.5 and Re = 0.54 the normal stress
distribution differences becomes noticeable. The normal
difference distribution effect with respect to flow rate Fig. 2 Comsol simulation showing first normal stress difference
distribution of non-Newtonian fluid in flow through straight rectan-
gular microchannel. The flow rate is varied from 6 µl/h (Re = 0.017) to
390 µl/h (Re = 1.082) at Weissenberg number (Wi): 0.2, 3.5, 5.2 and 7
respectively around 5 μl/min is in agreement with previous work of
Yang et al. [32].f Following, we evaluated experimentally the (i) effect
of different particle size and channel length; (ii) effect of
different concentrations (viscosities) of non-Newtonian
fluid (PEO); and (iii) effect of different microchannel Faridi et al. J Nanobiotechnol (2017) 15:3 Page 4 of 9 dimensions for particle focusing. For the channel geom-
etry, we tested different aspect ratio (approx. 1:1, 1:2 and
1:3) using channel height of 65 μm. For channel aspect
ratio 1:2 and above we could not observe particle focus-
ing at the centerline for 10 and 5 µm particles for the
channel length tested (=35 mm). Bacteria separation from whole blood Bacteria separation from whole blood
Before bacteria separation from whole blood, we fist
investigated the effect of non-Newtonian solution on
migration of the blood components. As can be seen in
Fig. 4a, when Newtonian fluid (1× PBS) is used, there
is no selective migration of blood cell components
towards the centerline. However, using PEO we could
achieve migration of the blood components, where
the blood cells are focused at the centerline and can be
extracted (Fig. 4b). As can be seen in Fig. 4c, while the
total volumes are similar, the color from the center out-
let indicates highly concentrated blood cell components. Furthermore, using Coulter counter we analyzed the
WBCs from the two fractions collected over a range of
flow rates and obtained a separation efficiency of 92%
into the middle outlet (Fig. 4d). The migration of the
blood cells is affected by the total flow rate as well as
the relative inlet fraction of the whole blood to the PEO
sheath buffer. For instance, when the flow rate increases,
insufficient migration of the cells results in reduced sepa-
ration efficiency through the middle outlet. These results
clearly show that non-Newtonian fluid can effectively be
used to separate blood components, and could find sev-
eral applications including plasma separation from blood. Here, we have applied this for bacteria separation from
whole blood.l Culture-independent, PCR based, detection of patho-
gens directly from the patient’s blood is attractive to
accelerate the diagnostic process. However, the use
of whole blood in assays designed to detect pathogen
nucleic acid is challenging. An excess of human DNA
may hamper the detection of pathogen genomic mate-
rial or inhibit the PCR reaction [36, 37]. Furthermore,
hemoglobin traces may also inhibit PCR-based ampli-
fication [38]. Therefore, molecular methods are often
forced to use a relatively small volume of blood, which
affects the sensitivity. As shown above (see Fig. 4c), our
continuous flow sample preparation strategy significantly
reduces the complexity by getting rid of majority RBCs
and WBSs. However, for sepsis diagnostics, the amount
of bacteria present in the blood is very low—in the order
of 10–100 cfu/ml. Hence, to be clinically relevant it is
imperative to further improve the method in order to
recover all bacteria cells as well as improve the sample
throughput. Elasto‑inertial based particle focusing and separationlf b Focusing profile of 5 µm particles with respect to particle Reynolds number Rp and channel length. The red, yellow and green
bars are corresponding to no lateral displacement, semi displacement and complete displacement into the center channel position respectively. c
Lateral position displacement of 5 µm particles at Rp = 0.008 as the function of length. d Separation of 2 and 5 µm particles at different outlets. The
yield of the 5 μm particles collected at the middle outlet was 95%, and 93% for the 2 μm particles in the side outlet Faridi et al. J Nanobiotechnol (2017) 15:3 Page 5 of 9 seen in Fig. 5a, the sample collected from side outlet con-
tains bacteria while there are no bacterial seen in sample
collected from the middle outlet. For bacteria spiked in
whole blood, the blood sample was introduced at 30 µl/h
and the 500 ppm PEO at 360 µl/h. To quantify bacteria
spiked in whole blood as well as in PBS, we used plat-
ing after collection of the fractions (Fig. 5b). For PBS, the
82% bacteria remained at the side outlet while for blood
sample 76% of bacteria remained at the side outlet. The
reduced bacteria recovery at the side outlets in whole
blood sample compared to the PBS sample is mainly
attributed to particle–particle interaction as well as the
complex nature of the whole blood that could potentially
make the bacteria stick to blood cells and migrate along
to the middle outlet. We tested a range of different flow
rates (covering those of Fig. 4c) and found the bacte-
ria recovery from the side outlets to be relatively stable,
about 81–82% for bacteria spiked in PBS and 75–76%
for whole blood (see Additional file 5: Figure S5). Inter-
estingly, the bacteria recovery remained high (73% at
side outlet) when the blood flow rate was doubled from
30–60 µl/h. the flow rate and PEO concentrations tested, indicating
strong Rp (and hence particle size) dependence for focus-
ing (Fe ∝ a3 and FL ∝ a4). Next, to mimic bacteria separation, we used the opti-
mized channel dimension, PEO concentration and Re to
separate 2 µm from 5 µm particles. The channel dimen-
sions 50 µm × 65 µm (width × height) and PEO con-
centration of 500 ppm was used to achieve size-based
separation of the particles. Elasto‑inertial based particle focusing and separationlf At the sample flow rate of
30 µl/h and PEO flow rate of 360 µl/h (corresponding
to Rp 0.008), the minimum channel length to fully focus
5 µm particles was found around 25 mm (Fig. 3c). Using
this channel length and flow conditions, we could suc-
cessfully separated 5 µm particles from 2 µm particles
(Fig. 3d). The yield of the 5 μm particles, calculated as
fraction of 5 μm particles recovered through the middle
outlet to the total count, was 95%, and the yield was 93%
for the 2 μm particles in the side outlet. Bacteria separation from whole blood In elasto-inertial microfluidics, the synergetic effect
of visco-elastic forces and inertial forces are harnessed
such that particles can migrate and occupy a single
focusing point. This focusing phenomenon has been
used for various applications, including sheathless cell
ordering [39, 40] size based cell separation [33, 41, 42]
and cell stretching measurements [43]. Nam et al. [33]
used similar channel geometry as ours to separate plate-
lets from diluted blood components with extremely high
purity (close to 99.9%). However, the relatively slow flow
rate combined with the use of diluted blood sample
(less than 1% solid content) makes it un-applicable for Based on the flow conditions, we tested how E. coli
bacteria will behave first using buffer and then spiked in
whole blood. For the tests in PBS, we used fluorescent-
tagged bacteria spiked at flow rates of 30 µl/h from side
inlet and 500 ppm PEO was introduced at flow rate of
360 µl/h. As expected, the bacteria kept flowing along
the channel wall and remain unaffected by normal stress
due to its size (see Additional file 4: Figure S4). As can be Faridi et al. J Nanobiotechnol (2017) 15:3 Page 6 of 9 Fig. 4 Elasto-inertial focusing of blood cells. Whole blood introduced from side inlet and Newtonian solution (a) or non-Newtonian (b) is intro-
duced from middle inlet. No lateral migration of blood cell is observed for Newtonian solution, while for the non-Newtonian (PEO) fluid, blood
cells are migrated away from the side channel towards the centerline of the channel. c The collected sample from middle (M) and side (S) outlets. d Quantification of the blood cells migration at different flow rates. The blood cells migration efficiency towards the middle outlet, analyzed by
Coulter counter, was between 76 and 92% depending on flow rate. When the blood flow rate was increased from 30 to 60 µl/h, the efficiency was
decreased to 71% Fig. 4 Elasto-inertial focusing of blood cells. Whole blood introduced from side inlet and Newtonian solution (a) or non-Newtonian (b) is intro-
duced from middle inlet. No lateral migration of blood cell is observed for Newtonian solution, while for the non-Newtonian (PEO) fluid, blood
cells are migrated away from the side channel towards the centerline of the channel. c The collected sample from middle (M) and side (S) outlets. d Quantification of the blood cells migration at different flow rates. Bacteria separation from whole blood The blood cells migration efficiency towards the middle outlet, analyzed by
Coulter counter, was between 76 and 92% depending on flow rate. When the blood flow rate was increased from 30 to 60 µl/h, the efficiency was
decreased to 71% of 60 µl/h tested in this work, it would take about 17 h
to process 1 ml blood. One way to improve through-
put is therefore through parallelization of the channels. Towards this, we have recently reported on a highly
scalable, parallel-channel, microfabrication method for
passive size-based particle separation [44]. Using the
16-channel parallel device [44], it would take only 1 h to
process 1 ml. While outside the scope of this paper, we
are currently working on combining the robust micro-
fluidic fabrication process of parallel channels (64 chan-
nels), with bacteria separation based on elasto-inertial bacteria separation applications. Here, we have demon-
strated bacteria separation from undiluted whole blood. While the method is immediately applicable as sample
preparation for MALDI-ToF MS based identification of
microorganisms from positive blood cultures, for blood
culture independent molecular diagnostics the method
needs to process ml volumes blood sample. The rela-
tive low volumetric flow rate is an inherent limitation of
elasto-inertial microfluidics since the synergetic effect of
the elastic forces and inertial forces are ideal at moder-
ate flow rates. For instance, even at volumetric flow rate Page 7 of 9 Faridi et al. J Nanobiotechnol (2017) 15:3 Fig. 5 Bacteria separation from PBS and whole blood. a Separation of
fluorescent tag expressed bacteria in PBS, bright field and fluorescent
images from middle (M) and side (S) outlets. b Quantitative separa-
tion efficiency of bacterial spiked PBS and whole blood. The yield
of bacteria collected at the side outlet was 82% for PBS and 76% for
bacteria spiked in whole blood by selectively migrating the larger blood cell components
from the sidewalls towards the centerline for separa-
tion. 76% of the bacteria were recovered at the side out-
let while 92% of the WBCs could be separated into the
middle outlet. The passive, label-free bacteria separa-
tion method is very promising and has great potential
as stand-alone sample preparation method or integrated
into lab-on-chip system for molecular and phenotypical
based sepsis diagnostics. Experimental Suspension of different fluorescent polystyrene particles
(2, 5 and 10 µm) were prepared in Phosphate Buffered
Saline (1× PBS). Poly (ethylene oxide) (Sigma Aldrich,
St Louis), Mw = 2,000,000 was prepared at different con-
centrations of 250, 500, 750 and 1000 ppm in 1× PBS. To
mimic the sepsis blood samples, blood samples obtained
from healthy blood donors were spiked with bacteria
(~106 cfu/ml). As a model strain, gram-negative E. coli
(strains BL21-A1) cultured in liquid medium, collected at
the mid-log phase, washed with PBS, was used to spike
the blood samples. Fig. 5 Bacteria separation from PBS and whole blood. a Separation of
fluorescent tag expressed bacteria in PBS, bright field and fluorescent
images from middle (M) and side (S) outlets. b Quantitative separa-
tion efficiency of bacterial spiked PBS and whole blood. The yield
of bacteria collected at the side outlet was 82% for PBS and 76% for
bacteria spiked in whole blood microfluidics. The method has potential value in clini-
cal sample preparation applications for both molecular
diagnostics as well as analysis by plating for antibiotic
susceptibility. For the particle based experiments, the solution con-
taining the particles were introduced into side inlet
using a syringe pump (Harvard apparatus PHD 2000,
Harvard Apparatus, USA) and Non-Newtonian fluid of
PEO was introduced into middle inlet by syringe pump
(NEMESYS, Cetoni Gmbh, Germany). The particles col-
lected from the side and center outlet were analyzed
using Coulter counter for quantification. For the bacteria
related work, initially PBS (1×) solution was spiked with
bacteria. This was followed by whole blood experiments
spiked with bacteria. The experimental procedure was
similar as for the particle suspension. Coulter counter
was used to quantify the white blood cells (WBCs) while
plating was used to quantify the bacteria. Microfluidic device fabricationh The polydimethylsiloxane (PDMS) microfluidic chips
with two inlets and two outlets were fabricated on a
master mould that was produced through photolithogra-
phy on a silicon wafer using SU-8 negative resist. PDMS
Sylgard 184 was then poured onto the SU-8 master in a
10:1 ratio, degassed, and cured at 65 °C overnight. The
PDMS slab was cut, the holes for inlets and outlet were
punched, and covalently bonded to glass slides using oxy-
gen plasma (CUTE Femto Science Co. Korea) treatment. The following cross section dimension (width × height)
were fabricated: 50 µm × 65 µm; 100 µm × 65 µm and
150 µm × 65 µm. Additional files MAF performed the experiments including the chip fabrication, preparation,
characterization, blood and bacteria separation experiment and analysis. HR
contributed in chip design and provide assistance in experiments and analysis. IB performed the Comsol simulations while SA and SZ contributed to bacteria
culturing. AR conceptualized, designed the chip and fabricated the master
mould. All authors provided scientific input during these studies and assisted
in preparation of the manuscript. All authors read and approved the final
manuscript. Additional file 1: Figure S1. Theoretical background of elasto-inertial
microfluidics. In non-Newtonian viscoelastic fluids, elastic forces (Fe) acts on
suspended particles directing them towards the centre and four corners of
microchannel. This migration of particle is the result of non-uniform normal
stress differences. For the shearing flow regime these stress are defined as
first (N1 = τxx − τyy) and second normal stress (N2 = τyy − τzz) differences. Here τ is the stress tensor and subscript x, y and z are denoting direction
of flow, direction of velocity gradient and z is the direction of vorticity
respectively. Since for most of the polymeric solution the N1/N2 ratio is less
than 0.1, the effect of N2 could be neglected while the elastic force, Fe, on
the particle is scaled with effect of N1 which varies depending upon the size
of particle (Fe ~ a3 ∇N1). This elastic effect could be defined by an important
dimensionless parameter known as Weissenberg number (Wi); which is the
ratio of viscous forces to elastic forces the fluid experiences. Wi, with some
simplifications, is defined as the product of shear rate (ϒ) to the relaxation
time (λ) of non-Newtonian fluid (λ): Wi = λ ϒ = λ2U/w = 2Q/hw2. Q is the
total flow rate and “w” and “h” are the width and height of microchannel
respectively. In flow through rectangular channels, one-way to reduce
the particle focusing points (away from the four corners of microchannel)
towards only the centre channel is by increasing the flow rate such that
inertial forces start to act on particles—and hence the term “elasto-inertial
microfluidics”. In inertial microfluidics, shear-induced lift force (FL,S) due to
shear gradient of the parabolic flow profile acts on suspended particles
towards the wall, while wall induces a lift force that directs the particles
away from the wall i.e. towards the centre of flow stream (FL,w). References 5 µm particle suspended in PBS (1×) is intro-
duced in micro channel with 500 ppm PEO non-Newtonian at (a) total flow
rate = 11 µl/min where 5 µm particle flow rate = 1 µl/min; Rp = 0.0132 (b)
total flow rate = 12 µl/min, 5 µm particle flow rate = 2 µl/min; Rp = 0.0144,
and (c) total flow = 13 µl/min, 5 µm particle flow rate = 3 µl/min;
Rp = 0.016, It could be seen that at (c) Rp 0.016 focused streams broadened
indicating the shear thinning effect and dilution of non-Newtonian fluid. Additional file 2: Figure S2. Particle defocussing due to shear thinning
effect as flow rate is increased. 5 µm particle suspended in PBS (1×) is intro-
duced in micro channel with 500 ppm PEO non-Newtonian at (a) total flow
rate = 11 µl/min where 5 µm particle flow rate = 1 µl/min; Rp = 0.0132 (b)
total flow rate = 12 µl/min, 5 µm particle flow rate = 2 µl/min; Rp = 0.0144,
and (c) total flow = 13 µl/min, 5 µm particle flow rate = 3 µl/min;
Rp = 0.016, It could be seen that at (c) Rp 0.016 focused streams broadened
indicating the shear thinning effect and dilution of non-Newtonian fluid. 7. Towns ML, Jarvis WR, Hsueh P-R. Guidelines on blood cultures. J Microbiol
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different concentrations of PEO (non-Newtonian fluid concentration of 250,
500, 750 and 1000 ppm), unfocussed (red), partially focused (yellow) and
focused (green) particle positions with respect to center of micro channel
are shown. Additional file 3: Figure S3. Conclusions We demonstrated bacteria separation from whole blood
based on elasto-inertial microfluidics. By harnessing
the synergetic effect of elastic and inertial forces, we
first demonstrate efficient particle separation where we
could separate 5 µm particles from 2 µm at a yield of
95% for 5 µm and 93% for 2 µm particles at the respec-
tive outlet fractions. Furthermore, we successfully dem-
onstrated bacteria isolation from undiluted whole blood Faridi et al. J Nanobiotechnol (2017) 15:3 Page 8 of 9 Competing interests The authors declare that they have no competing interests. Received: 22 July 2016 Accepted: 3 December 2016 Received: 22 July 2016 Accepted: 3 December 2016 Additional files Due to the
balance of these lift forces the particle attains a certain equilibrium position
within rectangular channel. The net lift force (FL) on particle is derived by
combination of both shear-induced lift force, FL,S and FL,W: FL = fc ρU2 a4/
Dh
2 = fc Rp2 µ2/ρ. Here, ρ is the density, U is the mean velocity and µ is
dynamic viscosity of the fluid. Particle diameter is denoted by a, and Dh
[=2wh/(w + h)] is the hydraulic diameter of microchannel. The coefficient
of inertial lift force, fc, is dependent on position of particle in the microchan-
nel and Reynolds number (Re = ρUDh/µ), which defines the scale of these
net forces within channel. Particle’s Reynolds number Rp is another impor-
tant dimensionless number [Rp = Re (a/Dh)2]. Elasto-inertial focusing is
achieved by optimizing the flow rate of the non-Newtonian fluid, such that
both inertial (FL) and elastic (Fe) forces act to focus relatively larger particle
into a single stream in 3D in flow through rectangular channels. Acknowledgementsi This work was financially supported by the Innovative Medicines Initiative, a
public–private partnership between the European Union, and the European
Federation of Pharmaceutical Industries and Associations (RAPP-ID project,
Grant agreement, No. 115153). References 5 µm particles focusing behavior mapped for
different concentrations of PEO (non-Newtonian fluid concentration of 250,
500, 750 and 1000 ppm), unfocussed (red), partially focused (yellow) and
focused (green) particle positions with respect to center of micro channel
are shown. 10. Mayeux PR. Pathobiology of lipopolysaccharide. J Toxicol Environ Health. 1997;51:415–35. 11. Ng AHC, Uddayasankar U, Wheeler AR. Immunoassays in microfluidic
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systems. Anal Bioanal Chem. 2010;397:991–1007. 12. Nguyen HB, Corbett SW, Steele R, Banta J, Clark RT, Hayes SR, et al. Imple-
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microchannel. The fluorescent tagged spiked bacteria in PBS was intro-
duced into PEO based elasto-inertial flow, and no displacement of bacteria
is observed close to the outlet in elasto inertial flow, presumably due to the
insignificant magnitude of forces on bacteria. Additional file 4: Figure S4. Confocal image of bacteria flowing inside
microchannel. The fluorescent tagged spiked bacteria in PBS was intro-
duced into PEO based elasto-inertial flow, and no displacement of bacteria
is observed close to the outlet in elasto inertial flow, presumably due to the
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(81–81% for PBS and 75–76% for whole blood) over the different flow rates. When the flow rate of the blood sample is doubled (from 30 to 60 µl/h), the
recovery rate of bacteria remained relatively high (73%). Additional file 5: Figure S5. Elasto-inertial microfluidics based bacteria
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(81–81% for PBS and 75–76% for whole blood) over the different flow rates. References 1. Engel C, Brunkhorst FM, Bone H-G, Brunkhorst R, Gerlach H, Grond S, et al. Epidemiology of sepsis in Germany: results from a national prospective
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rate = 11 µl/min where 5 µm particle flow rate = 1 µl/min; Rp = 0.0132 (b)
total flow rate = 12 µl/min, 5 µm particle flow rate = 2 µl/min; Rp = 0.0144,
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applications. Lab Chip. 2012;12:4644. • We accept pre-submission inquiries
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English
| null |
Effective nonvanishing of canonical Hecke $L$-functions
|
Proceedings of the American Mathematical Society
| 2,010
|
public-domain
| 3,123
|
PROCEEDINGS OF THE
AMERICAN MATHEMATICAL SOCIETY
Volume 138, Number 11, November 2010, Pages 3891–3897
S 0002-9939(2010)10430-6
Article electronically published on June 4, 2010 PROCEEDINGS OF THE
AMERICAN MATHEMATICAL SOCIETY
Volume 138, Number 11, November 2010, Pages 3891–3897
S 0002-9939(2010)10430-6
Article electronically published on June 4, 2010 (Communicated by Wen-Ching Winnie Li) Abstract. Motivated by work of Gross, Rohrlich, and more recently Kim,
Masri, and Yang, we investigate the nonvanishing of central values of L-
functions of “canonical” weight 2k −1 Hecke characters for Q(√−p), where
3 < p ≡3 (mod 4) is prime. Using the work of Rodriguez-Villegas and
Zagier, we show that there are nonvanishing central values provided that
p ≥6.5(k −1)2 and (−1)k+1
2
p
= 1. Moreover, we show that the num-
ber of such ψ ∈Ψp,k satisfies #{ψ ∈Ψp,k | L(ψ, k) ̸= 0} ≥
h(−p)
# Cl(K)[2k −1]. Received by the editors November 3, 2009 and, in revised form, February 4, 2010.
2010 Mathematics Subject Classification. Primary 11M99. ⃝
y
Reverts to public domain 28 years from publication c
⃝2010 American Mathematical Society
Reverts to public domain 28 years from publication 3891 c
⃝2010 American Mathematical Society 1. Introduction Throughout we suppose that 3 < p ≡3 (mod 4) is prime. Let K = Q(√−p)
be the corresponding imaginary quadratic field, and let OK be its ring of integers. A canonical Hecke character is a Hecke character ψ of K of weight 2k −1 and
conductor √−pOK satisfying ψ((α)) = ϵ(α)α2k−1
for (α, √−pOK) = 1, where ϵ is defined via the isomorphism OK/√−p ∼= Z/p and the Dirichlet character
−p
·
on Z/p. Since the canonical Hecke characters are determined on principal
ideals, there are precisely h(−p) of them, and if ψ is any one of them, they are
given by Ψp,k = {ψχ : χ ∈Cl(K)∧}. The L-function associated to a canonical Hecke character is L(ψ, s) =
0̸=a⊂OK
ψ(a)
N(a)s
for
ℜ(s) > k + 1
2. It has an analytic continuation to C and satisfies a functional equation under s →
k
2 It has an analytic continuation to C and satisfies a functional equation under s →
2k −s with root number (−1)k+1
2
(see [RVZ p 86]) 2k −s with root number (−1)k+1
2
p
(see [RVZ, p. 86]). 2k −s with root number (−1)k+1
2
p
(see [RVZ, p. 86]).
Our main result is the following effective nonvanishing theorem for the central
values L(ψ, k). 3891 GEORGE BOXER AND PETER DIAO 3892 Theorem 1. If k is a positive integer and 3 < p ≡3 (mod 4) is a prime for which
p ≥6.5(k −1)2 and (−1)k+1
2
p
= 1, then Theorem 1. If k is a positive integer and 3 < p ≡3 (mod 4) is a prime for which
Theorem 1. If k is a positive integer and 3 < p ≡3 (mod 4) is a prime for which
p ≥6.5(k −1)2 and (−1)k+1
2
p
= 1, then Theorem 1. If k is a positive integer and 3 < p ≡3 (mod 4) is a prime for which
p ≥6.5(k −1)2 and (−1)k+1
2
p
= 1, then
1
h(−p)
ψ∈Ψp,k
L(ψ, k) ≥
πkpk/2−3/4
18k−1(k −1)!e−π√p/4. p ≥6.5(k −1)2 and (−1)k+1
2
p
= 1, then 1
h(−p)
ψ∈Ψp,k
L(ψ, k) ≥
πkpk/2−3/4
18k−1(k −1)!e−π√p/4. In particular, there exists at least one ψ ∈Ψp,k such that L(ψ, k) > 0. 1. Introduction He uses a formula of Rodriguez-
Villegas and Zagier [RVZ] for L(ψ, k) as well as equidistribution of Heegner points
to obtain an asymptotic formula for the first moment 1
h(−p)
ψ∈Ψp,k
|L(ψ, k)| EFFECTIVE NONVANISHING OF CANONICAL HECKE L-FUNCTIONS 3893 as p tends to infinity. From here he uses a subconvexity bound of Duke, Friedlander,
and Iwaniec [DFI] to obtain a nonvanishing theorem of the form #{ψ ∈Ψp,k | L(ψ, k) ̸= 0} ≫pδ for some δ > 0 and all p sufficiently large. However, this result is ineffective due to
an application of Siegel’s theorem; thus one cannot say how large p must be taken
to guarantee the existence of a nonvanishing central value. The Rodriguez-Villegas and Zagier formula relates L(ψ, k) to a sum of a fixed
theta series evaluated at CM points. Our main observation in this paper is that we
can quantify the nonvanishing problem by studying the position of the CM point
corresponding to the trivial ideal class in the cusp. This idea of quantification in
the cusp also appears in work of Michel and Venkatesh [MV] on nonvanishing of
Rankin-Selberg L-functions. 1. Introduction In particular, there exists at least one ψ ∈Ψp,k such that L(ψ, k) > 0. Remark 2. One can obtain this result for general fundamental discriminants of
imaginary quadratic fields −3 > d ≡1 (mod 4) using Rodriguez-Villegas and Za-
gier formulae holding in that case (see [RV, p. 436] or [RVY, p. 561]). One obtains
the same lower bound in the same range. These central values first arose in connection with work of Gross [Gr] and
Rohrlich [R, R2] on ranks of elliptic Q-curves. More recently, Kim, Masri, and Yang
[KMY] have shown under some technical conditions that the nonvanishing of these
central values implies that certain Selmer groups associated to the characters ψ are
finite. The nonvanishing of the central values L(ψ, k) under the assumption that
(2k −1, h(−p)) = 1 has been studied extensively by Rohrlich, Rodriguez-Villegas,
Zagier, Yang, and many others (see for example [R, R2, MR, RVZ, MiY, Y, LX]). g
g
y
(
[
])
When (2k −1, h(−p)) > 1, Ψp,k breaks up into multiple Galois orbits. We recall
that the Galois group G = Gal(Q/K) acts on Ψp,k by ψσ(a) = ψ(a)σ
for
σ ∈G, where we have fixed an embedding K ⊂Q. By [R3] and [Ma2, Prop. 1.1], for any
ψ ∈Ψp,k, where we have fixed an embedding K ⊂Q. By [R3] and [Ma2, Prop. 1.1], for any
ψ ∈Ψp,k, #{ψσ | σ ∈G} =
h(−p)
# Cl(K)[2k −1], where Cl(K)[2k −1] is the (2k −1)-torsion subgroup of Cl(K). By an important
theorem of Shimura [Sh, p. 212], for σ ∈G and ψ ∈Ψp,k, L(ψσ, k) ̸= 0
⇐⇒
L(ψ, k) ̸= 0. The following corollary is now an immediate consequence of Theorem 1. Corollary 3. Under the same assumptions on p and k as in Theorem 1 we have #{ψ ∈Ψp,k | L(ψ, k) ̸= 0} ≥
h(−p)
# Cl(K)[2k −1]. Note that Siegel’s lower bound h(−p) ≫p1/2−ϵ and the widely believed upper
bound # Cl(K)[2k −1] ≪pϵ would imply #{ψ ∈Ψp,k | L(ψ, k) ̸= 0} ≫p1/2−ϵ. In [Ma], Masri proves nonvanishing theorems for canonical Hecke L–functions
which are valid even when (2k −1, h(−p)) > 1. 2. The formula of Rodriguez-Villegas and Zagier In this section we recall the Rodriguez-Villegas and Zagier formula for the central
values of canonical Hecke L-functions. In the notation of [RVZ], we first define the
real-analytic theta series θl+1/2 for l ≥0 by (1)
θl+1/2(z) =
1
(2πy)l/2
n≥1
n odd
−4
n
l
Hl(n
πy/2)eπin2z/4, (1) where Hl is the Hermite polynomial defined by (2)
Hl(z) =
0≤j≤l/2
l! j!(l −2j)!(−1)j(2z)l−2j. (2) Next, we define Heegner points suitable for evaluating modular forms of level a
power of 2. Given a primitive ideal a = [a, b+√−p
2
] prime to p, we are free to choose
b congruent to 1 modulo 16 and then we define (3)
z(2)
a
= b + √−p
2a
∈H. (3) With notation as above we recall the following theorem of Rodriguez-Villegas
and Zagier. Theorem 4 ([RVZ]). Let 3 < p ≡3 (mod 4) be a prime and let ψ be a canonical
Hecke character of weight 2k −1 associated to K with root number 1. Then one
has the following identity: L(ψ, k) = πkpk/2−3/4
2k−3(k −1)!
[a]∈Cl(K)
−4
N(a)
δ
ψ(a)
−1θk−1/2(z(2)
a2 )
2
,
here δ is defined by
2
p
= (−1)δ. L(ψ, k) = πkpk/2−3/4
2k−3(k −1)!
[a]∈Cl(K)
−4
N(a)
δ
ψ(a)
−1θk−1/2(z(2)
a2 )
2
, where δ is defined by
2
p
= (−1)δ. where δ is defined by
2
p
= (−1)δ. where δ is defined by
2
p
= (−1)δ. 3894 GEORGE BOXER AND PETER DIAO 3. Bounds for Hermite polynomials In this section we establish some facts concerning Hermite polynomials that will
be used in the proof of Theorem 1. First we need the following bound on the largest
zero of the Hermite polynomial Hl. Proposition 5 ([K, p. 36]). Let l ≥1. Then Hl(x) has no zeros for x ≥
√
2l −2. In particular, Hl(x) is positive in that range. Using this we can obtain upper and lower bounds for Hl(x). Lemma 6. For x >
√
2l we have Lemma 6. For x >
√
2l we have (2x/3)l ≤Hl(x) ≤(2x)l. Proof. First we prove the upper bound. It is clear for l = 0, 1 from the expressions
H0(x) = 1, H1(x) = 2x. For l ≥2 we proceed inductively using the standard
recurrence Hl(x) = 2xHl−1(x) −2lHl−2(x). For x >
√
2l, Proposition 5 implies Hl−2(x) ≥0 and so
Hl(x) ≤2xHl−1(x) ≤(2x)l Hl(x) = 2xHl−1(x) −2lHl−2(x). Hl(x) = 2xHl−1(x) −2lHl−2(x). on 5 implies Hl−2(x) ≥0 and so
l Hl(x) ≤2xHl−1(x) ≤(2x)l by induction. Similarly, the lower bound is immediately verified for l = 0, 1. For
l ≥2 we denote the largest real root of Hl(x) by rl. We use the standard fact that H′
l(x) = 2lHl−1(x). For y >
√
2l we can write Hl(y) =
y
rl
H′
l(x)dx =
y
rl
2lHl−1(x)dx ≥
y
√2l−2
2lHl−1(x)dx, where the last step is justified by Proposition 5 and rl > rl−1. Inductively using
the fact that Hl−1(x) ≥(2x/3)l−1 in the range x >
√
2l −2, we find that Hl(y) ≥
y
√2l−2
2l
2x
3
l−1
dx = 3
2
3
l
yl −(2l −2)l/2
. It remains to show that for y >
√
2l, It remains to show that for y >
√
2l, 3
2
3
l
yl −(2l −2)l/2
≥
2
3
l
yl, 2yl ≥3(2l −2)l/2. For y >
√
2l this follows from 2
3 > 1
√e >
1 −1
l
l/2
. □ Remark 7. Since Hl(x) ∼(2x)l as x tends to infinity, the lower bound in the above
lemma is far from optimal for x large. However, we are primarily interested in the
lower bound in the region where x is not much larger than the largest zero of Hl. Remark 7. 4. Proof of Theorem 1 4. Proof of Theorem 1 4. Proof of Theorem 1 We first use the Rodriguez-Villegas and Zagier formula to obtain an exact formula
for the first moment. By Theorem 4, we have that 1
h(−p)
ψ∈Ψp,k
L(ψ, k) =
Ck
h(−p)
ψ∈Ψp,k
[a]∈Cl(K)
−4
N(a)
δ
ψ(a)
−1θk−1/2(z(2)
a2 )
2
, where we denote Ck = πkpk/2−3/4
2k−3(k −1)!. 3. Bounds for Hermite polynomials Since Hl(x) ∼(2x)l as x tends to infinity, the lower bound in the above
lemma is far from optimal for x large. However, we are primarily interested in the
lower bound in the region where x is not much larger than the largest zero of Hl. EFFECTIVE NONVANISHING OF CANONICAL HECKE L-FUNCTIONS
3895 Recall that For (5)
π1/2p1/4
2
≥
√
2k −2 (5) we can use the estimates in Lemma 6 to obtain, for any integer m ≥0, a2m+3
a2m+1
=
Hk−1
(2m + 3)π1/2p1/4
2
e−π(2m+3)2√p/8
Hk−1
(2m + 1)π1/2p1/4
2
e−π(2m+1)2√p/8 a2m+3
a2m+1
=
Hk−1
(2m + 3)π1/2p1/4
2
e−π(2m+3)2√p/8
Hk−1
(2m + 1)π1/2p1/4
2
e−π(2m+1)2√p/8
≤
2m + 3
2m + 1
2k−2
32k−2e−π(m+1)√p
≤81k−1e−π√p. The above inequality holds for (5), which is true for p ≥6.5(k −1)2. Since p ≥7,
we uniformly obtain The above inequality holds for (5), which is true for p ≥6.5(k −1)2. Since p ≥7,
we uniformly obtain 81k−1e−π√p ≤1
3. Thus under these assumptions we have that |a2m+1| ≤|a1|
3m . Now we have Now we have |θk−1/2(z(2)
1 )| ≥
1
(π√p)
k−1
2
|a1| −
∞
m=1
|a2m+1|
≥
1
(π√p)
k−1
2
Hk−1
π1/2p1/4
2
e−π√p/8
1 −1
3 −1
32 −1
33 . . .
=
1
(π√p)
k−1
2
1
2
Hk−1
π1/2p1/4
2
e−π√p/8
≥
1
3k−1
1
2e−π√p/8 > 0, where we used the lower bound of Lemma 6 in the last step. For p ≥6.5(k −1)2,
substituting this lower bound into (4) yields where we used the lower bound of Lemma 6 in the last step. For p ≥6.5(k −1)2,
substituting this lower bound into (4) yields 1
h(−p)
ψ∈Ψp,k
L(ψ, k) ≥
πkpk/2−3/4
18k−1(k −1)!e−π√p/4. Recall that Ψp,k = {ψ0χ | χ ∈Cl(K)∧} Ψp,k = {ψ0χ | χ ∈Cl(K)∧} for a fixed ψ0 ∈Ψp,k. Squaring out the summand, interchanging orders of summa-
tion, and using the orthogonality relations yield 1
h(−p)
ψ∈Ψp,k
L(ψ, k) 1
h(−p)
ψ∈Ψp,k
L(ψ, k)
= Ck
[a],[b]∈Cl(K)
ψ0(ab)
−1
θk−1/2(z(2)
a2 )θk−1/2(z(2)
b2 )
−4
N(ab)
δ
1
h(−p)
χ∈Cl(K)∧
χ(ab)
= Ck
[a]∈Cl(K)
ψ0(aa)−1
θk−1/2(z(2)
a2 )
2
. = Ck
[a]∈Cl(K)
ψ0(aa)−1
θk−1/2(z(2)
a2 )
2
. Using the fact that ψ0(aa) = ψ0(N(a)) = N(a)2k−1, we obtain the following exact formula for the first moment: we obtain the following exact formula for the first moment: 1
h(−p)
ψ∈Ψp,k
L(ψ, k) = πkpk/2−3/4
2k−3(k −1)!
[a]∈Cl(K)
1
N(a)2k−1
θk−1/2(z(2)
a2 )
2
. Using positivity, we obtain the lower bound Using positivity, we obtain the lower bound 1
h(−p)
ψ∈Ψp,k
L(ψ, k) ≥πkpk/2−3/4
2k−3(k −1)! θk−1/2(z(2)
OK)
2
, (4) where w e e
z(2)
OK = 1 + √−p
2 z(2)
OK = 1 + √−p
2 is the Heegner point corresponding to the trivial ideal class. The Fourier expansion
of θk−1/2 at the cusp at infinity evaluated at the trivial Heegner point is is the Heegner point corresponding to the trivial ideal class. The Fourier expansion
of θk−1/2 at the cusp at infinity evaluated at the trivial Heegner point is θk−1/2
1 + √−p
2
=
1
(π√p)
k−1
2
n≥1
−4
n
k−1
Hk−1
nπ1/2p1/4
2
eπin2x/8e−πn2√p/8. For convenience, let an denote the nth term in this sum (note that an = 0 for n
even). The strategy is to bound the first term below in magnitude while comparing 3896
GEORGE BOXER AND PETER DIAO 896
GEORGE BOXER AND PETER DIAO GEORGE BOXER AND PETER DIAO 3896 3896 the rest of the terms to a geometric series by bounding the magnitude of the ratios
of consecutive terms. For the rest of the terms to a geometric series by bounding the magnitude of the ratios
of consecutive terms. References [DFI]
Duke, W., J. Friedlander, and H. Iwaniec, Bounds for Automorphic L-functions. II. Inventiones Mathematicae 115 (1994), 219–239. MR1258904 (95a:11044) [Gr]
B. Gross, Arithmetic on elliptic curves with complex multiplication. With an appendix
by B. Mazur. Lecture Notes in Mathematics, 776. Springer, Berlin, 1980. iii+95 pp. MR563921 (81f:10041) [KMY] B. D. Kim, R. Masri, and Tonghai Yang, Nonvanishing of Hecke L–functions and the
Bloch-Kato conjecture, preprint. [K]
Krasikov, Ilia, Nonnegative Quadratic Forms and Bounds on Orthogonal Polynomials. [K]
Krasikov, Ilia, Nonnegative Quadratic Forms and Bounds on Orthogonal Polyno
Journal of Approximation Theory 111 (2001), 31–49. MR1840019 (2002j:42034) [LX]
C. Liu and L. Xu, The vanishing order of certain Hecke L–functions of imaginary qua-
dratic fields. J. Number Theory 108 (2004), 76–89. MR2078658 (2005g:11221) (
)
(
)
[Ma]
R. Masri, Asymptotics for sums of central values of canonical Hecke L–series. Int. Math. R
N t IMRN 2007
19 A t ID
065 27
MR2359540 (2009b 11090) [Ma]
R. Masri, Asymptotics for sums of central values of canonical Hecke L–series. Int. Math. [Ma]
R. Masri, Asymptotics for sums of central values of canonical Hecke L–series. Int. M
Res. Not. IMRN 2007, no. 19, Art. ID rnm065, 27 pp. MR2359540 (2009b:11090) [Ma2]
R. Masri, Quantitative nonvanishing of L–series associated to canonical Hecke charac-
ters. Int. Math. Res. Not. IMRN 2007, no. 19, Art. ID rnm070, 16 pp. MR2359543
(2008j:11170) [MV]
P. Michel and A. Venkatesh, Heegner points and nonvanishing of Rankin-Selberg L–
functions. Proceedings of the Gauss-Dirichlet conference, Clay Math. Proc., 7, Amer. Math. Soc., Providence, RI, 2007. MR2362200 (2009a:11116) [MiY]
S. D. Miller and T. H. Yang, Nonvanishing of the central derivative of canonical Hecke
L–functions. Math. Res. Lett. 7 (2000), 263–277. MR1764321 (2001i:11058) [MR]
H. Montgomery and D. Rohrlich, On the L-functions of canonical Hecke characters of
imaginary quadratic fields. II . Duke Math. J. 49 (1982), 937–942. MR683009 (84e:12014) [MR]
H. Montgomery and D. Rohrlich, On the L-functions of canonical Hecke characters of
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[RV]
F. Rodriguez-Villegas, Square root formulas for central values of Hecke L-series II . Duke
M th J 72 (1993) 431 440 MR1248679 (95d 11156) imaginary quadratic fields. II . Duke Math. J. 49 (1982), 937 942. MR683009 (84e:12014)
[RV]
F. Rodriguez-Villegas, Square root formulas for central values of Hecke L-series II . Duke
Math. J. 72 (1993), 431–440. MR1248679 (95d:11156) [RVY]
F. Acknowledgements We are grateful to Riad Masri and Ken Ono for their guidance and support
throughout this project. We also thank the anonymous referee for pointing out
that our argument generalizes to general fundamental discriminant. References Rodriguez-Villegas and T. Yang, Central values of Hecke L–functions of CM number
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F. Rodriguez-Villegas and D. Zagier, Square roots of central values of Hecke L-series. Advances in number theory (Kingston, ON, 1991), 81–99, Oxford Sci. Publ., Oxford Univ. Press, New York, 1993. MR1368412 (96j:11069) [R]
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D. Rohrlich, On the L-functions of canonical Hecke characters of imaginary quadratic
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(57:3080) [Y] [Y]
T. H. Yang, Nonvanishing of central Hecke L-values and rank of certain elliptic curves. Compos. Math. 117 (1999), 337–359. MR1702416 (2001a:11093) Frist Center, Princeton University, Mailbox 2704, Princeton, New Jersey 08544 Frist Center, Princeton University, Mailbox 2868, Princeton, New Jersey 08544
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Cowpox with Severe Generalized Eruption, Finland
|
Emerging infectious diseases
| 2,003
|
cc-by
| 2,776
|
The Study Cowpox with a severe, generalized eruption was diag-
nosed in an atopic 4-year-old girl by electron microscopy,
virus
isolation,
polymerase
chain
reaction,
and
immunoglobulin (Ig) M and low-avidity IgG antibodies. The
hemagglutinin gene of the isolate clustered with a Russian
cowpox virus strain, and more distantly, with other cowpox
and vaccinia virus strains. The patient’s dog had
orthopoxvirus-specific antibodies, indicating a possible
transmission route. In Finnish wild rodents, orthopoxvirus
seroprevalences were 0%–92%, in humans the seropreva-
lence was 100% in the age group >50, decreasing towards
younger age groups. A 4-year-old girl from a small farm in eastern Finland
was hospitalized in September 2000 because of umbilicat-
ed vesicopapules, which developed over the previous 5
days (Figure 1), and unresponsiveness to cephalexin. She
had a past history of moderate atopic dermatitis. Animals
at her home farm included a horse, three dogs, and a rab-
bit, but she had no contact with cats because of allergy. On
examination, most lesions were located on her swollen red
extremities, a few were found on the side of her body, and
3-mm lesions were found on the face and vulva. All lesions
were at the same stage of development. She was febrile
with 38°C temperature and feeling unwell. A biopsy sam-
ple from a papule was sent to a virology laboratory, where
orthopoxvirus particles 230 × 300 nm in size were demon-
strated by electron microscopy with negative staining. C
owpox is a zoonotic dermatitis affecting, despite its
name, mainly cats and humans. The disease is caused
by cowpox virus, a close relative to vaccinia, smallpox
(variola),
and
monkeypox
viruses
within
the
Orthopoxvirus genus (1). The relationship between cow-
pox and vaccinia viruses has been unclear since Edward
Jenner used a virus isolate from cows for smallpox vacci-
nation (2,3). Orthopoxviruses, comprising a genus in the
family Poxviridae, are large, brick-shaped viruses with a
200-kbp DNA genome, and they replicate in the cytoplasm
(4). Because immunity to orthopoxviruses is cross-reac-
tive, smallpox vaccination might have suppressed cowpox
virus infections in the human population. Cowpox virus is
not highly infective for humans and usually produces a
localized lesion mainly on fingers, hands, or face (5). In
immunocompromised persons, however, the disease may
lead to death (6). The virus infects through skin abrasions,
resulting in successive lesions of macular, papular, vesicu-
lar, pustular, ulceral, and eschar stages for 2 weeks. Systemic symptoms are also common (5). DISPATCHES DISPATCHES *Faculty of Veterinary Medicine, Helsinki, Finland; †Haartman
Institute, Helsinki, Finland; ‡Central Hospital of North Karelia,
Joensuu, Finland; §Finnish Forest Research Institute, Vantaa,
Finland; and ¶HUCH Laboratory Diagnostics, Helsinki, Finland Cowpox with
Severe Generalized
Eruption, Finland
Paula M. Pelkonen,*† Kyllikki Tarvainen,‡
Arja Hynninen,‡ Eva R.K. Kallio,§
Heikki Henttonen,§ Airi Palva,* Antti Vaheri,†¶
and Olli Vapalahti*†¶ States by transport of animals indigenous to Africa (9), are
actually orthopoxviruses of their reservoir rodents and are
not well adapted to interhuman spread (1). The misleading
nomenclature is based on the hosts from which they were
first identified (1). Cowpox virus infections have been
detected in Europe and central and northern Asia (1). Nongeneralized infections in children have been previous-
ly characterized and diagnosed by electron microscopy
(10), virus isolation, polymerase chain reaction (PCR), and
restriction enzyme analysis (10,11). States by transport of animals indigenous to Africa (9), are
actually orthopoxviruses of their reservoir rodents and are
not well adapted to interhuman spread (1). The misleading
nomenclature is based on the hosts from which they were
first identified (1). Cowpox virus infections have been
detected in Europe and central and northern Asia (1). Nongeneralized infections in children have been previous-
ly characterized and diagnosed by electron microscopy
(10), virus isolation, polymerase chain reaction (PCR), and
restriction enzyme analysis (10,11). DISPATCHES forming
25,000
steps. The
HA
sequence
of
CPXV/FIN/T2000 formed a separate clade with cowpox
virus strain GRI-90 (Figure 2), isolated originally from
another 4-year-old girl, who contracted cowpox after play-
ing with a mole near Moscow (14). The reference cowpox
virus strain Brighton grouped with camelpox and variola
viruses instead of other cowpox or vaccinia viruses. However, because of the high homology of HA genes, this
finding should be interpreted with caution. The remaining
cowpox virus strains clustered together with vaccinia
viruses; this clustering may, in some cases, be explained by
origin from vaccinia virus strains that had escaped to
nature (13). This finding seems not to be the case in
Finland, since the HA nucleotide sequence of vaccinia
virus used in Finland differed 4% both from the strain
CPXV/FIN/T2000 and another Finnish cowpox virus iso-
late from 1989 with an identical HA sequence (data not
shown). Different alignment parameters and the neighbor-
joining method produced the same results with high sup-
port values. Scattered phylogenetic distribution of cowpox
virus strains is supported by data presented by other
researchers and may reflect an ancestral role of cowpox
viruses within the Orthopoxvirus genus; some strains clus-
ter with vaccinia viruses and others (including the refer-
ence Brighton strain) with variola virus (1). was seen after 2 days. The isolate, designated
CPXV/FIN/T2000, was shown to contain orthopoxvirus
by electron microscopy, and DNA samples from both the
infected cells and the original biopsy specimen were PCR-
positive for orthopoxvirus thymidine kinase gene (12). The
hemagglutinin (HA) gene of the isolate was amplified
(13), sequenced (948 nucleotides; accession no. AY366477), and compared to other orthopoxviruses. The
CPXV/FIN/T2000 strain differed 3% to 4% at the
nucleotide level from cowpox virus strains available in
GenBank. The sequences were further subjected to phylo-
genetic analysis: they were aligned by using Clustal X with
Gonnet protein matrix and analyzed by using the maxi-
mum likelihood phylogenetic software TREE-PUZZLE
5.0, applying the Hasegawa model of substitution and per- An immunofluorescence assay (IFA) to measure specif-
ic immunoglobulin (Ig) G, IgM, and avidity of IgG anti-
bodies
(15)
was
established
on
acetone-fixed,
CPXV/FIN/T2000-strain-infected Vero cells. The patient’s
serum had a high IgM-antibody titer at admission and low
avidity of specific IgG; after 60 days, the IgM level was
low and the IgG avidity high (Table 1). Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 11, November 2003
1459 The Study The reservoir
hosts of cowpox are wild rodents (7); wild rodents may
transmit the virus to humans through cats (5) or other pets
that roam outside. Direct transmission from a rodent to a
girl has been recently described (8). Both cowpox and
monkeypox, which was recently transmitted to the United The girl was treated in isolation at the hospital with
chlorhexidine washings and wound dressings with fusidic
acid. Intravenous dicloxacillin was administered to prevent
secondary bacterial infections. On day 12, skin lesions pro-
gressed to deep-seated, hard, black eschars. At the same
time the patient’s general condition improved. Two months
later, all lesions were healed with scars. A homogenized biopsy sample was used to infect Vero
cells, where a cytopathic effect typical of orthopoxviruses Figure 1. Cowpox lesions on patient’s forearm on day 7 after onset
of illness. Figure 1. Cowpox lesions on patient’s forearm on day 7 after onset
of illness. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 11, November 2003 1458 DISPATCHES Conclusions Cowpox virus (orthopoxvirus) infection was diagnosed
by electron microscopy, PCR, virus isolation, and serolog-
ic testing (positive IgM or low avidity of IgG antibodies). Cowpox virus strains show considerable genetic variations
with different positioning in the orthopoxvirus phylogenet-
ic tree. Circulation of cowpox virus in wild and domestic
animals, together with decreased immunity in humans,
may lead to increased occurrence of human cowpox, espe-
cially in atopic and immunocompromised persons who are
at risk for generalized infection. The described case further
suggests atopy to be a contraindication to smallpox vacci-
nation. Table 2. Orthopoxvirus antibodies in cat, horse, and wild rodent
panels, Finland
Panel
n
Positive
Prevalence (%)
Cats
77
3
3.9
Horses
127
2
1.6
Wild rodents
Southern Finland: Evo
36
33
91.7
Eastern Finland: Värtsilä
36
1
2.8
Western and central Finland:
Several localities
436
0
0
Lapland: several localities
394
7
1.8 (0.4–15.2)
Wild rodents altogether
902
41
4.5 Acknowledgments We thank the personnel of North Karelia Central Hospital
and the patient’s family for their efforts and cooperation; veteri-
narians from Tornio and Joensuu, the Central Laboratory of the
Veterinary Teaching Hospital, and the Helsinki and Porvoo
Animal Protection Societies for animal serum samples; and Tytti
Manni for excellent laboratory assistance. In addition, seroprevalence rates of 50% (7/14) and 1.4%
(1/73) have been found in foxes and lynxes, respectively,
from a limited geographic area in Finland (16). This work was funded by the University of Helsinki and
supported by the EU grant QLR2-CT-2002-01358, the Farmos
Foundation for Research and Science, and the Foundation for
Support of Veterinary Research. Sera collected at a Finnish Veterinary meeting in 2001
showed that every person >50 years had orthopoxvirus
antibodies, as measured by IFA. The seroprevalence
decreased gradually for younger age groups (Table 3),
reflecting the gradual cessation of smallpox vaccination
(the last vaccinations in Finland occurred in 1977). The
average population might have lower seroprevalence rates
than veterinarians because of veterinarians’ frequent con-
tact with animals that may harbor orthopoxvirus. Consequently, younger age groups are more susceptible to
smallpox and cowpox virus infections. Dr. Pelkonen is a doctor of veterinary medicine and a
researcher at the University of Helsinki, both in the Department
of Virology, Faculty of Medicine, and the Division of
Microbiology and Epidemiology, Faculty of Veterinary
Medicine. She is a doctoral student in virology and is developing
diagnostic methods and studying molecular epidemiology, dis-
ease associations, and reservoirs of orthopox and Borna disease
viruses. Although cowpox virus infection usually causes a sin-
gle, painful, ulcerated vesicopustule and local lym-
phadenopathy, immunocompromised patients and chil-
dren, especially those with atopic eczema, are susceptible DISPATCHES DISPATCHES We further studied orthopoxvirus antibodies by IFA in
Finnish fauna and humans. The seroprevalence rates for
cats and horses were 3.9% and 1.6%, respectively; the
rates for wild rodents (when either serum or lung or heart
extracts was used) were 0% to 92%, depending on the trap-
ping time and the location (Table 2). In Värtsilä, a rural dis-
trict in eastern Finland near where the patient lives, anti-
bodies were found in 1 (2.8%) of 36 rodents. However,
variation of seroprevalence rates in rodents is also influ-
enced by population dynamics: low prevalences are often
found in the increase phase and high prevalences in the
peak phase of rodent population fluctuations. The positive
rodents were mainly bank voles (Clethrionomys glareolus). The two seropositive horses were from a region near where
the patient lives in eastern Finland, and the three seroposi-
tive cats were free-roaming and found in southern Finland. to a generalized, even lethal, smallpoxlike infection (5,6). An early diagnosis and prompt recognition of the virus are
essential for treating and differentiating cowpox, from
other orthopoxvirus and herpesvirus infections, especially
in severe cases. 1. Esposito JJ, Fenner F. Poxviruses. In: Knipe DM, Howley PM, edi-
tors. Fields virology. 4th ed. Philadelphia: Lippincott Williams &
Wilkins; 2001. p. 2885–921. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 11, November 2003 2. Jenner E. An inquiry into the causes and effects of the variola vac-
cine, a disease discovered in some of the western counties of
England, particularly Gloucestershire, and known by the name of the
cowpox. London: Sampson Low; 1798. 3. Baxby D. Edward Jenner’s inquiry; a bicentenary analysis. Vaccine
1999;17:301–7. 1. Esposito JJ, Fenner F. Poxviruses. In: Knipe DM, Howley PM, edi-
tors. Fields virology. 4th ed. Philadelphia: Lippincott Williams &
Wilkins; 2001. p. 2885–921.
2. Jenner E. An inquiry into the causes and effects of the variola vac-
cine, a disease discovered in some of the western counties of
England, particularly Gloucestershire, and known by the name of the
cowpox. London: Sampson Low; 1798.
3. Baxby D. Edward Jenner’s inquiry; a bicentenary analysis. Vaccine
1999;17:301–7.
4. Moss B. Poxviridae: the viruses and their replication. In: Knipe DM,
Howley PM, editors. Fields virology. 4th ed. Philadelphia: Lippincott
Williams & Wilkins; 2001. p. 2849–83. 4. Moss B. Poxviridae: the viruses and their replication. In: Knipe DM,
Howley PM, editors. Fields virology. 4th ed. Philadelphia: Lippincott
Williams & Wilkins; 2001. p. 2849–83. DISPATCHES The serum samples from the patient’s pets were collect-
ed later and tested with IFA. A hunting dog had antibodies
with a titer of 320; another dog had a titer of 20. Thus, a
dog might have transmitted the infection from a wild
rodent to the patient, although cats are thought to be the
main source of human infection (5). Table 1. Orthopoxvirus antibodies in sera of the patient and her
petsa
Titer
Characteristic
IgM
IgG
IgG avidity (%)
Patient day 7
1280
2560
1.5
Patient day 14
640
2560
3.1
Patient day 60
10
640
25
Old-immunity controls (>50 y)
<10
80–640
50–100
Negative controls
<10
<10
—
Dog A
—
<20
—
Dog B
—
320
50
Dog C
—
20
50
Horse
—
<20
—
Rabbit
—
<20
—
aIg, immunoglobulin. Table 1. Orthopoxvirus antibodies in sera of the patient and her
petsa Figure 2. Phylogenetic tree of selected orthopoxvirus hemagglu-
tinin genes based on Clustal X alignment and the maximum likeli-
hood method TreePuzzle. Virus sequences used for the analysis
were raccoonpox as an outgroup (GenBank accession no. M94169); cowpoxvirus strains Brighton Red (AF482758),
FIN/T2000 (AY366477), GRI-90 (Z99047), Hamburg (Z99050),
and Turkmenia (Z99048); vaccinia virus strains IOC (AF229248),
Western Reserve (M93956), and Wyeth (Z99051); variola virus
strains Bangladesh (L22579), Harvey (X65516) and India
(X69198); camelpox virus (AF438165); Cantagalo orthopoxvirus
(AF229247); monkeypox virus (Z99049); and rabbitpox virus
(Z99049). Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 11, November 2003
1459 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 11, November 2003 DISPATCHES DISPATCHES 5. Baxby D, Bennett M, Getty B. Human cowpox 1969-93: a review
based on 54 cases. Br J Dermatol 1994;131:598–607. 13. Damaso CRA, Esposito JJ, Condit RC, Moussatché N. An emergent
poxvirus from humans and cattle in Rio de Janeiro state: Cantagalo
virus may derive from Brazilian smallpox vaccine. Virology
2000;277:439–49. 6. Czerny CP, Eis-Hübinger AM, Mayr A, Schneweis KE, Pfeiff B. Animal poxviruses transmitted from cat to man: current event with
lethal end. J Vet Med 1991;B38:421–31. 14. Marennikova SS, Gashnikov PV, Zhukova OA, Ryabchikova EI,
Streltsov VV, Ryazankina OI, et al. Biotype and genetic characteriza-
tion of isolate of cowpox virus having caused infection in a child. Zh
Mikrobiol Epidemiol Immunobiol.1996;4:6–10. 7. Chantrey J, Meyer H, Baxby D, Begon M, Bown KJ, Hazel SM, et al. Cowpox: reservoir hosts and geographic range. Epidemiol Infect
1999;122:455–60. Mikrobiol Epidemiol Immunobiol.1996;4:6–10. 8. Wolfs TFW, Wagenaar JA, Niesters HGM, Osterhaus ADME. Rat-to-
human transmission of cowpox infection. Emerg Infect Dis
2002;8:1495–6. 15. Hedman K, Vaheri A, Brummer-Korvenkontio M. Rapid diagnosis of
hantavirus
disease
with
an
IgG-avidity
assay. Lancet
1991;338:1353–6. 9. Larkin M. Monkeypox spreads as US public-health system plays
catch-up. Lancet Infect Dis 2003;3:461. 16. Tryland M, Sandvik T, Arnemo JM, Stuve G, Olsvik Ø, Traavik T. Antibodies against orthopoxviruses in wild carnivores from
Fennoscandia. J Wildl Dis 1998;34:443–50. 10. Wienecke R, Wolff H, Schaller M, Meyer H, Plewig G. Cowpox virus
infection in an 11-year-old girl. J Am Acad Dermatol 2000;42:892–4. 11. Schupp P, Pfeffer M, Meyer H, Burck G, Kölmel K, Neumann C. Cowpox virus in a 12-year-old boy: rapid identification by an
orthopoxvirus-specific polymerase chain reaction. Br J Dermatol
2001;145:146–50. Address for correspondence to: Paula Pelkonen, Department of Virology,
Haartman Institute, P.O.B. 21, FIN-00014 University of Helsinki,
Helsinki, Finland; fax: +358 9 191 26491; email: Paula.Pelkonen@
helsinki.fi 12. Sandvik T, Tryland M, Hansen H, Mehl R, Moens U, Olsvik O, et al. Naturally occurring orthopoxviruses: potential for recombination
with vaccine vectors. J Clin Microbiol 1998;36:2542–7. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 11, November 2003
1461
Search past issues of EID at www.cdc.gov/eid References 1. Esposito JJ, Fenner F. Poxviruses. In: Knipe DM, Howley PM, edi-
tors. Fields virology. 4th ed. Philadelphia: Lippincott Williams &
Wilkins; 2001. p. 2885–921. 1460
Emerging Infectious Diseases • www.cdc
Table 3. Orthopoxvirus antibodies in humans (veterinarians),
Finland
Humans (y)
n
Positive
Prevalence (%)
<25
19
2
10.5
26–30
23
4
17.4
31–50
78
46
59.0
>51
18
18
100.0 3. Baxby D. Edward Jenner’s inquiry; a bicentenary analysis. Vaccine
1999;17:301–7. 4. Moss B. Poxviridae: the viruses and their replication. In: Knipe DM,
Howley PM, editors. Fields virology. 4th ed. Philadelphia: Lippincott
Williams & Wilkins; 2001. p. 2849–83. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 11, November 2003 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 11, November 2003
1461
Search past issues of EID at www.cdc.gov/eid Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 9, No. 11, November 2003
14 1461
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KOMPAKT VE RİNG İPLİKLERDEN ELDE EDİLMİŞ ÖRME KUMAŞLARIN PATLAMA MUKAVEMETİ VE BONCUKLANMA PERFORMANSI ÜZERİNE BİR ARAŞTIRMA
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P A M U K K A L E Ü N İ V E R S İ T E S İ M Ü H E N D İ S L İ K F A K Ü L T E S İ
P A M U K K A L E U N I V E R S I T Y E N G I N E E R I N G C O L L E G E
MÜHENDİSLİK BİLİMLERİ DERGİSİ
J O U R N A L O F E N G I N E E R I N G S C I E N C E S
YIL
CİLT
SAYI
SAYFA
: 2005
: 11
: 3
: 357-360 P A M U K K A L E Ü N İ V E R S İ T E S İ M Ü H E N D İ S L İ K F A K Ü L T E S İ
P A M U K K A L E U N I V E R S I T Y E N G I N E E R I N G C O L L E G E
MÜHENDİSLİK BİLİMLERİ DERGİSİ
J O U R N A L O F E N G I N E E R I N G S C I E N C E S
YIL
CİLT
SAYI
SAYFA
: 2005
: 11
: 3
: 357-360 P A M U K K A L E U N I V E R S I T Y E N G I N E E R I N G C O L L E G E
MÜHENDİSLİK BİLİMLERİ DERGİSİ
J O U R N A L
O F
E N G I N E E R I N G
S C I E N C E S
CİLT
SAY
SAY MÜHENDİSLİK BİLİMLERİ DERGİSİ
J O U R N A L O F E N G I N E E R I N G S C I E N C E S
CİLT
SAYI
SAYFA
: 11
: 3
: 357 ÖZET Bu çalışmada; % 100 penye pamuk ring ve kompakt ipliklerden elde edilmiş süprem örgü kumaşların patlama
mukavemetleri ile boncuklanma eğilimleri incelenmiştir. Sonuçlara göre, ring ve kompakt iplik yapıları
arasındaki farklılıklar kumaş özellikleri üzerinde önemli rol oynamaktadır. Sonuç olarak; kompakt ipliklerden
elde edilmiş kumaşların daha yüksek patlama mukavemeti değerlerine ve daha düşük boncuklanma eğilimine
sahip oldukları görülmüştür. Anahtar Kelimeler : Ring, Kompakt, Suprem, Örgü kumaş, Patlama mukavemeti, Boncuklanma ABSTRACT In this paper, bursting strengths and pilling tendencies of a series of single jersey fabrics made from both 100 %
combed cotton ring and compact yarns are investigated. According to results, structural differences between
ring and compact spun yarns play significant role on fabric properties. As a result, it was seen that fabrics
knitted from compact yarns have higher bursting strength and lower pilling tendency than fabrics knitted from
ring spun yarns. Key Words : Ring, Compact, Single jersey, Knitted fabric, Burst strength, Pilling Sunay ÖMEROĞLU
Uludağ Üniversitesi, Mühendislik-Mimarlık Fakültesi, Tekstil Müh. Bölümü, 16059/Görükle /Bursa Sunay ÖMEROĞLU
Uludağ Üniversitesi, Mühendislik-Mimarlık Fakültesi, Tekstil Müh. Bölümü, 16059/Görükle /Bursa Geliş Tarihi : 01.11.2004 AN INVESTIGATION ABOUT BURSTING STRENGTH AND PILLING
PERFORMANCE OF KNITTED FABRICS MADE FROM RING AND COMPACT
YARNS ABSTRACT Anahtar Kelimeler : Ring, Kompakt, Suprem, Örgü kumaş, Patlama mukavemeti, Boncuklanma 1. GİRİŞ Ring ve kompakt iplikçilikte iplik oluşumu
ve eğirme üçgeninin durumu Kompakt ipliklerin
örme
işlemindeki
temel
avantajları;
kompakt
ipliklerin yapısını oluşturan liflerin sıyrılmaya ve
aşınmaya
karşı
olan
dirençlerinden
kaynaklanmaktadır (Kampen, 2000; Olbrich, 2000;
Rusch, 2002). Şekil 1. Ring ve kompakt iplikçilikte iplik oluşumu
ve eğirme üçgeninin durumu Kompakt ipliklerin
örme
işlemindeki
temel
avantajları;
kompakt
ipliklerin yapısını oluşturan liflerin sıyrılmaya ve
aşınmaya
karşı
olan
dirençlerinden
kaynaklanmaktadır (Kampen, 2000; Olbrich, 2000;
Rusch, 2002). Örme
kumaşların
boncuklanma
özelliklerinin
belirlenmesi için Atlas Random Tumble Pilling test
cihazında, ASTM D 3512-96 standardına göre
boncuklanma testi yapılmıştır. Buna göre; her bir
farklı tip kumaşa ait 3 numunenin 36000 devir
sonraki
görünümleri
tüy
sayısı,
büyüklüğü,
görünüşü, yüzeydeki aşınma, renk değişimi veya tüy
gelişimi gibi unsurların tümü göz önüne alınarak,
standart fotoğraflarla yapılan kıyaslama neticesinde;
1 en kötü 5 en iyi olacak şekilde, ara değerleri de
içeren bir değerlendirme yapılmıştır. Söz konusu
kumaşların
boncuklanma
testinden
sonraki
görüntülerinin (10 kat büyütülerek) fotograflanması
amacıyla;
Olympus
SZ6045TR
marka
üstten
aydınlatmalı binoküler mikroskop ve ona bağlı olan
Olympus
SC-35
marka
fotograf
makinesi
kullanılmıştır. 1. GİRİŞ ve Ömeroğlu, 1999). Şimdiye kadar “mükemmel”
olarak kabul edilen ring iplik yapısı, “ideal”
kompakt iplik yapısı karşısında bir alt sınıf olarak
değerlendirilmeye
başlanmıştır. Bu
iplikçilik
sistemiyle üretilen ipliklerin tamamen farklı olan
yapısı; yüksek mukavemet, düşük tüylülük ve
iyileştirilmiş düzgünsüzlük değerleriyle kendini
göstermektedir (Artzt, 1999). Aynı şekilde bu yeni
iplikçilik sistemi; iplik makinesindeki büküm
seviyesinin düşürülmesine, eğirme limitlerinin ve
çekimin arttırılabilmesine, daha parlak, daha net Kompakt iplikçilik sistemi; eğirme üçgeninin hemen
hemen ortadan kaldırılarak, kenar liflerininin iplik
yapısına entegre olması neticesinde dikkate değer
şekilde gelişmiş bir iplik yapısı sunmaktadır
(Smekal, 2001). Ring ve kompakt iplikçilik
sistemlerindeki iplik oluşumu ve eğirme üçgeninin
durumu Şekil 1.’de şematik olarak görülmektedir. Kompakt iplikçilik sisteminin geliştirilmesi, iplik
yapısı bakımından yeni standartlar sunmuştur (Ülkü 357 Kompakt ve Ring İpliklerden Elde Edilmiş Örme Kumaşların Patlama Mukavemeti ve Boncuklanma Performansı Üzerine…, S. Ömeroğlu ve Ring İpliklerden Elde Edilmiş Örme Kumaşların Patlama Mukavemeti ve Boncuklanma Performansı Üzerine…, S. Ömeroğlu Üretilen kompakt ve ring iplikler Ne 30, Ne 40 ve
Ne 50 olmak üzere 3 farklı numarada olup, tüm
ipliklerin üretiminde kullanılan büküm katsayısı
değeri αe = 3.75’dir. Üretilen ipliklerin mukavemet
ölçümleri
Uster
Tensorapid
3
cihazında,
düzgünsüzlük ölçümleri Uster Tester 3 cihazında,
tüylülük ölçümleri ise Zweigle G566 cihazında
gerçekleştirilmiştir. Tablo 1’de söz konusu ipliklere
ait özellikler verilmiştir. görünümlü bir yüzey yapısına sahip dokuma ve
örme kumaşların üretilmesine imkan vermektedir
(Egbers, 1999). Ring İplikçiliği
B : Çıkış silindirlerinin kıstırma hattında lif topluluğunun
genişliği Söz konusu iplikler kullanılarak 18” çapında 28
gauge
inceliğindeki
Marchisio
Jetrace
örme
makinasında süprem örme kumaşlar elde edilmiş,
ardından bu kumaşlara ön terbiye ve boyama
işlemleri
uygulanmıştır. Elde
edilen
örme
kumaşların kodlanması ve mamul kumaşların
yapısal özellikleri ile ilgili bilgiler Tablo 2’de
verilmiştir. B : Çıkış
genişliği Kompakt İplikçiliği
B : Eğirme üçgeninin genişliği Kompakt İplikçiliği Kompakt İplikçiliği
B : Eğirme üçgeninin genişliği
Şekil 1. Ring ve kompakt iplikçilikte iplik oluşumu
ve eğirme üçgeninin durumu Kompakt ipliklerin
örme
işlemindeki
temel
avantajları;
kompakt
ipliklerin yapısını oluşturan liflerin sıyrılmaya ve
aşınmaya
karşı
olan
dirençlerinden
kaynaklanmaktadır (Kampen, 2000; Olbrich, 2000;
Rusch, 2002). Örme kumaşların patlama mukavemeti testleri,
Messmer Buchel marka diyaframlı bir ölçüm
cihazında gerçekleştirilmiştir. Her bir farklı kumaş
tipi için 6 adet ölçüm yapılarak, ölçüm sonuçlarına
ait ortalama değerler ve değişim katsayıları tespit
edilmiştir. B : Eğirme üçgeninin genişliği Şekil 1. 3. TARTIŞMA kompakt ve ring iplikler arasındaki mukavemet
farklarının kumaş patlama mukavemeti değerlerine
tam olarak yansımadığı görülmektedir. Aynı büküm katsayısına sahip Ne 30, Ne 40 ve Ne
50 numara kompakt ve ring iplikler kullanılarak,
elde edilen 6 farklı tipteki süprem örme kumaşa
uygulanan patlama mukavemeti ve boncuklanma
testlerinden elde edilen verilerin değerlendirmesi şu
şekilde yapılabilir: Tablo 3. Örme Kumaşlara Ait Patlama Mukavemeti
Testlerinden Elde Edilen Veriler
Kumaş Kodu
Patlama Mukavemeti (kPa)
Ortalama
% CV
R30
230.8
7.2
K30
250.8
6.0
R40
185.8
5.7
K40
202.5
7.6
R50
127.5
7.3
K50
148.3
8.2 Tablo 3. Örme Kumaşlara Ait Patlama Mukavemeti
Testlerinden Elde Edilen Veriler Tablo 3’de kumaşlara ait patlama mukavemeti
testlerinden elde edilen veriler görülmektedir. Buna
göre
kompakt
ipliklerden
elde
edilen
örme
kumaşların patlama mukavemetinin, aynı numara ve
büküm değerine sahip ring ipliklerden elde edilen
kumaşların patlama mukavemetinden % 15.5 ile
% 8.7 arasında değişen oranlarda daha fazla olduğu
görülmektedir. Bununla beraber, ipliklerin kopma
mukavemeti sonuçları incelendiğinde; kompakt ve
ring iplikler arasındaki mukavemet farklılıklarının
% 32.7 ile % 20.7 arasında değiştiği dikkate alınırsa, Tablo 4’de verilmiş olan değerler, farklı tipteki
kumaşlardan
hazırlanmış
olan
numunelerin,
boncuklanma testi sonrasındaki görünümlerine ait
subjektif
değe
rlendirme
sonuçlarını
ifade
etmektedir. Tablo 4’de verilmiş olan değerler, farklı tipteki
kumaşlardan
hazırlanmış
olan
numunelerin,
boncuklanma testi sonrasındaki görünümlerine ait
subjektif
değe
rlendirme
sonuçlarını
ifade
etmektedir. Tablo 4. Örme Kumaşların Boncuklanma Testi Değerlendirme Sonuçları
Kumaş Tipi
Değerlendirme
Kumaş Tipi
Değerlendirme
Kumaş Tipi
Değerlendirme
R30
3
R40
3
R50
2
K30
4
K40
4
K50
3 durumun
oluşmasını
sağlamaktadır. Kompakt
ipliklerden oluşan kumaş, boncuklaşmaya sebebiyet
verecek bir etkiye maruz kaldığında; iplik yapısına
sıkı bir şekilde bağlanmış ve iplik gövdesinden çok
az dışarı çıkmış olan liflerin bu etki nedeniyle iplik
yapısından dışarı çekilmesi zor olmaktadır. Örme
kumaşların
orijinal
ve
boncuklanma
testi
sonrasındaki görünümlerine ait fotoğraflar Şekil 2,
3 ve 4’de görülmektedir. durumun
oluşmasını
sağlamaktadır. Kompakt
ipliklerden oluşan kumaş, boncuklaşmaya sebebiyet
verecek bir etkiye maruz kaldığında; iplik yapısına
sıkı bir şekilde bağlanmış ve iplik gövdesinden çok
az dışarı çıkmış olan liflerin bu etki nedeniyle iplik
yapısından dışarı çekilmesi zor olmaktadır. Örme
kumaşların
orijinal
ve
boncuklanma
testi
sonrasındaki görünümlerine ait fotoğraflar Şekil 2,
3 ve 4’de görülmektedir. Üretilmiş olan kumaşlara ait boncuklanma testi
sonuçlarından; kompakt iplikler kullanılarak elde
edilen örme kumaşların daha düşük boncuklanma
eğilimine sahip olduğu görülmektedir. 2. MATERYAL VE YÖNTEM Bu çalışmada, aynı % 100 penye pamuk fitilleri
kullanılarak Rieter K44 ve Rieter G33 iplik
makinalarında kompakt ve ring iplikler üretilmiştir. Daha sonra bu iplikler kullanılarak örme kumaşlar
elde
edilmiş
ve
bu
kumaşlara
ait
patlama
mukavemeti
ve
boncuklanma
gibi
özellikler
incelenmiştir. Bu çalışmada, aynı % 100 penye pamuk fitilleri
kullanılarak Rieter K44 ve Rieter G33 iplik
makinalarında kompakt ve ring iplikler üretilmiştir. Daha sonra bu iplikler kullanılarak örme kumaşlar
elde
edilmiş
ve
bu
kumaşlara
ait
patlama
mukavemeti
ve
boncuklanma
gibi
özellikler
incelenmiştir. Tablo 1. Örme Kumaşların Üretiminde Kullanılan İpliklere Ait Özellikler
İplik Tipi ( Ne / αe )
Test Cihazı
İplik Özellikleri
Ring
(30 / 3.75)
Kompakt
(30 / 3.75)
Ring
(40 / 3.75)
Kompakt
(40 / 3.75)
Ring
(50 / 3.75)
Kompakt
(50 / 3.75)
Uster Tensorapid 3
Mukavemet (cN/tex)
13.75
16.60
12.94
16.62
11.28
14.97
Uzama (%)
4.00
4.66
4.20
4.49
3.07
3.40
Düzgünsüzlük (%CVm)
11.78
11.30
13.80
13.75
14.59
14.51
Uster Tester 3
-%50 İnce yer
0
0
6
6
28
23
+%50 Kalın yer
8
14
48
78
75
114
+%200 Neps
24
9
113
81
136
103
1 mm Sınıfı
14393
14851
13705
11723
12514
10450
Zweigle G566
2 mm Sınıfı
2975
2506
2253
2088
2769
1561
S3- değeri (Σ ≥ 3mm)
2116
614
2045
306
1852
278 Tablo 1. Örme Kumaşların Üretiminde Kullanılan İpliklere Ait Özellikler dislik Bilimleri Dergisi 2005 11 (3) 357-360
358
Journal of Engineering Sciences 2005 11 (3) 357-3 Journal of Engineering Sciences 2005 11 (3) 357-360 Mühendislik Bilimleri Dergisi 2005 11 (3) 357-360 Kompakt ve Ring İpliklerden Elde Edilmiş Örme Kumaşların Patlama Mukavemeti ve Boncuklanma Performansı Üzerine…, S. Ömeroğlu Tablo 2. Elde Edilen Örme Kumaşların Kodlanması ve Yapısal Özellikleri
Kumaş Kodu
İplik Tipi
Sıra/cm
Çubuk/cm
Gramaj (g/m2)
R30
Ne30 Ring
18
17
151
K30
Ne30 Kompakt
18
17
162
R40
Ne40 Ring
18
19
117
K40
Ne40 Kompakt
18
19
114
R50
Ne50 Ring
15
22
87
K50
Ne50 Kompakt
15
22
92 Tablo 2. Elde Edilen Örme Kumaşların Kodlanması ve Yapısal Özellikleri 3. TARTIŞMA Kompakt
iplik yapısını oluşturan liflerin bir halat gibi, sıkı bir
şekilde bir arada bükülmüş olması ve iplik dış
yüzeyinde, iplik gövdesine gevşek bağlanmış uzun
tüy şeklinde liflerin çok az miktarda bulunması gibi
sebepler, bu R30 Orjinal R30 Boncuklanma Sonrası K30 Orjinal K30 Boncuklanma Sonrası
Şekil 2. Ne30 Numara ipliklerden elde edilmiş kumaşların orjinal ve boncuklanma testi sonrasındaki
görünümleri (Büyütme 10x10 ) R30 Orjinal R30 Boncuklanma Sonrası K30 Orjinal K30 Boncuklanma Sonrası
Şekil 2. Ne30 Numara ipliklerden elde edilmiş kumaşların orjinal ve boncuklanma testi sonrasındaki
görünümleri (Büyütme 10x10 ) Mühendislik Bilimleri Dergisi 2005 11 (3) 357-360 Journal of Engineering Sciences 2005 11 (3) 357-360 359 ve Ring İpliklerden Elde Edilmiş Örme Kumaşların Patlama Mukavemeti ve Boncuklanma Performansı Üzerine…, S. Ömeroğlu R40 Orjinal R40 Boncuklanma Sonrası K40 Orjinal K40 Boncuklanma Sonrası
Şekil 3. Ne40 Numara ipliklerden elde edilmiş kumaşların orjinal ve boncuklanma testi sonrasındaki
görünümleri (Büyütme 10 x 10) R50 OrjinalR50 Boncuklanma Sonrası K50 Orjinal K50 Boncuklanma Sonrası
Şekil 4. Ne50 Numara ipliklerden elde edilmiş kumaşların orjinal ve boncuklanma testi sonrasındaki
görünümleri (Büyütme 10 x 10) R50 OrjinalR50 Boncuklanma Sonrası K50 Orjinal K50 Boncuklanma Sonrası
Şekil 4. Ne50 Numara ipliklerden elde edilmiş kumaşların orjinal ve boncuklanma testi sonrasındaki
görünümleri (Büyütme 10 x 10) 6. KAYNAKLAR Artzt, P. 1999. Short Staple Spinning on the Way to
New Yarn Structures and Better Raw Material
Utilisation. ITB International Textile Bulletin, 4:
16-23. Egbers, G. 1999. ITMA 1999. Light at the end of the
Tunnel. ITB International Textile Bulletin, 4:11-15. Deneysel çalışmadan elde edilen sonuçlara göre,
kompakt
iplikler
kullanılarak
üretilen
örme
kumaşların boncuklanma oranında da bir azalma
görülmektedir. Bu
durum
kompakt
ipliklerin
yapısını oluşturan liflerin iplik yüzeyinde daha
düşük tüylülüğe sebep olmasından ve ayrıca bu
liflerin iplik yapısından sıyrılmaya karşı olan
dirençlerinden kaynaklanmaktadır. Çekilmiş olan
fotoğraflardan da görüldüğü gibi; kompakt ipliklerin
daha düşük tüylülüğe sahip olması aynı zamanda,
kompakt ipliklerden elde edilen örme kumaşlardaki
ilmek sırası ve çubukların, ring ipliklerden elde
edilen kumaşlardakine göre daha net bir görüntü
vermesini sağlamaktadır. Kampen, W. 2000. The Advantages of Condensed
Spinning. Melliand English, 4:58-59. Olbrich, A. 2000. The AIR-COM-TEX 700
Condenser Ring Spinning Machine. Melliand
International, 6 : 26-29. Rusch, B. 2002. COM4® Sayesinde Konfor–Sonraki
İşlemlerde
Yeni
Potansiyel. Rieter
Türkiye
Sempozyumu - 2002, Antalya, 45 s. Smekal, J. 2001. Air-Com-Tex 700 for Compact
Spinning Yarns. Melliand International, 7:18-19. 4. SONUÇ A.Ş./İstanbul
yetkililerine,
örme
kumaşların
üretilmesindeki yardımlarından dolayı Biesseci
Tekstil San.Tic. A.Ş./Bursa yetkililerine, kumaş
testlerinin gerçekleştirilmesindeki yardımlarından
dolayı
Yeşim
Tekstil
San.Tic. A.Ş./Bursa
yetkililerine teşekkürü bir borç bilirim. Aynı numara ve büküme sahip kompakt ipliklerden
elde edilen örme kumaşların patlama mukavemeti,
ring ipliklerden elde edilenlere göre daha yüksektir. Örme kumaşların üretiminde kullanılacak bir iplikte,
yüksek mukavemet özelliği dokuma kumaşlarda
olduğu kadar bir önem taşımamaktadır. Bu sebeple,
kompakt ipliklerin örme işlemlerindeki davranışı, bu
ipliklerin yüksek mukavemet özellikleri tarafından
pek
etkilenmez. Ancak,
yüksek
mukavemet
özelliğiyle bağlantılı olarak, kompakt ipliklerin ring
iplikleriyle ulaşılamayacak kadar düşük büküm
katsayısı seviyelerinde üretilebilmesi, daha yumuşak
tutumlu
örme
kumaşların
üretilmesine
imkan
verecektir. 5. TEŞEKKÜR Ülkü, Ş., Ömeroğlu, S. 1999. “ITMA 99’da Pamuk
İplikçiliği”, Tekstil Teknoloji Dergisi, Aralık 1999,
67-78. Bu çalışmada kullanılan ipliklerin üretilmesindeki
yardımlarından dolayı Topkapı İplik San. Tic. Mühendislik Bilimleri Dergisi 2005 11 (3) 357-360 Journal of Engineering Sciences 2005 11 (3) 357-360 360
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