identifier
stringlengths
11
32
pdf_url
stringlengths
17
4.62k
lang
stringclasses
120 values
error
stringclasses
1 value
title
stringlengths
2
500
source_name
stringlengths
1
435
publication_year
float64
1.9k
2.02k
license
stringclasses
3 values
word_count
int64
0
1.64M
text
stringlengths
1
9.75M
https://openalex.org/W2542496409
https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-016-3186-0
English
null
Dwarfism with joint laxity in Friesian horses is associated with a splice site mutation in B4GALT7
BMC genomics
2,016
cc-by
8,518
* 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 in Genie. J Comp Biol. 1997;4:311–23. 11. Lossos A, Stümpfig C, Stevanin G, Gaussen M, Zimmerman BE, Mundwiller E, Asulin M, Chamma L, Sheffer R, Misk A, Dotan S, Gomori JM, Ponger P, Brice A, Lerer I, Meiner V, Lill R. Fe/S protein assembly gene IBA57 mutation causes hereditary spastic paraplegia. Neurology. 2015;84:659–67. Abbreviations ECA14: Equus Caballus chromosome 14; EDSP1: Ehlers-Danlos syndrome, progeroid type 1; KFPS: Koninklijke Vereniging “Het Friesch Paarden-Stamboek” (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 Vet J. 2008;40:282–7. Additional file 2: Expression of B4GALT7 in relation to genotype. qPCR 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 foals. Vet Rec. 2009;165:373–6. RNA analysis 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). 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_ search.cgi?taxid=9796&build=101.0. Accessed 7 Dec 2015. 6. Doan R, Cohen ND, Sawyer J, Ghaffari N, Johnson CD, Dindot SV. Whole- genome sequencing and genetic variant analysis of a Quarter Horse mare. BMC Genomics. 2012;13:78. 7. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non- synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009;4:1073–81. Authors’ contributions 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 LHX3 intron 5 is associated with aberrant splicing and pituitary dwarfism in German shepherd dogs. PLoS One. 2011;6:e27940. RNA analysis Moreover, coverage > 10, a genotypequality of >30 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, Ruden DM. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012;6:80–92. 29. Stassen QE, Riemers FM, Reijmerink H, Leegwater PA, Penning LC. Reference genes for reverse transcription quantitative PCR in canine brain tissue. BMC Res Notes. 2015;8:761. 30. EMBL-EBI: European Nucleotide Archive. http://www.ebi.ac.uk/ena. Accessed 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 • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit 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 structure, and abnormal phenotype of the skin fibroblasts of an Ehlers- 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 DNA sequencing data. Genome Res. 2010;20:1297–303. 26. Van der Auwera GA, Carneiro M, Hartl C, Poplin R, del Angel G, Levy- Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella K, Altshuler D, Gabriel S, DePristo M. From FastQ Data to High-Confidence Variant Calls: The Genome Analysis Toolkit Best Practices Pipeline. Curr Protoc Bioinformatics. 2013;11:11.10.1–11.10.33. Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella K, Altshuler D, Gabriel S, DePristo M. From FastQ Data to High-Confidence Variant 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 and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries
https://openalex.org/W2329964189
http://www.mathnet.ru/php/getFT.phtml?jrnid=vsgtu&paperid=740&what=fullt&option_lang=rus
Russian
null
Параллельный алгоритм метода циклической редукции для периодической краевой задачи
Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ: Fiziko-matematičeskie nauki/Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ Fiziko-matematičeskie nauki
2,010
cc-by
3,367
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 согласны с пользовательским соглашением http://www.mathnet.ru/rus/agreement Параметры загрузки: IP: 195.242.1.96 24 октября 2024 г., 08:50:29 Параметры загрузки: IP: 195.242.1.96 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.
https://openalex.org/W1822236674
https://www.repository.cam.ac.uk/bitstream/1810/248588/5/Finch%20et%20al%202015%20%20Journal%20of%20Virology.pdf
English
null
Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus
Journal of virology
2,015
cc-by
11,625
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 http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIO http://jvi.asm.org/ 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 http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY / , 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 http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLO http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MO http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 Volume 89 Number 16 on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY http://jvi.asm.org/ 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 http://jvi.asm.org/ Downloaded from 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 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ 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 http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY http://jvi.asm.org/ 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 http://jvi.asm.org/ Downloaded from on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY http://jvi.asm.org/ Downloaded from 13. Brierley I, Gilbert R, Pennell S. 2010. Pseudoknot-dependent pro- grammed 1 ribosomal frameshifting: structures, mechanisms and mod- els, p 149–174. In Atkins J, Gesteland R (ed), Recoding: expansion of decoding rules enriches gene expression. Springer, Heidelberg, Germany. 14. Miller W, Giedroc D. 2010. Ribosomal frameshifting in decoding plant viral RNAs, p 193–220. In Atkins J, Gesteland R (ed), Recoding: expansion of decoding rules enriches gene expression. Springer, Heidelberg, Ger- many. on October 2, 2015 by MRC LAB OF MOLECULAR BIO http://jvi.asm.org/ om y 15. Takyar S, Hickerson RP, Noller HF. 2005. mRNA helicase activity of the ribosome. Cell 120:49–58. http://dx.doi.org/10.1016/j.cell.2004.11.042. 16. Qu X, Wen J-D, Lancaster L, Noller HF, Bustamante C, Tinoco I. 2011. TheribosomeusestwoactivemechanismstounwindmessengerRNAduring translation. Nature 475:118–121. http://dx.doi.org/10.1038/nature10126. 17. Plant EP, Dinman JD. 2005. Torsional restraint: a new twist on frame- shifting pseudoknots. Nucleic Acids Res 33:1825–1833. http://dx.doi.org /10.1093/nar/gki329. g 18. REFERENCES j pp 22. Loughran G, Libbey JE, Uddowla S, Scallan MF, Ryan MD, Fujinami RS, Rieder E, Atkins JF. 2013. Theiler’s murine encephalomyelitis virus contrasts with encephalomyocarditis and foot-and-mouth disease viruses in its functional utilization of the StopGo non-standard translation mech- anism. J Gen Virol 94:348–353. http://dx.doi.org/10.1099/vir.0.047571-0. 1. Palmenberg AC, Kirby EM, Janda MR, Drake NL, Duke GM, Potratz KF, Collett MS. 1984. The nucleotide and deduced amino acid sequences of the encephalomyocarditis viral polyprotein coding region. Nucleic Ac- ids Res 12:2969–2985. http://dx.doi.org/10.1093/nar/12.6.2969. 2. Jackson RJ. 1986. A detailed kinetic analysis of the in vitro synthesis and processing of encephalomyocarditis virus products. Virology 149:114– 127. http://dx.doi.org/10.1016/0042-6822(86)90092-9. 23. Yan S, Wen J-D, Bustamante C, Tinoco I. 2015. Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways. Cell 160:870–881. http://dx.doi.org/10.1016/j.cell.2015.02 .003. 3. Palmenberg AC. 1990. Proteolytic processing of picornaviral polyprotein. Annu Rev Microbiol 44:603–623. http://dx.doi.org/10.1146/annurev.mi .44.100190.003131. 24. Grentzmann G, Ingram JA, Kelly PJ, Gesteland RF, Atkins JF. 1998. A dual-luciferase reporter system for studying recoding signals. RNA 4:479–486. 4. Batson S, Rundell K. 1991. Proteolysis at the 2A/2B junction in Theiler’s murine encephalomyelitis virus. Virology 181:764–767. http://dx.doi.org /10.1016/0042-6822(91)90914-W. 25. Fixsen SM, Howard MT. 2010. Processive selenocysteine incorporation during synthesis of eukaryotic selenoproteins. J Mol Biol 399:385–396. http://dx.doi.org/10.1016/j.jmb.2010.04.033. 5. Palmenberg AC, Parks GD, Hall DJ, Ingraham RH, Seng TW, Pallai PV. 1992. Proteolytic processing of the cardioviral P2 region: primary 2A/2B cleavage in clone-derived precursors. Virology 190:754–762. http://dx.doi .org/10.1016/0042-6822(92)90913-A. 5. Palmenberg AC, Parks GD, Hall DJ, Ingraham RH, Seng TW, Pallai PV. 1992. Proteolytic processing of the cardioviral P2 region: primary 2A/2B cleavage in clone-derived precursors. Virology 190:754–762. http://dx.doi .org/10.1016/0042-6822(92)90913-A. 26. Girnary R, King L, Robinson L, Elston R, Brierley I. 2007. Structure- function analysis of the ribosomal frameshifting signal of two human im- munodeficiency virus type 1 isolates with increased resistance to viral protease inhibitors. J Gen Virol 88:226–235. http://dx.doi.org/10.1099/vir .0.82064-0. g 6. Atkins JF, Wills NM, Loughran G, Wu C-Y, Parsawar K, Ryan MD, Wang C-H, Nelson CC. 2007. A case for “StopGo”: reprogramming translation to augmentcodonmeaningofGGNbypromotingunconventionaltermination (Stop)afteradditionofglycineandthenallowingcontinuedtranslation(Go). RNA 13:803–810. http://dx.doi.org/10.1261/rna.487907. 27. Li Y, Treffers EE, Napthine S, Tas A, Zhu L, Sun Z, Bell S, Mark BL, van Veelen PA, van Hemert MJ, Firth AE, Brierley I, Snijder EJ, Fang Y. 2014. Transactivation of programmed ribosomal frameshifting by a viral protein. Proc Natl Acad Sci U S A 111:E2172–E2181. http://dx.doi.org/10 .1073/pnas.1321930111. p g 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- cleic Acids Res 18:6707–6708. http://dx.doi.org/10.1093/nar/18.22.6707. 2015 by MRC LAB OF MOLECULAR BIOLOGY We thank John Atkins and Mike Howard for the pDluc vector. We also thank John Atkins, Lawrence Tiley, David Brown, and Frédéric Sorgeloos for many useful discussions. 21. Sorgeloos F, Jha BK, Silverman RH, Michiels T. 2013. Evasion of antiviral innate immunity by Theiler’s virus L* protein through direct inhibition of RNase L. PLoS Pathog 9:e1003474. http://dx.doi.org/10.1371 /journal.ppat.1003474. DISCUSSION Namy O, Moran SJ, Stuart DI, Gilbert RJC, Brierley I. 2006. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frame- shifting. Nature 441:244–247. http://dx.doi.org/10.1038/nature04735. on October 2, 2015 by MRC LAB OF MOLECULAR BIOLOGY REFERENCES 1994. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J 13:928–933. g y 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 8589 Journal of Virology
W3138443974.txt
null
de
Gender Mainstreaming im Europäischen Parlament: Geschichte, Institutionalisierung, Hindernisse
Gender
2,021
cc-by
6,650
www.ssoar.info Gender Mainstreaming im Europäischen Parlament: Geschichte, Institutionalisierung, Hindernisse Ahrens, Petra Veröffentlichungsversion / Published Version Zeitschriftenartikel / journal article Zur Verfügung gestellt in Kooperation mit / provided in cooperation with: 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 Nutzungsbedingungen: Dieser Text wird unter einer CC BY Lizenz (Namensnennung) zur Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden Sie hier: https://creativecommons.org/licenses/by/4.0/deed.de Terms of use: This document is made available under a CC BY Licence (Attribution). For more Information see: https://creativecommons.org/licenses/by/4.0 Diese Version ist zitierbar unter / This version is citable under: https://nbn-resolving.org/urn:nbn:de:0168-ssoar-72146-3 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 https://doi.org/10.3224/gender.v13i1.02 2-Gender1-21_Ahrens.indd 11 GENDER Heft 1 | 2021, S. 11–26 09.02.2021 16:22:49 12 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 GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 12 09.02.2021 16:22:49 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- GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 13 09.02.2021 16:22:49 14 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. GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 14 09.02.2021 16:22:49 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. GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 15 09.02.2021 16:22:49 16 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]. GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 16 09.02.2021 16:22:49 Gender Mainstreaming im Europäischen Parlament 17 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, GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 17 09.02.2021 16:22:49 18 Petra Ahrens 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- GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 18 09.02.2021 16:22:49 Gender Mainstreaming im Europäischen Parlament 19 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- GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 19 09.02.2021 16:22:49 20 Petra Ahrens 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. GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 20 09.02.2021 16:22:49 Gender Mainstreaming im Europäischen Parlament 21 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 GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 21 09.02.2021 16:22:49 22 Petra Ahrens 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. GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 22 09.02.2021 16:22:49 Gender Mainstreaming im Europäischen Parlament 23 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 GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 23 09.02.2021 16:22:49 24 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 Abels, Gabriele (2020). „The beginning of a new chapter“? Eine Einordnung der neuen EUGleichstellungsstrategie 2020–2025. Integration, (2), 144–153. 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. Zugriff am 20. Juli 2020 unter www.europarl.europa.eu/sides/getDoc.do?pubRef=-//EP// 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)). GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 24 09.02.2021 16:22:49 Gender Mainstreaming im Europäischen Parlament 25 Europäisches Parlament (2014). Gender Mainstreaming in den Ausschüssen und Delegationen des Europäischen Parlaments. Studie für den FEMM-Ausschuss durch die Generaldirektion Interne Politikbereiche. Zugriff am 24. Juli 2020 unter https://www.europarl.europa.eu/ RegData/etudes/STUD/2014/493051/IPOL-FEMM_ET(2014)493051_DE.pdf. European Parliament (2018). Updating of the Study on Gender Mainstreaming in Committees and Delegations of the European Parliament. Policy Department for Citizens’ Rights and Constitutional Affairs. Study. Zugriff am 10. März 2020 unter http://www.europarl.europa. 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 Qualitative Research. Chicago: Aldine. Haastrup, Toni & Kenny, Meryl (2016). Gendering Institutionalism: A Feminist Institutionalist Approach to EU Integration Theory. In Gabriele Abels & Heather MacRae (Hrsg.), Gendering European Integration Theory (S. 197–216). Opladen, Berlin, Toronto: Verlag Barbara Budrich. Hubert, Agnès & Stratigaki, Maria (2016). Twenty Years of EU Gender Mainstreaming: Rebirth out of the Ashes? Femina Politica, 25(2), 21–36. Jacquot, Sophie (2015). Transformations in EU Gender Equality. From Emergence to Dismantling. Basingstoke: Palgrave Macmillan. Kantola, Johanna & Rolandsen Agustín, Lise (2016). Gendering Transnational Party Politics: The Case of European Union. Party Politics, 22(5), 641–651. 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 party groups. Journal of Common Market Studies, 57(4), 768–786. 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. In Charlie Jeffrey & James Mitchell (Hrsg.), The Scottish Parliament 1999–2009: The First Decade (S. 48–55). Edinburgh: Luath Press. Mackay, Fiona (2014). Nested Newness, Institutional Innovation, and the Gendered Limits of Change. Politics and Gender, 10(14), 549–571. Mackay, Fiona; Kenny, Meryl & Chappell, Louise (2010). New Institutionalism Through a Gender Lens: Towards a Feminist Institutionalism? International Political Science Review, 31(5), 573–588. MacRae, Heather & Weiner, Elaine (Hrsg.). (2017). Towards Gendering Institutionalism Equality in Europe. London: Rowman & Littlefield. Mamadouh, Virginie & Raunio, Tapio (2003). The committee system: powers, appointments and report allocation. Journal of Common Market Studies, 41(2), 333–351. Minto, Rachel & Mergaert, Lut (2018). Gender mainstreaming and evaluation in the EU: comparative perspectives from feminist institutionalism. International Feminist Journal of Politics, 20(2), 204–220. https://doi.org/10.1080/14616742.2018.1440181 Nugent, Mary (2019). “Feminist to Its Fingertips”? Gendered Divisions of Labour and the Committee on Women’s Rights and Gender Equality. In Petra Ahrens & Lise Rolandsen Agustín (Hrsg.), Gendering the European Parliament: Structures, Policies, and Practices (S. 123–140). London: Rowman & Littlefield. Rolandsen Agustín, Lise (2013). Gender Equality, Intersectionality, and Diversity in Europe. New York: Palgrave Macmillan. GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 25 09.02.2021 16:22:49 26 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.), Gendering the European Parliament: Structures, Policies, and Practices (S. 35–49). London: Rowman & Littlefield. Verloo, Mieke (2005). Displacement and empowerment: Reflections on the concept and practice of the Council of Europe approach to gender mainstreaming and gender equality. Social Politics, 12(3), 344–365. Verloo, Mieke & Paternotte, David (2018). The Feminist Project under Threat in Europe. Politics and Governance, 6(3), 1–5. Vromen, Ariadne (2010). Debating Methods: Rediscovering Qualitative Approaches. In David Marsh & Gerry Stoker (Hrsg.), Theory and Methods in Political Science (S. 249–266). New York: Palgrave Macmillan. Warasin, Markus; Rolandsen Agustín, Lise; Kantola, Johanna & Coughlan, Ciara (2019). Politicisation of gender equality in the European Parliament. Cohesion and Inter-Group Coalitions in plenary and committees. In Petra Ahrens & Lise Rolandsen Agustín (Hrsg.), Gendering the European Parliament: Structures, Policies, and Practices (S. 141–158). London: Rowman & Littlefield. Wiesner, Claudia (2018). The micro-politics of parliamentary powers: European Parliament strategies for expanding its influence in the EU institutional system. Journal of European 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 GENDER 1 | 2021 2-Gender1-21_Ahrens.indd 26 09.02.2021 16:22:49
https://openalex.org/W4298664203
https://periodicos.ufba.br/index.php/rvh/article/download/48150/26194
Portuguese
null
A “santa sabedoria” e a vanglória: dois textos da literatura sapiencial anglo-saxônica e sua tradução para o português
Veredas da História
2,015
cc-by
12,209
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.
https://openalex.org/W2934202356
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 the Amazon Rio Negro area, Brazil. Rev Inst Med Trop Sao Paulo. 2007;49(3):159-64. 14. Snounou G. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol. 1993;58(2):283-92. Conflict of Interest: The authors declare that there are no conflicts of interest. REFERENCES 1. Ferreira Mu & Castro MC 2016. Challenges for malaria elimination in Brazil. Malar J. 2015(1):284. 17. Greiner M, Pfeiffer D, Smith RD. Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Prev Vet Med. 2000;45(1-2):23-41. 2. De Pina-Costa A, Brasil P, Santi S. M, Araujo M. P, Suárez- Mutis M. C, Santelli A. C, et al. Malaria in Brazil: what happens outside the Amazonian endemic region. Mem Inst Oswaldo Cruz. 2014;109(5):618-33. 18. Oliveira-Ferreira J, Lacerda MV, Brasil P, Ladislau JL, Tauil PL, Daniel-Ribeiro CT. Malaria in Brazil: an overview. Malar J. 2010;9:115. 8/9 Rev Soc Bras Med Trop Vol.:52:e-20180537, 2019 19. Wanderley DM, Da Silva RA, De Andrade JC. Epidemiological aspects of malaria in the State of São Paulo, Brazil, 1983 to 1992. Rev Saude Publica. 1994;28(3):192-7. 32. Branquinho MS, Marrelli MT, Curado I, Natal D, Barata JM, Tubaki R et al. Infection of Anopheles (Kerteszia) cruzii by Plasmodium vivax and Plasmodium vivax variant VK247 in the municipalities of São Vicente and Juquitiba, São Paulo. Rev Panam Salud Publica. 1997;2(3):189-93. 20. Cerutti Jr C, Boulos M, Coutinho A. F, Hatab MCLD, Falqueto A, Rezende H. R, Duarte AMRC, Collins W, Malafronte RS. Epidemiologic aspects of the malaria transmission cycle in an area of very low incidence in Brazil. Malar J. 2007;6:33. 33. Yamasaki T, Summa ME, Neves DV, de Oliveira SG, Gomes AC. Detection of etiological agents of malaria in howler monkeys from Atlantic Forests, rescued in regions of São Paulo city, Brazil. J Med Primatol. 2011;40(6):392-400. 21. Couto RDA, Latorre MRDO, Di Santi S, Natal D. Malária autóctone notificada no estado de São Paulo: aspectos clínicos e epidemiológicos de 1980 a 2007. Rev Soc Bras Med Trop. 2010;43(1):52-8. 34. Costa DC, Cunha VP, Assis GMP, Souza Jr JC, Hirano ZMB, Arruda ME et al. Plasmodium simium/Plasmodium vivax infections in southern brown howler monkeys from the Atlantic forest. Mem Inst Oswaldo Cruz. 2014;109(5):641-3. 22. Costa A de P, Bressan C da S, Pedro RS, Valls-de-Souza R, Silva S, Souza PR, et al. Delayed diagnosis of malaria in a dengue endemic area in the Brazilian extra-Amazon: recent experience of a malaria surveillance unit in state of Rio de Janeiro. Rev Soc Bras Med Trop. 2010;43(5):571-4. 35. de Alvarenga DA, de Pina-Costa A, de Sousa TN, Pissinatti A, Zalis MG, Suaréz-Mutis et al. REFERENCES Simian malaria in the Brazilian Atlantic forest: first description of natural infection of capuchin monkeys (Cebinae subfamily) by Plasmodium simium. Malar J. 2015;14:81. 23. Marques GRAM, Condino MLF, Serpa LLN, Cursino TVM. Aspectos epidemiológicos de malária autóctone na mata atlântica, litoral norte, Estado de São Paulo, 1985 – 2006. Rev Soc Bras Med Trop. 2008;41(4):386-9. 36. Duarte A. M, Malafronte RS, Cerutti C, Curado I, de Paiva BR, Maeda AY, et al. Natural Plasmodium infections in Brazilian wild monkeys: reservoirs for human infections? Acta Trop. 2008;107:179- 85. 24. Curado I, Dos Santos Malafronte R, de Castro Duarte AM, Kirchgatter K, Branquinho MS, Bianchi Galati EA. Malaria epidemiology in low-endemicity areas of the Atlantic Forest in the Vale do Ribeira, São Paulo, Brazil. Acta Trop. 2006;100(1-2):54-62. 37. Duarte AM, Pereira DM, de Paula MB, Fernandes A, Urbinatti PR, Ribeiro AF et al. Natural infection in anopheline species and its implications for autochthonous  malaria  in the Atlantic Forest in Brazil. Parasit Vectors. 2013;6:58. 25. de Alencar FEC, Malafronte RDS, Cerutti Junior C, Natal Fernandes L, Buery JC, Fux B, et al. Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra- Amazonian region of Brazil. Malar J. 2018;17:113. 38. Marrelli MT, Malafronte RS, Sallum MA, Natal D. Kerteszia subgenus of Anopheles associated with the Brazilian  Atlantic  rainforest: current knowledge and future challenges. Malar J. 2007;6:127. 39. Rezende HR, Soares RM, Cerutti C Jr, Alves IC, Natal D, Urbinatti PR, et al. Entomological characterization and natural infection of anophelines in an area of the  Atlantic Forest  with autochthonous  malaria  cases in mountainous region of Espírito Santo State, Brazil. Neotrop Entomol. 2009;38(2):272-80. 26. Maselli LM, Levy D, Laporta GZ, Monteiro AM, Fukuya LA, Ferreira-da-Cruz MF, et al. Detection of Plasmodium falciparum and Plasmodium vivax subclinical infection in non-endemic region: implications for blood transfusion and malaria epidemiology. Malar J. 2014;13:224. 40. Rezende HR, Falqueto A, Urbinatti PR, De Menezes RM, Natal D, Cerutti C Jr. Comparative study of distribution of anopheline vectors (Diptera: Culicidae) in areas with and without malaria transmission in the highlands of an extra-Amazonian region in  Brazil. J Med Entomol. 2013;50(3):598-602. 27. Folegatti PM, Siqueira AM, Monteiro WM, Lacerda MV, Drakeley CJ, Braga ÉM. A systematic review on malaria sero-epidemiology studies in the Brazilian Amazon: insights into immunological markers for exposure and protection. Malar J. 2017;16:107. 28. REFERENCES Laporta GZ, Burattini MN, Levy D, Fukuya LA, de Oliveira TM, Maselli LM et al. Plasmodium falciparum in the southeastern Atlantic forest: a challenge to the bromeliad-malaria paradigm? Malar J. 2015;14:181. 41. Sallum MA, Daniel-Ribeiro CT, Laporta GZ, Ferreira-da-Cruz M de F, Maselli LM, Levy D et al. Finding connections in the unexpected detection of Plasmodium vivax and Plasmodium falciparum DNA in asymptomatic blood donors: a fact in the Atlantic Forest. Malar J. 2014;13:337. 29. Deane L. Simian Malaria in Brazil. Mem Inst Oswaldo Cruz. 1992;87:1-20. 42. World Health Organization (WHO). Guidelines on prevention of the reintroduction of malaria. Geneva: WHO; 2007. 49p. 30. Deane LM, Ferreira Neto J, Sitônio JG. A new natural host of Plasmodium simium and Plasmodium brasilianum: the woolly spider monkey. Rev Inst Med Trop Sao Paulo. 1968;10(5):287-8. 43. Albuquerque HG, Peiter PC, Toledo LM, Alencar JA, Sabroza PC, Dias CG et al. Geographical information system (GIS) modeling territory receptivity to strengthen entomological surveillance: Anopheles (Nyssorhynchus) case study in Rio de Janeiro State, 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 https://creativecommons.org/licenses/by/4.0/
W4361836448.txt
https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_3_from_i_WWOX_i_Gene_Expression_Abolishes_Ovarian_Cancer_Tumorigenicity_i_In_vivo_i_and_Decreases_Attachment_to_Fibronectin_via_Integrin_sub_3_sub_/22378149/1/files/39823500.pdf
en
Supplementary Figure 3 from &lt;i&gt;WWOX&lt;/i&gt; Gene Expression Abolishes Ovarian Cancer Tumorigenicity &lt;i&gt;In vivo&lt;/i&gt; and Decreases Attachment to Fibronectin via Integrin α&lt;sub&gt;3&lt;/sub&gt;
null
2,023
cc-by
0
https://openalex.org/W4280506896
https://boris.unibe.ch/169999/1/Beck__R.__Dibbern__J.___Wiener__M._Interview_with_Peter_Mertens_and_Wolfgang_K_nig.pdf
English
null
Interview with Peter Mertens and Wolfgang König: “From Reasonable Automation to (Sustainable) Autonomous Systems”
Business & information systems engineering
2,022
cc-by
3,654
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
https://openalex.org/W4280599546
https://aip.scitation.org/doi/pdf/10.1063/5.0083054
English
null
3D flow field measurements outside nanopores
Review of scientific instruments online/Review of scientific instruments
2,022
cc-by
3,660
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
https://openalex.org/W2771317382
https://www.cambridge.org/core/services/aop-cambridge-core/content/view/37CF3CA3B5669D2E558037D95C416747/S1047951117002517a.pdf/div-class-title-pediatric-cardiac-intensive-care-society-statement-caring-for-children-with-critical-cardiac-disease-across-the-globe-div.pdf
English
null
Pediatric Cardiac Intensive Care Society Statement: caring for children with critical cardiac disease across the globe
Cardiology in the young
2,017
cc-by
1,566
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
https://openalex.org/W1984241142
https://zenodo.org/records/1546436/files/article.pdf
German
null
Die Behandlung der Schlaflosigkeit
Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift
1,912
public-domain
3,321
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 *
https://openalex.org/W4280605595
https://www.frontiersin.org/articles/10.3389/fpubh.2022.886262/pdf
English
null
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
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 J, Tomek V, Skovránek J, Povysilová V, Samánek M. Prenatal ultrasound screening of congenital heart disease in an unselected national population: a 21-year experience. Heart. (2011) 97:124–30. doi: 10.1136/hrt.2010.206623 7. van Velzen CL, Clur SA, Rijlaarsdam ME, Bax CJ, Pajkrt E, Heymans MW, et al. Prenatal detection of congenital heart disease–results of a national screening program. Bjog-Int J Obstet Gy. (2016) 123:400– 7. doi: 10.1111/1471-0528.13274 23. Yang XY Li XF, Lv XD, Liu YL. Incidence of congenital heart disease in Beijing, China. Chin med J (Engl). (2009) 122:1128–32. 24. Qu YJ, Chen JJ, Han FZ, Lin S, Bell EM, Pan W, et al. Can we improve the perinatal outcomes and early postnatal survival of fetuses with congenital heart disease by initiating specialized prenatal consultation service? Clinics Mother Child Health. (2020) 17:360. 8. Health Department of Guangdong Province in China. Guideline for the performance of obstetric ultrasound examinations. Bulletin People’s Gover Guangdong Provinc. (2006) 17:16–31. 9. Stumpflen I, Stumpflen A, Wimmer M, Bernaschek G. Effect of detailed fetal echocardiography as part of routine prenatal ultrasonographic screening on detection of congenital heart disease. Lancet. (1996) 348:854– 7. doi: 10.1016/S0140-6736(96)04069-X 25. Quartermain MD, Pasquali SK, Hill KD, Goldberg DJ, Huhta JC, Jacobs JP, et al. Variation in prenatal diagnosis of congenital heart disease in infants. Pediatrics. (2015) 136:e378–85. doi: 10.1542/peds.201 4-3783 10. Ou YQ, Mai JZ, Zhuang J, Liu XQ, Wu Y, Gao XM, et al. Risk factors of different congenital heart defects in Guangdong, China. Pediatr Res. (2016) 79:549–58. doi: 10.1038/pr.2015.264 26. Peterson C, Ailes E, Riehle-Colarusso T, Oster ME, Olney RS, Cassell CH, et al. Late detection of critical congenital heart disease among US infants: estimation of the potential impact of proposed universal screening using pulse oximetry. JAMA Pediatr. (2014) 168:361–70. doi: 10.1001/jamapediatrics.2013.4779 11. Qu YJ, Liu XQ, Zhuang J, Chen GC, Mai JZ, Guo XL, et al. Incidence of congenital heart disease: the 9-year experience of the Guangdong registry of congenital heart disease, China. PLoS ONE. (2016) 11:e0159257. doi: 10.1371/journal.pone.0159257 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. 12. Chinese Doctor Association Council on Ultrasound. Guidelines of prenatal ultrasound examinations. Chin J Med Ultrasound. (2012) 9:574–80. 13. Guangdong Provincial People’s Hospital, Guangdong Cardiovascular Institute, Guangdong Eugenics Association Council on Congenital Heart Diseases. REFERENCES 17. Ewigman BG, Crane JP, Frigoletto FD, LeFevre ML, Bain RP, McNellis D. Effect of prenatal ultrasound screening on perinatal outcome: RADIUS study group. N Engl J Med. (1993) 329:821–7. doi: 10.1056/NEJM1993091632 91201 1. Donofrio MT, Moon-Grady AJ, Hornberger LK, Copel JA, Sklansky MS, Abuhamad A, et al. Diagnosis and treatment of fetal cardiac disease: a scientific statement from the American heart association. Circulation. (2014) 129:2183–242. doi: 10.1161/01.cir.0000437597.44550.5d 1. Donofrio MT, Moon-Grady AJ, Hornberger LK, Copel JA, Sklansky MS, Abuhamad A, et al. Diagnosis and treatment of fetal cardiac disease: a scientific statement from the American heart association. Circulation. (2014) 129:2183–242. doi: 10.1161/01.cir.0000437597.44550.5d 18. Garne E1, Stoll C, Clementi M. Evaluation of prenatal diagnosis of congenital heart diseases by ultrasound: experience from 20 European registries. Ultrasound Obstet Gynecol. (2001) 17:386–91. doi: 10.1046/j.1469-0705.2001.00385.x 2. Zeng ZD, Zhang HW, Liu FL, Zhang NN. Current diagnosis and treatments for critical congenital heart defects. Exp Ther Med. (2016) 11:1550– 4. doi: 10.3892/etm.2016.3167 19. Tegnander E, Eik-Nes SH, Johansen OJ, Linker DT. Prenatal detection of heart defects at the routine fetal examination at 18 weeks in a nonselected population. Ultrasound Obstet Gynecol. (1995) 5:372– 80. doi: 10.1046/j.1469-0705.1995.05060372.x 3. Chang RK, Gurvitz M, Rodriguez S. Missed diagnosis of critical congenital heart disease. Arch Pediatr Adolesc Med. (2018) 162:969–74. doi: 10.1001/archpedi.162.10.969 20. Del Bianco A, Russo S, Lacerenza N, Rinaldi M, Rinaldi G, Nappi L, et al. Four chamber view plus three-vessel and trachea view for a complete evaluation of the fetal heart during the second trimester. J Perinat Med. (2006) 34:309– 12. doi: 10.1515/JPM.2006.059 4. Liberman RF, Getz KD, Lin AE, Higgins CA, Sekhavat S, Markenson GR, et al. Delayed diagnosis of critical congenital heart defects: trends and associated factors. Pediatrics. (2014) 134:e373–81. doi: 10.1542/peds.2013-3949 5. Peterson C, Dawson A, Grosse SD, Riehle-Colarusso T, Olney RS, Tanner JP, et al. Hospitalizations, costs, and mortality among infants with critical congenital heart disease: How important is timely detection? Birth Defects Res A Clin Mol Teratol. (2013) 97:664–72. doi: 10.1002/bdra.23165 21. Kirk JS, Riggs TW, Comstock CH, Lee W, Yang SS, Weinhouse E. Prenatal screening for cardiac anomalies: the value of routine addition of the aortic root to the four-chamber view. Obstet Gynecol. (1994) 84:427–31. 6. Nelle M, Raio L, Pavlovic M, Carrel T, Surbek D, Meyer-Wittkopf M. Prenatal 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
https://openalex.org/W368873271
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0128028&type=printable
English
null
Angiogenesis Dysregulation in Term Asphyxiated Newborns Treated with Hypothermia
PloS one
2,015
cc-by
8,410
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 May 21, 2015 References 1. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003; 9: 653–660. PMID: 12778163 2. Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, et al. VEGF-induced neuroprotection, neurogen- esis, and angiogenesis after focal cerebral ischemia. J Clin Invest. 2003; 111: 1843–1851. PMID: 12813020 2. Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, et al. VEGF-induced neuroprotection, neurogen- esis, and angiogenesis after focal cerebral ischemia. J Clin Invest. 2003; 111: 1843–1851. PMID: 12813020 3. Wang L, Zhang Z, Wang Y, Zhang R, Chopp M. Treatment of stroke with erythropoietin enhances neu- rogenesis and angiogenesis and improves neurological function in rats. Stroke. 2004; 35: 1732–1737. PMID: 15178821 4. Taguchi A, Soma T, Tanaka H, Kanda T, Nishimura H, Yoshikawa H, et al. (2004) Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model. J Clin Invest. 2004; 114: 330–338. PMID: 15286799 5. Krupinski J, Kaluza J, Kumar P, Kumar S, Wang JM. Role of angiogenesis in patients with cerebral is- chemic stroke. Stroke. 1994; 25: 1794–1798. PMID: 7521076 6. Dilenge ME, Majnemer A, Shevell MI. Long-term developmental outcome of asphyxiated term neo- nates. J Child Neurol. 2001; 16: 781–792. PMID: 11732762 7. Jacobs SE, Berg M, Hunt R, Tarnow-Mordi WO, Inder TE, Davis PG. Cooling for newborns with hypox- ic-ischemic encephalopathy. Cochrane Database Syst Rev. 2013; 1: CD003311. doi: 10.1002/ 14651858.CD003311.pub3 PMID: 23440789 8. Tagin MA, Woolcott CG, Vincer MJ, Whyte RK, Stinson DA. Hypothermia for neonatal hypoxic ischemic encephalopathy: an updated systematic review and meta-analysis. Arch Pediatr Adolesc Med. 2012; 166: 558–566. doi: 10.1001/archpediatrics.2011.1772 PMID: 22312166 9. Xiong Y, Mahmood A, Chopp M. Angiogenesis, neurogenesis and brain recovery of function following injury. Curr Opin Investig Drugs. 2010; 11: 298–308. PMID: 20178043 10. Yenari MA, Han HS. Neuroprotective mechanisms of hypothermia in brain ischaemia. Nat Rev Neu- rosci. 2012; 13: 267–278. doi: 10.1038/nrn3174 PMID: 22353781 11. Ara J, Shukla P, Frank M. Enhanced expression of the Flt-1 and Flk-1 receptor tyrosine kinases in a newborn piglet model of ischemic tolerance. J Neurochem. 2013; 124: 735–746. doi: 10.1111/jnc. 12110 PMID: 23199256 12. Li L, Xiong Y, Qu Y, Mao M, Mu W, Wang H, et al. The requirement of extracellular signal-related protein kinase pathway in the activation of hypoxia inducible factor 1 alpha in the developing rat brain after hyp- oxia-ischemia. Acta Neuropathol. 2008; 115: 297–303. Supporting Information Additionally, IGF-BP-1 and IGF-BP-4 were decreased, reflecting decreased expres- 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 May 21, 2015 References doi: 10.1007/s00401-008-0339-5 PMID: 18210138 13. Li L, Qu Y, Mao M, Xiong Y, Mu D. The involvement of phosphoinositid 3-kinase/Akt pathway in the acti- vation of hypoxia-inducible factor-1alpha in the developing rat brain after hypoxia-ischemia. Brain Res. 2008; 1197: 152–158. doi: 10.1016/j.brainres.2007.12.059 PMID: 18241842 14. Li L, Qu Y, Li J, Xiong Y, Mao M, Mu D. Relationship between HIF-1alpha expression and neuronal apo- ptosis in neonatal rats with hypoxia-ischemia brain injury. Brain Res. 2007; 1180: 133–139. PMID: 17920049 15. Mu D, Jiang X, Sheldon RA, Fox CK, Hamrick SE, Vexler ZS, et al. Regulation of hypoxia-inducible fac- tor 1α and induction of vascular endothelial growth factor in a rat neonatal stroke model. Neurobiol Dis. 2003; 14: 524–534. PMID: 14678768 12 / 15 PLOS ONE | DOI:10.1371/journal.pone.0128028 May 21, 2015 Angiogenesis Dysregulation following Birth Asphyxia 16. Gluckman PD, Wyatt JS, Azzopardi D, Ballard R, Edwards AD, Ferriero DM, et al. Selective head cool- ing with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial. Lancet 2005; 365: 663–670. PMID: 15721471 17. Shankaran S, Laptook AR, Ehrenkranz RA, Tyson JE, McDonald SA, Donovan EF, et al.; National Insti- tute of Child Health and Human Development Neonatal Research Network. Whole-body hypothermia for neonates with hypoxic-ischemic encephalopathy. N Engl J Med. 2005; 353: 1574–1584. PMID: 16221780 18. Azzopardi DV, Strohm B, Edwards AD, Dyet L, Halliday HL, Juszczak E, et al. Moderate hypothermia to treat perinatal asphyxial encephalopathy. N Engl J Med. 2009; 361: 1349–1358. doi: 10.1056/ NEJMoa0900854 PMID: 19797281 19. [No authors listed]. Executive summary: Neonatal encephalopathy and neurologic outcome, second edition. Report of the American College of Obstetricians and Gynecologists’ Task Force on Neonatal Encephalopathy. Obstet Gynecol. 2014; 123: 896–901. doi: 10.1097/01.AOG.0000445580.65983.d2 PMID: 24785633 20. American Academy of Pediatrics. Neonatal Encephalopathy and Neurologic Outcome, Second Edition. Pediatrics. 2014; 133: e1482–1488. 21. Boudes E, Tan X, Saint-Martin C, Shevell M, Wintermark P. Magnetic resonance imaging obtained dur- ing versus after hypothermia in asphyxiated Newborns. Arch Dis Child Fetal Neonatal Ed. 2015; [Epub ahead of print]. 22. Barkovich AJ, Hajnal BL, Vigneron D, Sola A, Partridge JC, Allen F, et al. Prediction of neuromotor out- come in perinatal asphyxia: evaluation of MR scoring systems. AJNR Am J Neuroradiol. 1998; 19: 143–149. PMID: 9432172 23. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 2009; 4: 44–57. doi: 10.1038/nprot.2008.211 PMID: 19131956 24. References Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehen- sive functional analysis of large gene lists. Nucleic Acids Res. 2009; 37: 1–13. doi: 10.1093/nar/gkn923 PMID: 19033363 25. Beck H, Acker T, Püschel AW, Fujisawa H, Carmeliet P, Plate KH. Cell type-specific expression of neu- ropilins in an MCA-occlusion model in mice suggests a potential role in post-ischemic brain remodeling. J Neuropathol Exp Neurol. 2002; 61: 339–350. PMID: 11939589 26. Jin K, Sun Y, Xie L, Childs J, Mao XO, Greenberg DA. Post-ischemic administration of heparin-binding epidermal growth factor-like growth factor (HB-EGF) reduces infarct size and modifies neurogenesis after focal cerebral ischemia in the rat. J Cereb Blood Flow Metab. 2004; 24: 399–408. PMID: 15087709 27. Sugiura S, Kitagawa K, Tanaka S, Todo K, Omura-Matsuoka E, Sasaki T, et al. Adenovirus-mediated gene transfer of heparin-binding epidermal growth factor-like growth factor enhances neurogenesis and angiogenesis after focal cerebral ischemia in rats. Stroke. 2005; 36: 859–864. PMID: 15746462 28. Oyagi A, Morimoto N, Hamanaka J, Ishiguro M, Tsuruma K, Shimazawa M, et al. Forebrain specific heparin-binding epidermal growth factor-like growth factor knockout mice show exacerbated ischemia and reperfusion injury. Neuroscience. 2011; 185: 116–24. doi: 10.1016/j.neuroscience.2011.04.034 PMID: 21524692 29. Doverhag C, Hedtjärn M, Poirier F, Mallard C, Hagberg H, Karlsson A, et al. Galectin-3 contributes to neonatal hypoxic-ischemic brain injury. Neurobiol Dis. 2010; 38: 36–46. doi: 10.1016/j.nbd.2009.12. 024 PMID: 20053377 30. Savman K, Heyes MP, Svedin P, Karlsson A. Microglia/macrophage-derived inflammatory mediators galectin-3 and quinolinic acid are elevated in cerebrospinal fluid from newborn infants after birth as- phyxia. Transl Stroke Res. 2013; 4: 228–235. doi: 10.1007/s12975-012-0216-3 PMID: 23807898 31. Delafontaine P, Song YH, Li Y. Expression, regulation, and function of IGF-1, IGF-1R, and IGF-1 bind- ing proteins in blood vessels. Arterioscler Thromb Vasc Biol. 2004; 24: 435–444. PMID: 14604834 32. Contois LW, Nugent DP, Caron JM, Cretu A, Tweedie E, Akalu A, et al. Insulin-like growth factor binding protein-4 differentially inhibits growth factor-induced angiogenesis. J Biol Chem. 2012; 287: 1779– 1789. doi: 10.1074/jbc.M111.267732 PMID: 22134921 33. Rho SB, Dong SM, Kang S, Seo SS, Yoo CW, Lee DO, et al. Insulin-like growth factor-binding protein-5 (IGFBP-5) acts as a tumor suppressor by inhibiting angiogenesis. Carcinogenesis. 2008; 29: 2106– 2111. doi: 10.1093/carcin/bgn206 PMID: 18775916 34. Clawson TF, Vannucci SJ, Wang GM, Seaman LB, Yang XL, Lee WH. Hypoxia-ischemia-induced apo- ptotic cell death correlates with IGF-I mRNA decrease in neonatal rat brain. PLOS ONE | DOI:10.1371/journal.pone.0128028 May 21, 2015 References Biol Signals Recept. 1999; 8:281–293. PMID: 10494014 13 / 15 PLOS ONE | DOI:10.1371/journal.pone.0128028 May 21, 2015 Angiogenesis Dysregulation following Birth Asphyxia 35. Lee WH, Wang GM, Seaman LB, Vannucci SJ. Coordinate IGF-I and IGFBP5 gene expression in peri- natal rat brain after hypoxia-ischemia. J Cereb Blood Flow Metab. 1996; 16: 227–236. PMID: 8594054 36. Gustafson K, Hagberg H, Bengtsson BA, Brantsing C, Isgaard J. Possible protective role of growth hor- mone in hypoxia-ischemia in neonatal rats. Pediatr Res. 1999; 45: 318–323. PMID: 10088648 37. Beresewicz M, Majewska M, Makarewicz D, Vayro S, Zabłocka B, Gorecki DC. Changes in the expres- sion of insulin-like growth factor 1 variants in the postnatal brain development and in neonatal hypoxia- ischaemia. Int J Dev Neurosci. 2010; 28: 91–97. doi: 10.1016/j.ijdevneu.2009.09.002 PMID: 19766709 38. Satar M, Ozcan K, Yapicioğlu H, Narli N. Serum insulin-like growth factor 1 and growth hormone levels of hypoxic-ischemic newborns. Biol Neonate. 2004; 85: 15–20. PMID: 14631161 39. Cunningham LA, Wetzel M, Rosenberg GA. Multiple roles for MMPs and TIMPs in cerebral ischemia. Glia. 2005; 50: 329–339. PMID: 15846802 40. Svedin P, Hagberg H, Sävman K, Zhu C, Mallard C. Matrix metalloproteinase-9 gene knock-out pro- tects the immature brain after cerebral hypoxia-ischemia. J Neurosci. 2007; 27: 1511–1518. PMID: 17301159 41. Dragun P, Makarewicz D, Wójcik L, Ziemka-Nałecz M, Słomka M, Zalewska T. Matrix metaloprotei- nases activity during the evolution of hypoxic-ischemic brain damage in the immature rat. The effect of 1-methylnicotinamide (MNA). J Physiol Pharmacol. 2008; 59: 441–455. PMID: 19218668 42. Bednarek N, Svedin P, Garnotel R, Favrais G, Loron G, Schwendiman L, et al. Increased MMP-9 and TIMP-1 in mouse neonatal brain and plasma and in human neonatal plasma after hypoxia-ischemia: a potential marker of neonatal encephalopathy. Pediatr Res. 2012; 71: 63–70. doi: 10.1038/pr.2011.3 PMID: 22289852 43. Sunagawa S, Ichiyama T, Honda R, Fukunaga S, Maeba S, Furukawa S. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in perinatal asphyxia. Brain Dev. 2009; 31: 588–593. doi: 10. 1016/j.braindev.2008.09.001 PMID: 18849127 44. Aimes RT, Zijlstra A, Hooper JD, Ogbourne SM, Sit ML, Fuchs S, et al. Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis. Thromb Haemost. 2003; 89: 561–572. PMID: 12624642 45. Wei L, Keogh CL, Whitaker VR, Theus MH, Yu SP. Angiogenesis and stem cell transplantation as po- tential treatments of cerebral ischemic stroke. Pathophysiology. 2005; 12: 47–62. PMID: 15927824 46. PLOS ONE | DOI:10.1371/journal.pone.0128028 May 21, 2015 References Zhang ZG, Zhang L, Jiang Q, Zhang R, Davies K, Powers C, et al. VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest. 2000; 106: 829–838. PMID: 11018070 47. Abumiya T, Yokota C, Kuge Y, Minematsu K. Aggravation of hemorrhagic transformation by early intra- arterial infusion of low-dose vascular endothelial growth factor after transient focal cerebral ischemia in rats. Brain Res. 2005; 1049: 95–103. PMID: 15935998 48. Kimura R, Nakase H, Tamaki R, Sakaki T. Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. Stroke. 2005; 36: 1259–1263. PMID: 15879344 49. Rosenkranz K, Kumbruch S, Tenbusch M, Marcus K, Marschner K, Dermietzel R, et al. Transplantation of human umbilical cord blood cells mediated beneficial effects on apoptosis, angiogenesis and neuro- nal survival after hypoxic-ischemic brain injury in rats. Cell Tissue Res. 2012; 348: 429–438. doi: 10. 1007/s00441-012-1401-0 PMID: 22526623 50. Feng Y, Rhodes PG, Bhatt AJ. Neuroprotective effects of vascular endothelial growth factor following hypoxic ischemic brain injury in neonatal rats. Pediatr Res. 2008; 64: 370–374. doi: 10.1203/PDR. 0b013e318180ebe6 PMID: 18535483 51. Dzietko M, Derugin N, Wendland MF, Vexler ZS, Ferriero DM. Delayed VEGF treatment enhances an- giogenesis and recovery after neonatal focal rodent stroke. Transl Stroke Res. 2013; 4: 189–200. doi: 10.1007/s12975-012-0221-6 PMID: 23926451 52. Aly H, Hassanein S, Nada A, Mohamed MH, Atef SH, Atiea W. Vascular endothelial growth factor in ne- onates with perinatal asphyxia. Brain Dev. 2009; 31: 600–604. doi: 10.1016/j.braindev.2008.09.004 PMID: 18926648 53. Vasiljevic B, Maglajlic-Djukic S, Gojnic M, Stankovic S, Ignjatovic S, Lutovac D. New insights into the pathogenesis of perinatal hypoxic-ischemic brain injury. Pediatr Int. 2011; 53: 454–462. doi: 10.1111/j. 1442-200X.2010.03290.x PMID: 21077993 54. Ergenekon E, Gücüyener K, Erbaş D, Aral S, Koç E, Atalay Y. Cerebrospinal fluid and serum vascular endothelial growth factor and nitric oxide levels in newborns with hypoxic ischemic encephalopathy. Brain Dev. 2004; 26: 283–286. PMID: 15165666 55. Okazaki K, Kusaka T, Kondo M, Kozawa K, Yoshizumi M, Kimura H. Temporal alteration of serum G- CSF and VEGF levels in perinatal asphyxia treated with head cooling. Cytokine. 2012; 60: 812–814. doi: 10.1016/j.cyto.2012.08.001 PMID: 22944463 PLOS ONE | DOI:10.1371/journal.pone.0128028 May 21, 2015 14 / 15 Angiogenesis Dysregulation following Birth Asphyxia 56. Bader M. Kallikrein—Kinin System in Neovascularization. Arterioscler Thromb Vasc Biol. 2009; 29: 617–619. doi: 10.1161/ATVBAHA.109.184549 PMID: 19369653 57. Imam SS, Gad GI, Atef SH, Shawky MA. PLOS ONE | DOI:10.1371/journal.pone.0128028 May 21, 2015 References Cord blood brain derived neurotrophic factor: diagnostic and prognostic marker in fullterm newborns with perinatal asphyxia. Pak J Biol Sci. 2009; 12: 1498–1504. PMID: 20180326 58. Liu F, Yang S, Du Z, Guo Z. Dynamic changes of cerebral-specific proteins in full-term newborns with hypoxic-ischemic encephalopathy. Cell Biochem Biophys. 2013; 66: 389–396. doi: 10.1007/s12013- 012-9478-3 PMID: 23203841 59. Korhonen L, Riikonen R, Nawa H, Lindholm D. Brain derived neurotrophic factor is increased in cere- brospinal fluid of children suffering from asphyxia. Neurosci Lett. 1998; 240:151–154. PMID: 9502226 60. Wang Y, Cao M, Liu A, Di W, Zhao F, Tian Y, et al. Changes of inflammatory cytokines and neurotro- phins emphasized their roles in hypoxic-ischemic brain damage. Int J Neurosci. 2013; 123:191–195. doi: 10.3109/00207454.2012.744755 PMID: 23110519 61. Graham EM, Sheldon RA, Flock DL, Ferriero DM, Martin LJ, O'Riordan DP, et al. Neonatal mice lacking functional Fas death receptors are resistant to hypoxic-ischemic brain injury. Neurobiol Dis. 2004; 17: 89–98. PMID: 15350969 62. Muramatsu K, Fukuda A, Togari H, Wada Y, Nishino H. Vulnerability to cerebral hypoxic-ischemic insult in neonatal but not in adult rats is in parallel with disruption of the blood-brain barrier. Stroke. 1997; 28: 2281–2288, discussion 2288–2289. PMID: 9368577 15 / 15
https://openalex.org/W4391560123
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0290929&type=printable
English
null
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
2,024
cc-by
9,706
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 //d i /10 1371/j l 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 //d i /10 1371/j l 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- leui Jung, Yonggyun Kim. Project administration: Yonggyun Kim. References 1. Klein AM, Vaissiere BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C, et al. Importance of pollinators in changing landscapes for world crops. Proc. R. Soc. B Biol. Sci. 2007; 274:303–313. https://doi.org/10.1098/rspb.2006.3721 PMID: 17164193 2. Ricketts TH, Regetz J, Steffan-Dewenter I, Cunningham SA, Kremen C, Bogdanski A, et al. Landscape effects on crop pollination services: are there general patterns? Ecol Lett. 2008; 11:499–515. https:// doi.org/10.1111/j.1461-0248.2008.01157.x PMID: 18294214 3. Ollerton J, Winfree R, Tarrant S. How many flowering plants are pollinated by animals? Oikos 2011; 120:321–326. 4. Delaplane KS, Mayer DF. Crop pollination by bees. CABI Press, Wallingford, CT. 2000. 5. Jung C. Economic value of honeybee pollination on major fruit and vegetable crops in Korea. Korean J Apic. 2008; 23:147–152. 6. Winston ML, Dropkin JA, Taylor OR. Demography and life history characteristics of two honey bee races (Apis mellifera). Oecologia. 1981; 48:407–413. https://doi.org/10.1007/BF00346502 PMID: 28309760 7. Yasuda M, Sakamoto Y, Goka K, Nagamitsu T, Taki H. Insecticide susceptibility in Asian honey bees (Apis cerana (Hymenoptera: Apidae)) and implications for wild honey bees in Asia. J Econ Entomol. 2017; 110:447–452. https://doi.org/10.1093/jee/tox032 PMID: 28334064 8. Ai H, Yan X, Han R. Occurrence and prevalence of seven bee viruses in Apis mellifera and Apis cerana apiaries in China. J Invertebr Pathol. 2012; 109:160–164. 9. Lo´pez-Uribe MM, Ricigliano VA, Simone-Finstrom M. Defining pollinator health: a holistic approach based on ecological, genetic, and physiological factors. Annu Rev Anim Biosci. 2020; 8:269–294. https://doi.org/10.1146/annurev-animal-020518-115045 PMID: 31618045 10. Favaro R, Bauer LM, Rossi M, D’Ambrosio L, Bucher E, Angeli S. Botanical origin of pesticide residues in pollen loads collected by honeybees during and after apple bloom. Front Physiol. 2019; 10:1069. https://doi.org/10.3389/fphys.2019.01069 PMID: 31620006 11. Zhao K, Wu H, Hou R, Wu J, Wang Y, Huang S, et al. Effects of sublethal azadirachtin on the immune response and midgut microbiome of Apis cerana cerana (Hymenoptera: Apidae). Ecotoxicol Environ Saf. 2022; 229:113089. https://doi.org/10.1016/j.ecoenv.2021.113089 PMID: 34929506 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929 February 6, 2024 16 / 19 PLOS ONE Honeybee immunity against the American founbrood disease 12. Genersch E, Ashiralieva A, Fries I. Strain- and genotype-specific differences in virulence of Paenibacil- lus larvae subsp. larvae, a bacterial pathogen causing American foulbrood disease in honeybees. Appl Environ Microbiol. 2005; 71:7551–7555. 13. Genersch E. American foulbrood in honeybees and its causative agent, Paenibacillus larvae. J Inver- tebr Pathol. 2010; 103:S10–S19. 14. Yue D, Nordhoff M, Wieler LH, Genersch E. References Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foul- brood of honeybees (Apis mellifera). Environ Microbiol. 2008; 10:1612–1620. https://doi.org/10.1111/j. 1462-2920.2008.01579.x PMID: 18331334 15. Lindstro¨m A, Korpela S, Fries I. The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies. J Invertebr Pathol. 2008; 99:82–86. https://doi.org/10. 1016/j.jip.2008.06.010 PMID: 18640122 16. Evans JD, Aronstein K, Chen YP, Hetru C, Imler JL, Jiang H, et al. Immune pathways and defence mechanisms in honey bees Apis mellifera. Insect Mol Biol. 2006; 15:645–656. 17. Alaux C, Kemper N, Kretzschmar A, Le Conte Y. Brain, physiological and behavioral modulation induced by immune stimulation in honeybees (Apis mellifera): a potential mediator of social immunity? Brain Behav Immun. 2012; 26:1057–1060. https://doi.org/10.1016/j.bbi.2012.04.004 PMID: 22542419 18. Evans JD, Spivak M. Socialized medicine: individual and communal disease barriers in honey bees. J Invertebr Pathol. 2010; 103:S62–72. https://doi.org/10.1016/j.jip.2009.06.019 PMID: 19909975 19. Richard FJ, Aubert A, Grozinger CM. Modulation of social interactions by immune stimulation in honey bee, Apis mellifera, workers. BMC Biol. 2008; 6:50. https://doi.org/10.1186/1741-7007-6-50 PMID: 19014614 20. Doublet V, Poeschl Y, Gogol-Do¨ring A, Alaux C, Annoscia D, Aurori C, et al. Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens. BMC Genomics. 2017; 18:207. https://doi.org/10.1186/s12864-017-3597-6 PMID: 28249569 21. El-Seedi HR, Ahmed HR, El-Wahed AAA, Saeed A, Algethami AF, Attia NF, et al. Bee stressors from an immunological perspective and strategies to improve bee health. Vet Sci. 2022; 9:199. https://doi. org/10.3390/vetsci9050199 PMID: 35622727 22. Brandt A, Gorenflo A, Siede R, Meixner M, Bu¨chler R. The neonicotinoids thiacloprid, imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.). J Insect Physiol. 2016; 86:40–47. https://doi.org/10.1016/j.jinsphys.2016.01.001 PMID: 26776096 23. Brandt A, Grikscheit K, Siede R, Grosse R, Meixner MD, Bu¨chler R. Immunosuppression in honeybee queens by the neonicotinoids thiacloprid and clothianidin. Sci Rep. 2017; 7:4673. https://doi.org/10. 1038/s41598-017-04734-1 PMID: 28680118 24. Lo´pez JH, Krainer S, Engert A, Schuehly W, Riessberger-Galle´ U, Crailsheim K. Sublethal pesticide doses negatively affect survival and the cellular responses in American foulbrood-infected honeybee larvae. Sci Rep. 2017; 7:40853. https://doi.org/10.1038/srep40853 PMID: 28145462 25. Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu Rev Immunol. 2007; 25:697–743. 26. Mollah MMI, Choi HW, Yeam I, Lee JM, Kim Y. References Interaction of group IA phospholipase A2 with metal ions and phospholipid vesicles probed with deuterium exchange mass spectrometry. Bio- chemistry 2008; 47:6451–6459. 41. Winstead MV, Balsinde J, Dennis EA. Calcium-independent phospholipase A2: structure and function. Biochim Biophys Acta 2000; 1488:28–39. 42. Shayman JA, Tesmer JJG. Lysosomal phospholipase A2. Biochim Biophys Acta 2019; 1864:932–944. 43. Annoscia D, Brown SP, Di Prisco G, De Paoli E, Del Fabbro S, et al. Haemolymph removal by Varroa mite destabilizes the dynamical interaction between immune effectors and virus in bees, as predicted by Volterra’s model. Proc Biol Sci. 2019; 286:20190331. https://doi.org/10.1098/rspb.2019.0331 PMID: 30991929 44. Cotter SC, Simpson SJ, Raubenheimer D, Wilson K. Macronutrient balance mediates trade-offs between immune function and life history traits. Funct Ecol. 2011; 25:186–198. 45. Krongdang S, Evans JD, Chen Y, Mookhploy W, Chantawannakul P. Comparative susceptibility and immune responses of Asian and European honey bees to the American foulbrood pathogen, Paeniba- cillus larvae. Insect Sci. 2019; 26:831–842. https://doi.org/10.1111/1744-7917.12593 PMID: 29578641 46. Yoshiyama M, Kimura K. Bacteria in the gut of Japanese honeybee, Apis cerana japonica, and their antagonistic effect against Paenibacillus larvae, the causal agent of American foulbrood. J Invertebr Pathol. 2009; 102:91–96. 47. Nazzi F, Annoscia D, Caprio E, Di Prisco G, Pennacchio F. Honeybee immunity and colony losses. Entomologia 2014; 2:80–87. 48. Negri P, Maggie M, Ramirez L, Szawarski N, De Feudis L, Lamattina L, et al. Cellular immunity in Apis mellifera: studying hemocytes brings light about bees skills to confront threats. Apidologie 2016; 47:379–388. 49. Ga´bor E, Cinege G, Csorda´s G, Rusvai M, Honti V, Kolics B, et al. Identification of reference markers for characterizing honey bee (Apis mellifera) hemocyte classes. Dev Comp Immunol. 2020; 109:103701. https://doi.org/10.1016/j.dci.2020.103701 PMID: 32320738 50. Gao L, Wang H, Liu Z, Liu S, Zhao G, Xu B. et al. The initial analysis of a serine proteinase gene (AccSp10) from Apis cerana cerana: possible involvement in pupal development, innate immunity and abiotic stress responses. Cell Stress Chaperones 2017; 22:867–877. https://doi.org/10.1007/s12192- 017-0818-5 PMID: 28695333 51. Zou Z, Lopez DL, Kanost MR, Evans JD, Jiang H. Comparative analysis of serine protease-related genes in the honey bee genome: possible involvement in embryonic development and innate immunity. Insect Mol Biol. 2006; 15:603–614. https://doi.org/10.1111/j.1365-2583.2006.00684.x PMID: 17069636 52. Kim BY, Jin BR. Apolipophorin III from honeybees (Apis cerana) exhibits antibacterial activity. Comp Biochem Physiol B Biochem Mol Biol. 2015; 182:6–13. https://doi.org/10.1016/j.cbpb.2014.11.010 PMID: 25483322 53. References Salicylic acid, a plant hormone, suppresses phytopha- gous insect immune response by interrupting HMG-like DSP1. Front Physiol. 2021; 12:744272. https:// doi.org/10.3389/fphys.2021.744272 PMID: 34671276 27. Kim CY, Ahmed S, Stanley D, Kim Y. HMG-like DSP1 is a damage signal to mediate the western flower thrips, Frankliniella occidentalis, immune responses to tomato spotted wilt virus infection. Dev Comp Immunol. 2023; 144:104706. 28. Gillespie JP, Kanost MR, Trenczek T. Biological mediators of insect immunity. Annu Rev Entomol. 1997; 42:611–643. https://doi.org/10.1146/annurev.ento.42.1.611 PMID: 9017902 29. Kim Y, Ahmed S, Stanley D, An C. Eicosanoid-mediated immunity in insects. Dev Comp Immunol. 2018; 83:130–143. https://doi.org/10.1016/j.dci.2017.12.005 PMID: 29225005 30. Kim Y, Stanley D. Eicosanoid signaling in insect immunology: new genes and unresolved issues. Genes 2021; 12:211. https://doi.org/10.3390/genes12020211 PMID: 33535438 31. Bedick JC, Tunaz H, Nor Aliza AR, Putnam SM, Ellis MD, Stanley DW. Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older for- agers. Comp Biochem Physiol C Toxicol Pharmacol. 2001; 130:107–117. 32. Dennis E, Cao J, Hsu YH, Magrioti V, Kokotos G. Phospholipase A2 enzymes: physical structure, bio- logical function, disease implication, chemical inhibition, and therapeutic intervention. Chem Rev. 2011; 111:6130–6185. 17 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929 February 6, 2024 PLOS ONE Honeybee immunity against the American founbrood disease 33. Schaloske RH, Dennis EA. The phospholipase A2 superfamily and its group numbering system. Bio- chim Biophys Acta 2006; 1761:1246–1259. 34. Vasquez AM, Mouchlis VD, Dennis EA. Review of four major distinct types of human phospholipase A2. Adv Biol Regul. 2018; 67:212–218. https://doi.org/10.1016/j.jbior.2017.10.009 PMID: 29248300 35. Lavine MD, Strand MR. Insect hemocytes and their role in immunity. Insect Biochem Mol Biol. 2002; 32:1295–1309. https://doi.org/10.1016/s0965-1748(02)00092-9 PMID: 12225920 36. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: mini- mum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55:611– 622. https://doi.org/10.1373/clinchem.2008.112797 PMID: 19246619 37. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods. 2001; 25:402–408. 38. Schmehl DR, Tome´ HVV, Mortensen AN, Martins GF, Ellis JD. Protocol for the in vitro rearing of honey bee (Apis mellifera L.) workers. J Apic Res. 2016; 55:113–129. 39. SAS Institute Inc. SAS/STAT user’s guide, Release 6.03, Ed. Cary, NC. 1989. 40. Burke JE, Karbarz MJ, Deems RA, Li S, Woods VL Jr, Dennis EA. PLOS ONE | https://doi.org/10.1371/journal.pone.0290929 February 6, 2024 References Prado A, Brunet JL, Peruzzi M, Bonnet M, Bordier C, Crauser D, et al. Warmer winters are associated with lower levels of the cryoprotectant glycerol, a slower decrease in vitellogenin expression and reduced virus infections in winter honeybees. J Insect Physiol. 2022; 136:104348. https://doi.org/10. 1016/j.jinsphys.2021.104348 PMID: 34906562 54. Park HG, Lee KS, Kim BY, Yoon HJ, Choi YS, Lee KY, et al. Honeybee (Apis cerana) vitellogenin acts as an antimicrobial and antioxidant agent in the body and venom. Dev Comp Immunol. 2018; 85:51– 60. https://doi.org/10.1016/j.dci.2018.04.001 PMID: 29621531 PLOS ONE | https://doi.org/10.1371/journal.pone.0290929 February 6, 2024 18 / 19 PLOS ONE Honeybee immunity against the American founbrood disease 55. Stanley DW. Eicosanoids in invertebrate signal transduction systems. Princeton University Press, Princeton, NJ. 2000. 56. Burke JE, Dennis EA. Phospholipase A2 structure/function, mechanism, and signaling. J Lipid Res. 2009; 50:S237–S242. 57. Habermann E. Bee and wasp venoms. Science 1972; 177:314–322. https://doi.org/10.1126/science. 177.4046.314 PMID: 4113805 58. Kuchler K, Gmachl M, Sippl MJ. Analysis of the cDNA for phospholipase A2 from honeybee venom glands. The deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes. Eur J Biochem. 1989; 184:249–254. 59. Defferrari MS, Lee DH, Fernandes CL, Orchard I, Carlini CR. A phospholipase A2 gene is linked to Jack bean urease toxicity in the Chagas’ disease vector Rhodnius prolixus. Biochim Biophys Acta. 2014; 1840:396–405. 60. Hrithik MTH, Hong J, Kim Y. Identification of four secretory phospholipase A2s in a lepidopteran insect, Acrolepiopsis sapporensis, and their functional association with cellular immune responses. Front Endocrinol. 2023; 14:1190834. 61. Schneider BE, Behrends J, Hagens K, Harmel N, Shayman JA, Schaible UE. Lysosomal phospholipase A2: a novel player in host immunity to Mycobacterium tuberculosis. Eur J Immunol. 2014; 44:2394– 2404. 62. Mollah MMI, Ahmed S, Kim Y. Immune mediation of HMG-like DSP1 via Toll-Spa¨tzle pathway and its specific inhibition by salicylic acid analogs. PLoS Pathog. 2021; 17:e1009467. 63. Ahmed S, Roy MC, Choi D, Kim Y. HMG-Like DSP1 Mediates immune responses of the western flower thrips (Frankliniella occidentalis) against Beauveria bassiana, a fungal pathogen. Front Immunol. 2022; 13:875239. https://doi.org/10.3389/fimmu.2022.875239 PMID: 35450074 19 / 19
https://openalex.org/W2485554478
https://research-information.bris.ac.uk/ws/files/145380589/Full_text_PDF_final_published_version_.pdf
English
null
Is there a role for <scp>IGF</scp>‐1 in the development of second primary cancers?
Cancer medicine
2,016
cc-by
12,300
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 survivors. Nature reviews. Clin. Oncol. (R. Coll. Radiol.) 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- stand the role of IGF-­1 in the development of specific SPCs, as well as to determine which pathways downstream of the IGF-­1R are involved in this process. 7. Bianchini, F., R. Kaaks, and H. Vainio. 2002. Overweight, obesity, and cancer risk. Lancet Oncol 3:565–574. 8. Tsugane, S., and M. Inoue. 2010. Insulin resistance and cancer: epidemiological evidence. Cancer Sci. 101:1073–1079. 9. Trevisan, M., J. Liu, P. Muti, G. Misciagna, A. Menotti, F. Fucci, et  al. 2001. Markers of insulin resistance and colorectal cancer mortality. Cancer Epidemiol. Biomark. Prev. 10:937–941. 10. Travis, L. B., C. S. Rabkin, L. M. Brown, J. M. Allan, B. P. Alter, C. B. Ambrosone, et  al. 2006. Cancer survivorship–genetic susceptibility and second primary cancers: research strategies and recommendations. J. Natl Cancer Inst. 4:15–25. References Serum levels of insulin growth factor (IGF-­I) and IGF-­binding protein predict risk of second primary tumors in patients with head and neck cancer. Clin. Cancer Res. 10(12 Pt 1):3988–3995. 35. Yu, H., and T. Rohan. 2000. Role of the insulin-­like growth factor family in cancer development and progression. J. Natl Cancer Inst. 92:1472–1489. 21. Yu, H., M. R. Spitz, J. Mistry, J. Gu, W. K. Hong, and X. Wu. 1999. Plasma levels of insulin-­like growth factor-­I and lung cancer risk: a case-­control analysis. J. Natl Cancer Inst. 20:151–156. 36. Pollak, M. N., E. S. Schernhammer, and S. E. Hankinson. 2004. Insulin-­like growth factors and neoplasia. Nat. Rev. Cancer 4:505–518. 37. Hartog, H., H. M. Boezen, M. M. de Jong, M. Schaapveld, J. Wesseling, and W. T. van der Graaf. 2013. Prognostic value of insulin-­like growth factor 1 and insulin-­like growth factor binding protein 3 blood levels in breast cancer. Breast 22:1155–1160. 22. Hankinson, S. E., W. C. Willett, G. A. Colditz, D. J. Hunter, D. S. Michaud, B. Deroo, et  al. 1998 May. Circulating concentrations of insulin-­like growth factor-­I and risk of breast cancer. Lancet 9:1393–1396. 23. Ma, J., M. N. Pollak, E. Giovannucci, J. M. Chan, Y. Tao, C. H. Hennekens, et  al. 1999. Prospective study of colorectal cancer risk in men and plasma levels of insulin-­like growth factor (IGF)-­I and IGF-­binding protein-­3. J. Natl Cancer Inst. 91:620–625. 38. Yakar, S., D. Leroith, and P. Brodt. 2005. The role of the growth hormone/insulin-­like growth factor axis in tumor growth and progression: lessons from animal models. Cytokine Growth Factor Rev. 16(4–5):407–420. 39. Laron, Z. 2001. Insulin-­like growth factor 1 (IGF-­1): a growth hormone. Mol. Pathol. 54:311–316. 24. Chan, J. M., M. J. Stampfer, E. Giovannucci, P. H. Gann, J. Ma, P. Wilkinson, et  al. 1998. Plasma insulin-­like growth factor-­I and prostate cancer risk: a prospective study. Science 279:563–566. 40. Sklar, C. A., A. C. Mertens, P. Mitby, G. Occhiogrosso, J. Qin, G. Heller, et  al. 2002. Risk of disease recurrence and second neoplasms in survivors of childhood cancer treated with growth hormone: a report from the childhood cancer survivor study. J. Clin. Endocrinol. Metab. 87:3136–3141. 25. Brooks, A. J., and M. J. Waters. 2010. The growth hormone receptor: mechanism of activation and clinical implications. Nat. Rev. Endocrinol. 6:515–525. 26. Laron, Z. 2015. Lesson from 50 years of study of laron syndrome. Endocr. Pra. 12:1395–1402. 41. Peyrat, J. P., J. References 1. Morris, L. G., A. G. Sikora, S. G. Patel, R. B. Hayes, and I. Ganly. 2011. Second primary cancers after an index head and neck cancer: subsite-­specific trends in the era of human papillomavirus-­associated oropharyngeal cancer. J. Clin. Oncol. 29:739–746. 15. Li, X., and K. Hemminki. 2003. Familial and second lung cancers: a nation-­wide epidemiologic study from Sweden. Lung Cancer 39:255–263. 2. Travis, L. B. 2006. The epidemiology of second primary cancers. Cancer Epidemiol. Biomarkers Prev. 15:2020–2026. 16. Dong, C., and K. Hemminki. 2001. Second primary neoplasms in 633,964 cancer patients in Sweden, 1958-­1996. Int. J. Cancer 93:155–161. 17. Gao, X., S. G. Fisher, and B. Emami. 2003. Risk of second primary cancer in the contralateral breast in women treated for early-­stage breast cancer: a population-­based study. Int. J. Radiat. Oncol. Biol. Phys. 15:1038–1045. 3. Cancer Facts & Figures.2015. Atlanta, Georgia: American Cancer Society; 2015; Available from: http://www.cancer. org/acs/groups/content/@editorial/documents/document/ acspc-044552.pdf (accessed date: 7 February 2016). 3. Cancer Facts & Figures.2015. Atlanta, Georgia: American Cancer Society; 2015; Available from: http://www.cancer. org/acs/groups/content/@editorial/documents/document/ acspc-044552.pdf (accessed date: 7 February 2016). 4. Sasco, A. J., M. B. Secretan, and K. Straif. 2004. Tobacco smoking and cancer: a brief review of recent epidemiological evidence. Lung Cancer 45(Suppl 2):S3–S9. 18. Neglia, J. P., D. L. Friedman, Y. Yasui, A. C. Mertens, S. Hammond, M. Stovall, et  al. 2001. Second malignant neoplasms in five-­year survivors of childhood cancer: childhood cancer survivor study. J. Natl Cancer Inst. 93:618–629. 5. Lee, P. N., B. A. Forey, and K. J. Coombs. 2012. Systematic review with meta-­analysis of the epidemiological evidence in the 1900s relating smoking to lung cancer. BMC Cancer 12:385. 19. Van Hemelrijck, M., A. Feller, H. Garmo, F. Valeri, D. Korol, S. Dehler, et  al. 2014. Incidence of second malignancies for prostate cancer. PLoS ONE 9:e102596. 6. Wolk, A., G. Gridley, M. Svensson, O. Nyren, J. K. McLaughlin, J. F. Fraumeni, et  al. 2001. A prospective 3362 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd T. Shanmugalingam et al. IGF-1 in the development of first and second primary cancers functions for insulin-­like growth factors. Physiol. Rev. 76:1005–1026. 20. Wu, X., H. Zhao, K. A. Do, M. M. Johnson, Q. Dong, W. K. Hong, et  al. 2004. References Bonneterre, B. Hecquet, P. Vennin, M. M. Louchez, C. Fournier, et  al. 1993. Plasma insulin-­like growth factor-­1 (IGF-­1) concentrations in human breast cancer. Eur. J. Cancer 29A:492–497. 27. NIH National Cancer Institute. Available from: http:// dceg.cancer.gov/research/what-we-study/second-cancers (accessed date: 22 February 2015). 28. Sachdev, D., and D. Yee. 2001. The IGF system and breast cancer. Endocr. Relat. Cancer 8:197–209. 42. Endogenous, H.,Breast Cancer Collaborative G; T. J. Key, P. N. Appleby, G. K. Reeves, and A. W. Roddam. 2010. Insulin-­like growth factor 1 (IGF1), IGF binding protein 3 (IGFBP3), and breast cancer risk: pooled individual data analysis of 17 prospective studies. Lancet Oncol. 11:530–542. 29. Sara, V. R., and K. Hall. 1990. Insulin-­like growth factors and their binding proteins. Physiol. Rev. 70:591–614. 30. Rosenfeld, R. G., G. Lamson, H. Pham, Y. Oh, C. Conover, D. D. De Leon, et  al. 1990. Insulinlike growth factor-­binding proteins. Recent Prog. Horm. Res. 46:99–159; discussion -63. 43. Rinaldi, S., P. H. Peeters, F. Berrino, L. Dossus, C. Biessy, A. Olsen, et  al. 2006. IGF-­I, IGFBP-­3 and breast cancer risk in women: the European Prospective Investigation into Cancer and Nutrition (EPIC). Endocr. Relat. Cancer 13:593–605. 31. El-Shewy, H. M., M. H. Lee, L. M. Obeid, A. A. Jaffa, and L. M. Luttrell. 2007. The insulin-­like growth factor type 1 and insulin-­like growth factor type 2/mannose-­6-­ phosphate receptors independently regulate ERK1/2 activity in HEK293 cells. J. Biol. Chem. 282:26150–26157. 44. Kaaks, R., T. Johnson, K. Tikk, D. Sookthai, A. Tjonneland, N. Roswall, et  al. 2014 Jun. Insulin-­like growth factor I and risk of breast cancer by age and hormone receptor status-­A prospective study within the EPIC cohort. Int. J. Cancer 134:2683–2690. 32. Bach, L. A., and L. J. Hale. 2015. Insulin-­like growth factors and kidney disease. Am. J. Kidney Dis. 65:327–336. 45. Baglietto, L., D. R. English, J. L. Hopper, H. A. Morris, W. D. Tilley, and G. G. Giles. 2007. Circulating insulin-­like growth factor-­I and binding protein-­3 and the risk of breast cancer. Cancer Epidemiol. Biomark. Prev. 16:763–768. 33. Kaplan, R. C., H. D. Strickler, T. E. Rohan, R. Muzumdar, and D. L. Brown. 2005. Insulin-­like growth factors and coronary heart disease. Cardiol. Rev. 13:35–39. 46. Renehan, A. G., M. Zwahlen, C. Minder, S. T. O’Dwyer, S. M. Shalet, and M. Egger. 2004. Insulin-­like growth factor (IGF)-­I, IGF binding protein-­3, and 34. Stewart, C. E., and P. Rotwein. 1996. References Growth, differentiation, and survival: multiple physiological 3363 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. T. Shanmugalingam et al. IGF-1 in the development of first and second primary cancers cancer risk: systematic review and meta-­regression analysis. Lancet 363:1346–1353. cancer risk: systematic review and meta-­regression analysis. Lancet 363:1346–1353. screening. Evidence from the BUPA study and meta-­ analysis of prospective epidemiological studies. Br. J. Cancer 95:112–117. 47. Shi, R., H. Yu, J. McLarty, and J. Glass. 2004. IGF-­I and breast cancer: a meta-­analysis. Int. J. Cancer 111:418–423. 58. Chen, B., S. Liu, W. Xu, X. Wang, W. Zhao, and J. Wu. 2009. IGF-­I and IGFBP-­3 and the risk of lung cancer: a meta-­analysis based on nested case-­control studies. J. Exp. Clin. Cancer Res. 28:89. 48. Sugumar, A., Y. C. Liu, Q. Xia, Y. S. Koh, and K. Matsuo. 2004. Insulin-­like growth factor (IGF)-­I and IGF-­binding protein 3 and the risk of premenopausal breast cancer: a meta-­analysis of literature. Int. J. Cancer 111:293–297. 59. Cao, H., G. Wang, L. Meng, H. Shen, Z. Feng, Q. Liu, et  al. 2012. Association between circulating levels of IGF-­1 and IGFBP-­3 and lung cancer risk: a meta-­ analysis. PLoS ONE 7:e49884. 49. Schernhammer, E. S., J. M. Holly, D. J. Hunter, M. N. Pollak, and S. E. Hankinson. 2006. Insulin-­like growth factor-­I, its binding proteins (IGFBP-­1 and IGFBP-­3), and growth hormone and breast cancer risk in the nurses health study II. Endocr. Relat. Cancer 13:583–592. 60. Jaques, G., K. Noll, B. Wegmann, S. Witten, E. Kogan, R. T. Radulescu, et  al. 1997. Nuclear localization of insulin-­like growth factor binding protein 3 in a lung cancer cell line. Endocrinology 138:1767–1770. 50. Kaaks, R., E. Lundin, S. Rinaldi, J. Manjer, C. Biessy, S. Soderberg, et  al. 2002. Prospective study of IGF-­I, IGF-­binding proteins, and breast cancer risk, in northern and southern Sweden. Cancer Causes Control 13:307–316. 61. Favoni, R. E., A. de Cupis, F. Ravera, C. Cantoni, P. Pirani, A. Ardizzoni, et  al. 1994. Expression and function of the insulin-­like growth factor I system in human non-­small-­cell lung cancer and normal lung cell lines. Int. J. Cancer 56:858–866. 51. Maor, S., D. Mayer, R. I. Yarden, A. V. Lee, R. Sarfstein, H. Werner, et  al. 2006. Estrogen receptor regulates insulin-­like growth factor-­I receptor gene expression in breast tumor cells: involvement of transcription factor Sp1. J. Endocrinol. 191:605–612. 62. Cohen, P., D. M. Peehl, T. References A. Stamey, K. F. Wilson, D. R. Clemmons, and R. G. Rosenfeld. 1993. Elevated levels of insulin-­like growth factor-­binding protein-­2 in the serum of prostate cancer patients. J. Clin. Endocrinol. Metab. 76:1031–1035. 52. Stewart, A. J., M. D. Johnson, F. E. May, and B. R. Westley. 1990. Role of insulin-­like growth factors and the type I insulin-­like growth factor receptor in the estrogen-­stimulated proliferation of human breast cancer cells. J. Biol. Chem. 265:21172–21178. 63. Kanety, H., Y. Madjar, Y. Dagan, J. Levi, M. Z. Papa, C. Pariente, et  al. 1993. Serum insulin-­like growth factor-­binding protein-­2 (IGFBP-­2) is increased and IGFBP-­3 is decreased in patients with prostate cancer: correlation with serum prostate-­specific antigen. J. Clin. Endocrinol. Metab. 77:229–233. 53. Ahn, J., S. J. Weinstein, K. Snyder, M. N. Pollak, J. Virtamo, and D. Albanes. 2006. No association between serum insulin-­like growth factor (IGF)-­I, IGF-­binding protein-­3, and lung cancer risk. Cancer Epidemiol. Biomark. Prev. 15:2010–2012. 64. Mantzoros, C. S., A. Tzonou, L. B. Signorello, M. Stampfer, D. Trichopoulos, and H. O. Adami. 1997. Insulin-­like growth factor 1 in relation to prostate cancer and benign prostatic hyperplasia. Br. J. Cancer 76:1115–1118. 54. Spitz, M. R., M. J. Barnett, G. E. Goodman, M. D. Thornquist, X. Wu, and M. Pollak. 2002. Serum insulin-­like growth factor (IGF) and IGF-­binding protein levels and risk of lung cancer: a case-­control study nested in the beta-­carotene and retinol efficacy trial cohort. Cancer Epidemiol. Biomark. Prev. 11:1413–1418. 65. Dunn, S. E., F. W. Kari, J. French, J. R. Leininger, G. Travlos, R. Wilson, et  al. 1997. Dietary restriction reduces insulin-­like growth factor I levels, which modulates apoptosis, cell proliferation, and tumor progression in p53-­deficient mice. Cancer Res. 57:4667–4672. 55. London, S. J., J. M. Yuan, G. S. Travlos, Y. T. Gao, R. E. Wilson, R. K. Ross, et  al. 2002. Insulin-­like growth factor I, IGF-­binding protein 3, and lung cancer risk in a prospective study of men in China. J. Natl Cancer Inst. 94:749–754. 66. Pollak, M., W. Beamer, and J. C. Zhang. 1998. Insulin-­like growth factors and prostate cancer. Cancer Metastasis Rev. 17:383–390. 67. Saikali, Z., H. Setya, G. Singh, and S. Persad. 2008. Role of IGF-­1/IGF-­1R in regulation of invasion in DU145 prostate cancer cells. Cancer Cell Int. 8:10. 56. Lukanova, A., P. Toniolo, A. Akhmedkhanov, C. Biessy, N. J. Haley, R. E. Shore, et  al. 2001. References A prospective study of insulin-­like growth factor-­I, IGF-­binding proteins-­1, -­2 and -­3 and lung cancer risk in women. Int. J. Cancer 92:888–892. 68. Grimberg, A., and P. Cohen. 1999. Growth hormone and prostate cancer: guilty by association? J. Endocrinol. Invest. 22(5 Suppl):64–73. 57. Morris, J. K., L. M. George, T. Wu, and N. J. Wald. 2006. Insulin-­like growth factors and cancer: no role in 69. Nomura, A. M., G. N. Stemmermann, J. Lee, and M. N. Pollak. 2003. Serum insulin-­like growth factor I and 3364 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. T. Shanmugalingam et al. IGF-1 in the development of first and second primary cancers IGF-1 in the development of first and second primary cancers Kingdom Acromegaly study group. J. Clin. Endocrinol. Metab. 83:2730–2734. subsequent risk of colorectal cancer among Japanese-­ American men. Am. J. Epidemiol. 158:424–431. Kingdom Acromegaly study group. J. Clin. Endocrinol. Metab. 83:2730–2734. 70. Palmqvist, R., G. Hallmans, S. Rinaldi, C. Biessy, R. Stenling, E. Riboli, et  al. 2002. Plasma insulin-­like growth factor 1, insulin-­like growth factor binding protein 3, and risk of colorectal cancer: a prospective study in northern Sweden. Gut 50:642–646. 81. Jenkins, P. J., V. Frajese, A. M. Jones, C. Camacho- Hubner, D. G. Lowe, P. D. Fairclough, et  al. 2000. Insulin-­like growth factor I and the development of colorectal neoplasia in acromegaly. J. Clin. Endocrinol. Metab. 85:3218–3221. 82. Zecha, H., H. P. Schmid, A. Tschopp, T. Sulser, and D. S. Engeler. 2011. High incidence of independent second malignancies after non-­muscle-­invasive bladder cancer. Scand. J. Urol. Nephrol. 45:245–250. 71. Tripkovic, I., A. Tripkovic, M. Strnad, V. Capkun, and L. Zekan. 2007. Role of insulin-­like growth factor-­1 in colon cancerogenesis: a case-­control study. Arch. Med. Res. 38:519–525. 72. Kaaks, R., P. Toniolo, A. Akhmedkhanov, A. Lukanova, C. Biessy, H. Dechaud, et  al. 2000. Serum C-­peptide, insulin-­like growth factor (IGF)-­I, IGF-­binding proteins, and colorectal cancer risk in women. J. Natl Cancer Inst. 92:1592–1600. 83. Ge, J., H. F. Gou, Y. Chen, K. Cheng, L. H. Li, H. Dong, et  al. 2013. Clinical characteristics of patients with solitary pulmonary mass after radical treatment for primary cancers: pulmonary metastasis or second primary lung cancer? Cancer Invest. 31:397–403. 84. Brenner, D. J. 2010. Contralateral second breast cancers: prediction and prevention. J. Natl Cancer Inst. 102:444–445. 73. Giovannucci, E., M. N. Pollak, E. A. Platz, W. C. Willett, M. J. Stampfer, N. References Majeed, et  al. 2000. A prospective study of plasma insulin-­like growth factor-­1 and binding protein-­3 and risk of colorectal neoplasia in women. Cancer Epidemiol. Biomark. Prev. 9:345–349. 85. Rusner, C., K. Wolf, U. Bandemer-Greulich, J. Engel, C. Stegmaier, B. Holleczek, et  al. 2014. Risk of contralateral second primary breast cancer according to hormone receptor status in Germany. Breast Cancer Res. 16:452. 74. Suzuki, S., M. Kojima, S. Tokudome, K. Suzuki, K. Ozasa, Y. Ito, et  al. 2009. Insulin-­like growth factor (IGF)-­I, IGF-­II, IGF binding protein-­3, and risk of colorectal cancer: a nested case-­control study in the Japan Collaborative Cohort study. Asian Pac. J. Cancer Prev. 10(Suppl):45–49. 86. Crump, M., and D. Hodgson. 2009. Secondary breast cancer in Hodgkin’s lymphoma survivors. J. Clin. Oncol. 27:4229–4231. 87. Bhatia, S., L. L. Robison, O. Oberlin, M. Greenberg, G. Bunin, F. Fossati-Bellani, et  al. 1996. Breast cancer and other second neoplasms after childhood Hodgkin’s disease. N. Engl. J. Med. 334:745–751. 75. Max, J. B., P. J. Limburg, A. Ogunseitan, R. Z. Stolzenberg-Solomon, R. A. Vierkant, M. J. Pollak, et  al. 2008. IGF-­I, IGFBP-­3, and IGF-­I/IGFBP-­3 ratio: no association with incident colorectal cancer in the alpha-­tocopherol, beta-­carotene cancer prevention study. Cancer Epidemiol. Biomark. Prev. 17:1832–1834. 88. Swerdlow, A. J., R. Cooke, A. Bates, D. Cunningham, S. J. Falk, D. Gilson, et  al. 2012. Breast cancer risk after supradiaphragmatic radiotherapy for Hodgkin’s lymphoma in England and Wales: a National Cohort Study. J. Clin. Oncol. 30:2745–2752. 76. Peters, G., S. Gongoll, C. Langner, M. Mengel, P. Piso, J. Klempnauer, et  al. 2003. IGF-­1R, IGF-­1 and IGF-­2 expression as potential prognostic and predictive markers in colorectal-­cancer. Virchows Arch. 443:139–145. 89. Cooke, R., M. E. Jones, D. Cunningham, S. J. Falk, D. Gilson, B. W. Hancock, et  al. 2013. Breast cancer risk following Hodgkin lymphoma radiotherapy in relation to menstrual and reproductive factors. Br. J. Cancer 108:2399–2406. 77. Freier, S., O. Weiss, M. Eran, A. Flyvbjerg, R. Dahan, I. Nephesh, et  al. 1999. Expression of the insulin-­like growth factors and their receptors in adenocarcinoma of the colon. Gut 44:704–708. 90. Bernstein, J. L., R. H. Lapinski, S. S. Thakore, J. T. Doucette, and W. D. Thompson. 2003. The descriptive epidemiology of second primary breast cancer. Epidemiology 14:552–558. 78. Hakam, A., T. J. Yeatman, L. Lu, L. Mora, G. Marcet, S. V. Nicosia, et  al. 1999. Expression of insulin-­like growth factor-­1 receptor in human colorectal cancer. Hum. Pathol. 30:1128–1133. 91. References Storm, H. H., and O. M. Jensen. 1986. Risk of contralateral breast cancer in Denmark 1943-­80. Br. J. Cancer 54:483–492. 79. Shiratsuchi, I., Y. Akagi, A. Kawahara, T. Kinugasa, K. Romeo, T. Yoshida, et  al. 2011. Expression of IGF-­1 and IGF-­1R and their relation to clinicopathological factors in colorectal cancer. Anticancer Res. 31:2541–2545. 92. Hancock, S. L., M. A. Tucker, and R. T. Hoppe. 1993. Breast cancer after treatment of Hodgkin’s disease. J. Natl Cancer Inst. 85:25–31. 93. Kleinberg, D. L., M. Feldman, and W. Ruan. 2000. IGF-­I: an essential factor in terminal end bud formation and ductal morphogenesis. J. Mammary Gland Biol. Neoplasia 5:7–17. 80. Orme, S. M., R. J. McNally, R. A. Cartwright, and P. E. Belchetz. 1998. Mortality and cancer incidence in acromegaly: a retrospective cohort study. United 3365 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. IGF-1 in the development of first and second primary cancers 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. 94. Frasca, F., G. Pandini, L. Sciacca, V. Pezzino, S. Squatrito, A. Belfiore, et  al. 2008. The role of insulin receptors and IGF-­I receptors in cancer and other diseases. Arch. Physiol. Biochem. 114:23–37. according to estrogen receptor status. Cancer Causes Control 23:1721–1728. 106. Kim, W. Y., M. J. Kim, H. Moon, P. Yuan, J. S. Kim, J. K. Woo, et  al. 2011. Differential impacts of insulin-­like growth factor-­binding protein-­3 (IGFBP-­3) in epithelial IGF-­induced lung cancer development. Endocrinology 152:2164–2173. 95. Yee, D., S. Paik, G. S. Lebovic, R. R. Marcus, R. E. Favoni, K. J. Cullen, et  al. 1989. Analysis of insulin-­ like growth factor I gene expression in malignancy: evidence for a paracrine role in human breast cancer. Mol. Endocrinol. 3:509–517. 107. Levi, F., L. Randimbison, V. C. Te, G. Erler, and C. La Vecchia. 1999. Second primary tumors after prostate carcinoma. Cancer 86:1567–1570. 96. Allen, N. E., A. W. Roddam, D. S. Allen, I. S. Fentiman, Silva. I. Dos Santos, J. Peto, et  al. 2005. A prospective study of serum insulin-­like growth factor-­I (IGF-­I), IGF-­II, IGF-­binding protein-­3 and breast cancer risk. Br. J. Cancer 92:1283–1287. 108. Davis, E. J., J. L. Beebe-Dimmer, C. L. Yee, and K. A. Cooney. 2014. Risk of second primary tumors in men diagnosed with prostate cancer: a population-­based cohort study. Cancer 120:2735–2741. 97. References Smith, J., D. Axelrod, B. Singh, and D. Kleinberg. 2011. Prevention of breast cancer: the case for studying inhibition of IGF-­1 actions. Ann. Oncol. 22(Suppl 1):i50–i52. 109. Abern, M. R., A. M. Dude, M. Tsivian, and C. L. Coogan. 2013. The characteristics of bladder cancer after radiotherapy for prostate cancer. Urologic Oncol. 31:1628–1634. 98. Coyte, A., D. S. Morrison, and P. McLoone. 2014. Second primary cancer risk -­ the impact of applying different definitions of multiple primaries: results from a retrospective population-­based cancer registry study. BMC Cancer 14:272. 110. Anderson, J. K., S. Alanee, B. Lindgren, and J. Slaton. 2013. The risk of bladder cancer in patients diagnosed with other primary neoplasms: analysis of the SEER database. Urologic Oncol. 31:862–865. 111. Braisch, U., M. Meyer, and M. Radespiel-Troger. 2012. Risk of subsequent primary cancer among prostate cancer patients in Bavaria, Germany. Eur. J. Cancer Prev. 21:552–559. 99. Kaufman, E. L., J. S. Jacobson, D. L. Hershman, M. Desai, and A. I. Neugut. 2008. Effect of breast cancer radiotherapy and cigarette smoking on risk of second primary lung cancer. J. Clin. Oncol. 26:392–398. 112. Zhang, H., J. L. Bermejo, J. Sundquist, and K. Hemminki. 2009. Prostate cancer as a first and second cancer: effect of family history. Br. J. Cancer 101:935–939. 100. Senthi, S., C. J. Haasbeek, F. J. Lagerwaard, W. F. Verbakel, P. F. de Haan, B. J. Slotman, et  al. 2013. Radiotherapy for a second primary lung cancer arising post-­pneumonectomy: planning considerations and clinical outcomes. J. Thorac. Dis. 5:116–122. 113. Chrouser, K., B. Leibovich, E. Bergstralh, H. Zincke, and M. Blute. 2005. Bladder cancer risk following primary and adjuvant external beam radiation for prostate cancer. J. Urol. 174:107–110; discussion 10-1. 101. Argiris, A., B. E. Brockstein, D. J. Haraf, K. M. Stenson, B. B. Mittal, M. S. Kies, et  al. 2004. Competing causes of death and second primary tumors in patients with locoregionally advanced head and neck cancer treated with chemoradiotherapy. Clin. Cancer Res. 10:1956–1962. 114. Kok, D. E., S. A. van de Schans, L. Liu, E. Kampman, J. W. Coebergh, L. A. Kiemeney, et  al. 2013. Risk of prostate cancer among cancer survivors in the Netherlands. Cancer Epidemiol. 37:140–145. 102. Boyle, J. M., D. J. Tandberg, J. P. Chino, T. A. D’Amico, N. E. Ready, and C. R. Kelsey. 2015. Smoking history predicts for increased risk of second primary lung cancer: a comprehensive analysis. Cancer 121:598–604. 115. References Kellen, E., M. P. Zeegers, M. Dirx, S. Houterman, J. Droste, G. Lawrence, et  al. 2007. Occurrence of both bladder and prostate cancer in five cancer registries in Belgium, The Netherlands and the United Kingdom. Eur. J. Cancer 43:1694–1700. 103. Richardson, G. E., M. A. Tucker, D. J. Venzon, R. I. Linnoila, R. Phelps, J. C. Phares, et  al. 1993. Smoking cessation after successful treatment of small-­cell lung cancer is associated with fewer smoking-­related second primary cancers. Ann. Intern. Med. 119:383–390. 116. Kurokawa, K., K. Ito, T. Yamamoto, H. Takechi, S. Miyamoto, K. Suzuki, et  al. 2004. Comparative study on the prevalence of clinically detectable prostate cancer in patients with and without bladder cancer. Urology 63:268–272. 117. Bradford, P. T., D. M. Freedman, A. M. Goldstein, and M. A. Tucker. 2010. Increased risk of second primary cancers after a diagnosis of melanoma. Arch. Dermatol. 146:265–272. 104. Johnson, B. E., P. Cortazar, and J. P. Chute. 1997. Second lung cancers in patients successfully treated for lung cancer. Semin. Oncol. 24:492–499. 105. Schonfeld, S. J., R. E. Curtis, W. F. Anderson, and A. Berrington de Gonzalez. 2012. The risk of a second primary lung cancer after a first invasive breast cancer 118. Chen, C., Q. Zhang, S. Liu, K. R. Parajuli, Y. Qu, J. Mei, et  al. 2015. IL-­17 and insulin/IGF1 enhance 3366 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. T. Shanmugalingam et al. IGF-1 in the development of first and second primary cancers IGF-1 in the development of first and second primary cancers System in colorectal cancer development and resistance to therapy. Front. Oncol. 5:230. adhesion of prostate cancer cells to vascular endothelial cells through CD44-­VCAM-­1 interaction. Prostate 75:883–895. System in colorectal cancer development and resistance to therapy. Front. Oncol. 5:230. 125. Grannis, F. W. Jr. 2013. Minimizing over-­diagnosis in lung cancer screening. J. Surg. Oncol. 108:289–293. 119. Pickles, T., and N. Phillips. 2002. The risk of second malignancy in men with prostate cancer treated with or without radiation in British Columbia, 1984-­2000. Radiother. Oncol. 65:145–151. 126. Tworoger, S. S., and S. E. Hankinson. 2006. Use of biomarkers in epidemiologic studies: minimizing the influence of measurement error in the study design and analysis. Cancer Causes Control 17:889–899. 120. McCredie, M., G. J. Macfarlane, J. Stewart, and M. Coates. 1996. Second primary cancers following cancers of the kidney and prostate in New South Wales (Australia), 1972-­91. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. References Cancer Causes Control 7:337–344. 127. LeRoith, D. 2012. Insulin-like growth factors and cancer: from basic biology to therapeutics. Development CDDa, editor: Springer, Springer US. 121. Brenner, D. J., R. E. Curtis, E. J. Hall, and E. Ron. 2000. Second malignancies in prostate carcinoma patients after radiotherapy compared with surgery. Cancer 88:398–406. 128. Bokarewa, M. I., M. C. Erlandsson, J. Bjersing, M. Dehlin, and K. Mannerkorpi. 2014. Smoking is associated with reduced leptin and neuropeptide Y levels and higher pain experience in patients with fibromyalgia. Mediators Inflamm. 2014:627041. 122. Baxter, N. N., J. E. Tepper, S. B. Durham, D. A. Rothenberger, and B. A. Virnig. 2005. Increased risk of rectal cancer after prostate radiation: a population-­ based study. Gastroenterology 128:819–824. 129. Lin, I. H., M. L. Ho, H. Y. Chen, H. S. Lee, C. C. Huang, Y. H. Chu, et  al. 2012. Smoking, green tea consumption, genetic polymorphisms in the insulin-­like growth factors and lung cancer risk. PLoS ONE 7:e30951. 123. Chaturvedi, A. K. E., E. A. Engels, E. S. Gilbert, B. E. Chen, H. Storm, C. F. Lynch, et  al. 2007. Second cancers among 104 760 survivors of cervical cancer: evaluation of long-­term risk. J. Natl Cancer Inst. 99:1634–1643. 130. Schisterman, E. F., S. R. Cole, and R. W. Platt. 2009. Overadjustment bias and unnecessary adjustment in epidemiologic studies. Epidemiology 20:488–495. 124. Vigneri, P. G., E. Tirro, M. S. Pennisi, M. Massimino, S. Stella, C. Romano, et  al. 2015. The Insulin/IGF 3367
https://openalex.org/W3111403837
https://rodin.uca.es/bitstream/10498/24395/1/2021_040.pdf
English
null
Sustainability in the Aerospace, Naval, and Automotive Supply Chain 4.0: Descriptive Review
Materials
2,020
cc-by
15,274
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 i t f b th d i ti l it f th i t ti f I d t 4 0 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 gy q p [ ] 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 th d t i l t C b it th h th i t ti f S l Ch i 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 d t t bli h i b t 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., Eds.; Pearson Educación, S.A.: México City, Mexico, 2013; ISBN 978-607-32-2133-7. 1. Chopra, S.; Meindl, P. Administración de la Cadena de Suministro; Cruz Castillo, L.M., Gutierrez Hernández, B., Eds.; Pearson Educación, S.A.: México City, Mexico, 2013; ISBN 978-607-32-2133-7. 2. Heizer, J.; Render, B. How China Sees America; Cañizal, A., Bazaco, E., Eds.; Pearson Educación, S.A.: Madrid, Spain, 2012; Volume 91, ISBN 9788578110796. 2. Heizer, J.; Render, B. How China Sees America; Cañizal, A., Bazaco, E., Eds.; Pearson Educación, S.A.: Madrid, Spain, 2012; Volume 91, ISBN 9788578110796. 3. Ballou, R.H. The evolution and future of logistics and supply chain management. Eur. Bus. Rev. 2007, 19, 332–348. [CrossRef] 3. Ballou, R.H. The evolution and future of logistics and supply chain management. Eur. Bus. Rev. 2007, 19, 332–348. [CrossRef] 4 Sanchez Loppacher J Compitiendo a Través de la Cadena del Negocio Supply Chain Management; ScarfiJ Ed ; 4. Sanchez Loppacher, J. Compitiendo a Través de la Cadena del Negocio. Supply Chain Management; Scarfi Temas Grupo Editorial: Buenos Aires, Argentina, 2013; ISBN 9789871826568. 5. Biswal, J.N.; Muduli, K.; Satapathy, S. Critical analysis of drivers and barriers of sustainable supply chain management in Indian thermal sector. Int. J. Procure. Manag. 2017, 10, 411–430. [CrossRef] 5. Biswal, J.N.; Muduli, K.; Satapathy, S. Critical analysis of drivers and barriers of sustainable supply chain management in Indian thermal sector. Int. J. Procure. Manag. 2017, 10, 411–430. [CrossRef] 6. Pirvulescu, P.; Enevoldsen, P. Supply Chain management in the age of digitalization. Int. J. Supply Chain Manag. 2019, 8, 414–428. 6. Pirvulescu, P.; Enevoldsen, P. Supply Chain management in the age of digitalization. Int. J. Supply Chain Manag. 2019, 8, 414–428. 7. Felsberger, A.; Reiner, G. Sustainable Industry 4.0 in Production and Operations Management: A Systematic Literature Review. Sustainability 2020, 12, 7982. [CrossRef] 8. Vera Carrasco, O. Cómo escribir artículos de revisión. Rev. Med. Paz 2009, 15, 63–69. 9. Briner, R.B.; Denyer, D.; Rousseau, D. Systematic Review and Evidence Synthesis as a Practice and Scholarship Tool. In The Oxford Handbook of Evedence-Based Management; Press, O.U., Ed.; Oxford University Press: New York, NY, USA, 2012; pp. 112–129. 10. Müller, J.M.; Voigt, K.I. The Impact of Industry 4.0 on Supply Chains in Engineer-to-Order Industries— An Exploratory Case Study. IFAC PapersOnLine 2018, 51, 122–127. [CrossRef] 11. Haddud, A.; Khare, A. 4. Conclusions It seems that the sector is committed to the implementation of the technologies in a collaborative 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 supported this work. 16 of 23 16 of 23 Materials 2020, 13, 5625 Conflicts of Interest: The authors declare no conflict of interest. References Digitalizing supply chains potential benefits and impact on lean operations. Int. J. Lean Six Sigma 2020, 11, 731–765. [CrossRef] 12. Duarte, S.; Cruz-Machado, V. An investigation of lean and green supply chain in the Industry 4.0. Proc. Int. Conf. Ind. Eng. Oper. Manag. 2017, 2017, 255–265. 13. Masudin, I.; Wastono, T.; Zulfikarijah, F. The effect of managerial intention and initiative on green supply chain management adoption in Indonesian manufacturing performance. Cogent Bus. Manag. 2018, 5, 1–19. [CrossRef] 14. Manavalan, E.; Jayakrishna, K. A review of Internet of Things (IoT) embedded sustainable supply chain for industry 4.0 requirements. Comput. Ind. Eng. 2019, 127, 925–953. [CrossRef] 15. Esmaeilian, B.; Sarkis, J.; Lewis, K.; Behdad, S. Blockchain for the future of sustainable supply chain management in Industry 4.0. Resour. Conserv. Recycl. 2020, 163, 105064. [CrossRef] 16. Kiel, D. What do we know about “Industry 4.0” so far? [Rewarded with IAMOT Best Student Paper Award]. Int. Assoc. Manag. Technol. 2017, 2, 9–22. 17. Omar, Y.M.; Minoufekr, M.; Plapper, P. Business analytics in manufacturing: Current trends, challenges and pathway to market leadership. Oper. Res. Perspect. 2019, 6, 100127. [CrossRef] 18. Franco, D.; Miller Devós Ganga, G.; de Santa-Eulalia, L.A.; Godinho Filho, M. Consolidated and inconclusive effects of additive manufacturing adoption: A systematic literature review. Comput. Ind. Eng. 2020, 148, 106713. [CrossRef] 19. Nayak, G.; Dhaigude, A.S. A conceptual model of sustainable supply chain management in small and medium enterprises using blockchain technology. Cogent Econ. Financ. 2019, 7. [CrossRef] 20. Núñez-Merino, M.; Maqueira-Marín, J.M.; Moyano-Fuentes, J.; Martínez-Jurado, P.J. Information and digital technologies of Industry 4.0 and Lean supply chain management: A systematic literature review. Int. J. Prod. Res. 2020, 58, 5034–5061. [CrossRef] 21. Zaman, I.; Pazouki, K.; Norman, R.; Younessi, S.; Coleman, S. Challenges and opportunities of big data analytics for upcoming regulations and future transformation of the shipping industry. Procedia Eng. 2017, 194, 537–544. [CrossRef] 22. Yan, X.; Wang, K.; Yuan, Y.; Jiang, X.; Negenborn, R.R. Energy-efficient shipping: An application of big data analysis for optimizing engine speed of inland ships considering multiple environmental factors. Ocean Eng. 2018, 169, 457–468. [CrossRef] 17 of 23 Materials 2020, 13, 5625 23. Anan, T.; Higuchi, H.; Hamada, N. New artificial intelligence technology improving fuel efficiency and reducing CO2 emissions of ships through use of operational big data. Fujitsu Sci. Tech. J. 2017, 53, 23–28. 24. References Hamedifar, H.; Spitzenberger, C.; Stahl, C.; Brown, A.; Nilberg, B.; Demay, V.; Aspholm, O. Terminal and transportation risk assessment for LNG export in North America. In Proceedings of the Petroleum Abstracts, Houston, TX, USA, 4–7 May 2015; Volume 55, pp. 112–113. y pp 25. Ramirez-Peña, M.; Fraga, F.J.A.; Salguero, J.; Batista, M. Assessing sustainability in the shipbuilding supply chain 4.0: A systematic review. Sustainability 2020, 12, 6373. [CrossRef] 26. Riahi, M.; Krichen, S. A multi-objective decision support framework for virtual machine placement in cloud data centers: A real case study. J. Supercomput. 2018, 74, 2984–3015. [CrossRef] 27. Carbone, R.; Montella, R.; Narducci, F.; Petrosino, A. DeepNautilus: A Deep Learning Based System for Nautical Engines’ Live Vibration Processing. In Proceedings of the Lecture Notes in Computer Science (Including Subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics); Springer: Berlin/Heidelberg, Germany, 2019; Volume 11679, pp. 120–131. 28. Ju, D.; Shen, B. Sustainable development of marine economy guided by knowledge cloud services. In Proceedings of the 2nd International Conference on Networking and Distributed Computing (ICNDC 2011), Beijing, China, 21–24 September 2011; pp. 235–239. 29. McGillivary, P. Why maritime cybersecurity is an ocean policy priority and how it can be add Mar. Technol. Soc. J. 2018, 52, 44–57. [CrossRef] 30. Koštál, K.; Krupa, T.; Gembec, M.; Vereš, I.; Ries, M.; Kotuliak, I. On transition between PoW and PoS. In Proceedings of the International Symposium Electronics in Marine, Zadar, Croatia, 16–19 September 2018; Volume 2018, pp. 207–210. 31. Wu, D.; Ren, A.; Zhang, W.; Fan, F.; Liu, P.; Fu, X.; Terpenny, J. Cybersecurity for digital manufacturing. J. Manuf. Syst. 2018, 48, 3–12. [CrossRef] 32. Fernández-Caramés, T.M.; Fraga-Lamas, P.; Suárez-Albela, M.; Díaz-Bouza, M.A. A fog computing based cyber-physical system for the automation of pipe-related tasks in the industry 4.0 shipyard. Sensors 2018, 18, 1961. [CrossRef] [PubMed] 33. Malik, S.; Azur, K.M.; Rouf, R.; Kontsos, A. The industry internet of things (IIoT) as a methodology for autonomous diagnostics, prognostics in aerospace structural health monitoring. Struct. Health Monit. 2019, 1, 1007–1015. [CrossRef] 34. Mastos, T.D.; Nizamis, A.; Vafeiadis, T.; Alexopoulos, N.; Ntinas, C.; Gkortzis, D.; Papadopoulos, A.; Ioannidis, D.; Tzovaras, D. Industry 4.0 sustainable supply chains: An application of an IoT enabled scrap metal management solution. J. Clean. Prod. 2020, 269, 122377. [CrossRef] 35. Ante, G.; Facchini, F.; Mossa, G.; Digiesi, S. Developing a key performance indicators tree for lean and smart production systems. References IFAC PapersOnLine 2018, 51, 13–18. [CrossRef] 36. Raji, I.O.; Rossi, T. Exploring industry 4.0 technologies as drivers of lean and agile supply chain strategies. In Proceedings of the International Conference on Industrial Engineering and Operations Management, Toronto, ON, Canada, 23–25 October 2019; pp. 292–303. 37. De Giovanni, P.; Cariola, A. Process innovation through industry 4.0 technologies, lean practices and green supply chains. Res. Transp. Econ. 2020, 100869. [CrossRef] 38. Tortorella, G.; Miorando, R.; Mac Cawley, A.F. The moderating effect of Industry 4.0 on the relationship between lean supply chain management and performance improvement. Supply Chain Manag. 2019, 24, 301–314. [CrossRef] 39. Yu, H.; Solvang, W.D. Enhancing the competitiveness of manufacturers through Small-scale Intelligent Manufacturing System (SIMS): A supply chain perspective. In Proceedings of the 6th International Conference on Industrial Technology & Management (ICITM 2017), Cambridge, UK, 7–10 March 2017; pp. 101–107. [CrossRef] 40. Wei, D.; Solvang, B.S.; Deng, Z. A Closed-loop Supply Chain Model for Managing Overall Optimization of Eco-efficiency. In Proceedings of the POMS 18th Annual Conference, Dallas, TX, USA, 4–7 May 2007. 41. Takhar, S.S.; Liyanage, K. The impact of Industry 4.0 on sustainability and the circular economy reporting requirements. Int. J. Integr. Supply Manag. 2020, 13, 107–139. [CrossRef] 42. Ozkan-Ozen, Y.D.; Kazancoglu, Y.; Kumar Mangla, S. Synchronized Barriers for Circular Supply Chains in Industry 3.5/Industry 4.0 Transition for Sustainable Resource Management. Resour. Conserv. Recycl. 2020, 161, 104986. [CrossRef] 18 of 23 Materials 2020, 13, 5625 43. Kumar, P.; Singh, R.K.; Kumar, V. Managing supply chains for sustainable operations in the era of industry 4.0 and circular economy: Analysis of barriers. Resour. Conserv. Recycl. 2021, 164, 105215. [CrossRef] 43. Kumar, P.; Singh, R.K.; Kumar, V. Managing supply chains for sustainable operations in the era of industry 4.0 and circular economy: Analysis of barriers. Resour. Conserv. Recycl. 2021, 164, 105215. [CrossRef] 44. Luthra, S.; Mangla, S.K. Evaluating challenges to Industry 4.0 initiatives for supply chain sustainability in emerging economies. Process Saf. Environ. Prot. 2018, 117, 168–179. [CrossRef] 45. Avilés-Sacoto, S.V.; Avilés-González, J.F.; García-Reyes, H.; Bermeo-Samaniego, M.C.; Cañizares-Jaramillo, A.K.; Izquierdo-Flores, S.N. A glance of industry 4.0 at supply chain and inventory management. Int. J. Ind. Eng. Theory Appl. Pract. 2019, 26, 486–506. 46. Bhagawati, M.T.; Manavalan, E.; Jayakrishna, K.; Venkumar, P. Identifying Key Success Factors of Sustainability in Supply Chain Management for Industry 4.0 Using Dematel Method. References In Proceedings of the International Conference on Intelligent Manufacturing and Automation, Lecture Notes in Mechanical Engineering, Tokyo, Japan, 27–30 September 2019; Springer: Singapore, 2019. [CrossRef] 47. Ivanov, D.; Das, A.; Choi, T.M. New flexibility drivers for manufacturing, supply chain and service operations. Int. J. Prod. Res. 2018, 56, 3359–3368. [CrossRef] 48. Schluter, F.; Hetterscheid, E. A Simulation Based Evaluation Approach for Supply Chain Risk Management Digitalization Scenarios. In Proceedings of the 2017 International Conference on Industrial Engineering, Management Science and Application (ICIMSA 2017), Seoul, Korea, 3–15 June 2017; pp. 1–5. [CrossRef] g pp pp 9. Hoffa-Dabrowska, P.; Grzybowska, K. Simulation modeling of the sustainable supply chain. Sustainab 2020, 12, 6007. [CrossRef] 50. Fathi, M.; Nourmohammadi, A.; Ghobakhloo, M.; Yousefi, M. Production sustainability via supermarket location optimization in assembly lines. Sustainability 2020, 12, 4728. [CrossRef] 51. Vieira, A.A.; Dias, L.M.; Santos, M.Y.; Pereira, G.A.; Oliveira, J.A. On the use of simulation as a Big Data semantic validator for supply chain management. Simul. Model. Pract. Theory 2020, 98, 101985. [CrossRef] 51. Vieira, A.A.; Dias, L.M.; Santos, M.Y.; Pereira, G.A.; Oliveira, J.A. On the use of simulation as a Big Data semantic validator for supply chain management. Simul. Model. Pract. Theory 2020, 98, 101985. [CrossRef] 52. Bag, S.; Pretorius, J.H.C. Relationships between industry 4.0, sustainable manufacturing and circular economy: 51. Vieira, A.A.; Dias, L.M.; Santos, M.Y.; Pereira, G.A.; Oliveira, J.A. On the use of simulation as a Big Data semantic validator for supply chain management. Simul. Model. Pract. Theory 2020, 98, 101985. [CrossRef] 52. Bag, S.; Pretorius, J.H.C. Relationships between industry 4.0, sustainable manufacturing and circular economy: Proposal of a research framework. Int. J. Organ. Anal. 2020. [CrossRef] 52. Bag, S.; Pretorius, J.H.C. Relationships between industry 4.0, sustainable manufacturing and circular economy: Proposal of a research framework. Int. J. Organ. Anal. 2020. [CrossRef] 53. Ardito, L.; Petruzzelli, A.M.; Panniello, U.; Garavelli, A.C. Towards Industry 4.0: Mapping digital technologies for supply chain management-marketing integration. Bus. Process Manag. J. 2019, 25, 323–346. [CrossRef] 54. Garay-Rondero, C.L.; Martinez-Flores, J.L.; Smith, N.R.; Caballero Morales, S.O.; Aldrette-Malacara, A. Digital supply chain model in Industry 4.0. J. Manuf. Technol. Manag. 2019. [CrossRef] 55. Pandey, S.; Singh, R.K.; Gunasekaran, A.; Kaushik, A. Cyber security risks in globalized supply chains: Conceptual framework. J. Glob. Oper. Strateg. Sourc. 2020, 13, 103–128. [CrossRef] 56. Bodkhe, U.; Tanwar, S.; Parekh, K.; Khanpara, P.; Tyagi, S.; Kumar, N.; Alazab, M. References Blockchain for Industry 4.0: A comprehensive review. IEEE Access 2020, 8, 79764–79800. [CrossRef] 57. Raut, R.D.; Gotmare, A.; Narkhede, B.E.; Govindarajan, U.H.; Bokade, S.U. Enabling Technologies for Industry 4.0 Manufacturing and Supply Chain: Concepts, Current Status, and Adoption Challenges. IEEE Eng. Manag. Rev. 2020, 48, 83–102. [CrossRef] 58. Liu, B.; De Giovanni, P. Green process innovation through Industry 4.0 technologies and supply chain coordination. Ann. Oper. Res. 2019. [CrossRef] 59. Ghosh, D.; Sant, T.G.; Kuiti, M.R.; Swami, S.; Shankar, R. Strategic decisions, competition and cost-sharing contract under industry 4.0 and environmental considerations. Resour. Conserv. Recycl. 2020, 162, 105057. [CrossRef] 0. Horváth, D.; Szabó, R.Z. Driving forces and barriers of Industry 4.0: Do multinational and small and medium-s companies have equal opportunities? Technol. Forecast. Soc. Chang. 2019, 146, 119–132. [CrossRef] 61. Bag, S.; Telukdarie, A.; Pretorius, J.H.C.; Gupta, S. Industry 4.0 and supply chain sustainability: Framework and future research directions. Benchmarking 2018. [CrossRef] 62. Abdirad, M.; Krishnan, K. Industry 4.0 in Logistics and Supply Chain Management: A Systematic Literature Review. EMJ Eng. Manag. J. 2020, 15. [CrossRef] 63. Yadav, G.; Luthra, S.; Jakhar, S.K.; Mangla, S.K.; Rai, D.P. A framework to overcome sustainable supply chain challenges through solution measures of industry 4.0 and circular economy: An automotive case. J. Clean. Prod. 2020, 254, 120112. [CrossRef] 4. Princes, E. Facing disruptive challenges in supply chain 4.0. Int. J. Supply Chain Manag. 2020, 9, 52–57. 65. Schniederjans, D.G.; Curado, C.; Khalajhedayati, M. Supply chain digitisation trends: An integration of knowledge management. Int. J. Prod. Econ. 2020, 220, 107439. [CrossRef] Materials 2020, 13, 5625 19 of 23 19 of 23 66. Fatorachian, H.; Kazemi, H. Impact of Industry 4.0 on supply chain performance. Prod. Plan. Control 2020, 1–19. [CrossRef] 67. Tabaklar, T.; Yildirim, C. The development of servitization concept in the era of industry 4.0 through SCM perspective. Handb. Res. Strateg. Fit Des. Bus. Ecosyst. 2019, 593–615. [CrossRef] 68. Shao, X.F.; Liu, W.; Li, Y.; Chaudhry, H.R.; Yue, X.G. Multistage implementation framework for smart supply chain management under industry 4.0. Technol. Forecast. Soc. Chang. 2021, 162. [CrossRef] Shao, X.F.; Liu, W.; Li, Y.; Chaudhry, H.R.; Yue, X.G. Multistage implementation framework for smart supply chain management under industry 4.0. Technol. Forecast. Soc. Chang. 2021, 162. [CrossRef] 69. Ivascu, L. Measuring the implications of sustainable manufacturing in the context of industry 4.0. Processes 2020, 8, 585. [CrossRef] 70. Ruiz-Benitez, R.; López, C.; Real, J.C. References Achieving sustainability through the lean and resilient management of the supply chain abstract. Int. J. Phys. Distrib. Logist. Manag. 2019, 49, 122–155. [CrossRef] 71. Manville, G.; Papadopoulos, T.; Garengo, P. Twenty-first century supply chain management: A multiple case study analysis within the UK aerospace industry. Total Qual. Manag. Bus. Excell. 2019, 2020. [CrossRef] 72. Ruiz-Benítez, R.; López, C.; Real, J.C. The lean and resilient manage on performance. Int. J. Prod. Econ. 2018, 203, 190–202. [CrossRef] 72. Ruiz-Benítez, R.; López, C.; Real, J.C. The lean and resilient management of the supply chain and its impact on performance. Int. J. Prod. Econ. 2018, 203, 190–202. [CrossRef] 73. Ruiz-Benitez, R.; López, C.; Real, J.C. Environmental benefits of lean, green and resilient supply chain management: The case of the aerospace sector. J. Clean. Prod. 2017, 167, 850–862. [CrossRef] 74. Martínez-Jurado, P.J.; Moyano-Fuentes, J. Lean management and supply chain management: Interrelationships in the aerospace sector. In Operations and Service Management: Concepts, Methodologies, Tools, and Applications; IGI Global: Hershey, PA, USA, 2017; pp. 1208–1242. ISBN 9781522539100. 75. Mastrocinque, E.; Mondragon, A.E.C.; Hogg, P.J. A four-element framework for research on the composite materials supply chain. In Proceedings of the Engineering Technology, Engineering Education and Engineering Management—International Conference on Engineering Technology, Engineering Education and Engineering Management (ETEEEM 2014), Guangzhou, China, 15–16 November 2014; pp. 423–426. 76. Beelaerts van Blokland, W.W.A.; Fiksi´nski, M.A.; Amoa, S.O.B.; Santema, S.C.; van Silfhout, G.J.; Maaskant, L. Measuring value-leverage in aerospace supply chains. Int. J. Oper. Prod. Manag. 2012, 32, 982–1007. [CrossRef] 77. Hallam, C.R.A. Lean supply chain management techniques for complex aerospace systems: Using discrete event simulation to mitigate programmatic cost and schedule risk. In Proceedings of the PICMET’10—Portland International Center for Management of Engineering and Technology, Proceedings—Technology Management for Global Economic Growth, Phuket, Thailand, 18–22 July 2010; pp. 2565–2573. y pp 78. Lai, E.T.H.; Yun, F.N.J.; Arokiam, I.C.; Joo, J.H.A. Barriers affecting successful lean implementation in Singapore’s shipbuilding industry: A case study. Oper. Supply Chain Manag. 2020, 13, 166–175. [CrossRef] y pp 78. Lai, E.T.H.; Yun, F.N.J.; Arokiam, I.C.; Joo, J.H.A. Barriers affecting successful lean implementation in Singapore’s shipbuilding industry: A case study. Oper. Supply Chain Manag. 2020, 13, 166–175. [CrossRef] 79. Hameri, A.P.; McKay, K.N.; Wiers, V.C.S. A maturity model for industrial supply chains. Supply Chain Forum 2013, 14, 2–15. [CrossRef] 79. Hameri, A.P.; McKay, K.N.; Wiers, V.C.S. A maturity model for industrial supply chains. Supply Chain Forum 2013, 14, 2–15. 86. Desai, P.; Saremi, R.; Hoffenson, S.; Lippizi, C. Agile and affordable: A survey of supply chain management methods in long lifecycle products. In Proceedings of the SysCon 2019—13th Annual IEEE International Systems Conference, Orlando, FL, USA, 8–11 April 2019. 85. Fleischer, M.; Kohler, R.; Lamb, T.; Bongiorni, H.B. Marine supply chain management. J. Sh. Prod. 1999, 15, 233–252. References Policy Manag. 2016, 43, 1005–1020. [CrossRef] 93. Feng, J.; Gong, Z. Integrated linguistic entropy weight method and multi-objective programming model for supplier selection and order allocation in a circular economy: A case study. J. Clean. Prod. 2020, 277, 2020. [CrossRef] 94. Gholipour, S.; Ashoftehfard, A.; Mina, H. Green supply chain network design considering inventory-l routing problem: A fuzzy solution approach. Int. J. Logist. Syst. Manag. 2020, 35, 436–452. [CrossRef] 95. Stevenson, M.; Busby, J. An exploratory analysis of counterfeiting strategies: Towards counterfeit-resilient supply chains. Int. J. Oper. Prod. Manag. 2015, 35, 110–144. [CrossRef] 96. Ramirez-Peña, M.; Sánchez Sotano, A.J.; Pérez-Fernandez, V.; Abad, F.J.; Batista, M. Achieving a sustainable shipbuilding supply chain under I4.0 perspective. J. Clean. Prod. 2020, 244. [CrossRef] 97. Carvalho, H.; Duarte, S.; Machado, V.C. Lean, agile, resilient and green: Divergencies and synergies. Int. J. Lean Six Sigma 2011, 2, 151–179. [CrossRef] 98. Pramanik, D.; Mondal, S.C.; Haldar, A. Resilient supplier selection to mitigate uncertainty: Soft-computing approach. J. Model. Manag. 2020, 15, 1339–1361. [CrossRef] 99. Quariguasi Frota Neto, J.; Dutordoir, M. Mapping the market for remanufacturing: An application of “Big Data” analytics. Int. J. Prod. Econ. 2020, 230, 2020. [CrossRef] 100. Bhatia, M.S.; Jakhar, S.K.; Mangla, S.K.; Gangwani, K.K. Critical factors to environment management in a closed loop supply chain. J. Clean. Prod. 2020, 255, 120239. [CrossRef] 101. Gilbert, P.; Wilson, P.; Walsh, C.; Hodgson, P. The role of material efficiency to reduce CO2 emissions during ship manufacture: A life cycle approach. Mar. Policy 2017, 75, 227–237. [CrossRef] 102. Teresko, J. Making a pitch for PLM. Ind. Week 2004, 253, 57–62. 103. Mason, H.; Jahadi, M.R. Computer-aided enterprise solutions. Aerosp. Am. 2008, 46, 101. 104. Barbarin, P.; Swallow, C.; Deeley, M. Leveraging Plm Capabilities to Manage Quality and Reliability through the Warship’s Life Cycle. RINA, Royal Institution of Naval Architects—Warship 2010: Advanced Technologies in Naval Design and Construction—Papers. 2010, pp. 171–181. Available online: https: //www.scimagojr.com/journalsearch.php?q=19700180705&tip=sid&clean=0 (accessed on 8 December 2020). 105. Fahhama, L.; Zamma, A.; Mansouri, K.; Elmajid, Z. Towards a mixed method model and simulation of the automotive supply chain network connectivity. In Proceedings of the 2017 International Colloquium on Logistics and Supply Chain Management: Competitiveness and Innovation in Automobile and Aeronautics Industries (LOGISTIQUA 2017), Rabat, Morocco, 27–28 April 2017; pp. 13–18. 106. Zammit, J.P.; Gao, J.; Evans, R. The challenges of adopting PLM tools involving diversified technologies in the automotive supply chain. References [CrossRef] 80. Qamar, A.; Hall, M.A.; Chicksand, D.; Collinson, S. Quality and flexibility performance trade-offs between lean and agile manufacturing firms in the automotive industry. Prod. Plan. Control 2020, 31, 723–738. [CrossRef] 81. Verma, A.K.; Hirkannawar, H. Lean supply chain integration and assessment—A simulation based training program. In Proceedings of the 26th Annual National Conference of the American Society for Engineering Management 2005—Organizational Transformation: Opportunities and Challenges (ASEM 2005), Virginia Beach, VA, USA, 26–29 October 2005; pp. 475–482. 82. Ramos, E.; Pettit, T.J.; Flanigan, M.; Romero, L.; Huayta, K. Inventory management model based on lean supply chain to increase the service level in a distributor of automotive sector. Int. J. Supply Chain Manag. 2020, 9, 113–131. 83. Barbosa, C.; Cunha, N.F.E.; Malarranha, C.; Pinto, T.; Carvalho, A.; Amorim, P.; Carvalho, M.S.; Azevedo, A.; Relvas, S.; Pinto-Varela, T.; et al. Towards an Integrated Framework for Aerospace Supply Chain Sustainability. In Springer Proceedings in Mathematics and Statistics; Springer: Berlin/Heidelberg, Germany, 2019; pp. 1–13. 84. Armoutis, N.; Maropoulos, P.G.; Matthews, P.; Lomas, C. Establishing agile supply networks through competence profiling. Int. J. Comput. Integr. Manuf. 2008, 21, 166–173. [CrossRef] 85. Fleischer, M.; Kohler, R.; Lamb, T.; Bongiorni, H.B. Marine supply chain management. J. Sh. Prod. 1999, 15, 233–252. 86. Desai, P.; Saremi, R.; Hoffenson, S.; Lippizi, C. Agile and affordable: A survey of supply chain management methods in long lifecycle products. In Proceedings of the SysCon 2019—13th Annual IEEE International Systems Conference, Orlando, FL, USA, 8–11 April 2019. 20 of 23 Materials 2020, 13, 5625 87. Chandak, A.; Gangele, A. Conceptual and statistical modeling for identifying linkages among supply chain strategy, flexibility and performance. Int. J. Mech. Prod. Eng. Res. Dev. 2019, 9, 993–1002. [CrossRef] 88. Kumar, M.; Garg, D.; Agarwal, A. Fuzzy DEMATEL approach for agile supplier selections performance criteria. J. Phys. Conf. Ser. 2019, 1240, 012157. [CrossRef] 89. Roehrich, J.K.; Hoejmose, S.U.; Overland, V. Driving green supply chain management performance through supplier selection and value internalisation: A self-determination theory perspective. Int. J. Oper. Prod. Manag. 2017, 37, 489–509. [CrossRef] 90. Vieira, D.R.; Vieira, R.K.; Chain, M.C. Elements of managerial integration for sustainable product lifecycle management. Int. J. Prod. Lifecycle Manag. 2016, 9, 87–107. [CrossRef] 91. Lee, J.J. Greener manufacturing, maintenance and disposal—Towards the ACARE targets. Aeronaut. J. 2006, 110, 567–571. [CrossRef] 92. Caniëls, M.C.J.; Cleophas, E.; Semeijn, J. Implementing green supply chain practices: An empirical investigation in the shipbuilding industry. Marit. References E-Kanban hybrid model for Malaysian automotive component suppliers with IoT solution. In Proceedings of the International Conference on Industrial Engineering and Operations Management, Dubai, UAE, 10–12 March 2020; pp. 728–738. 116. Alvarado, K.; Rabelo, L.; Moraga, R.; Quijada, S.; Gruber, F.; Sepulveda, J. Application of SCOR to e-government: A case study. Int. J. Simul. Process Model. 2007, 3, 99–114. [CrossRef] 117. Alexander, D. Collaboration software (2003). Aerosp. Eng. 2003, 23, 25–27. 118. Chryssolouris, G.; Makris, S.; Xanthakis, V.; Mourtzis, D. Towards the internet-based supply chain management for the ship repair industry. Int. J. Comput. Integr. Manuf. 2004, 17, 45–57. [CrossRef] 119. Makris, S.; Mourtzis, D.; Papakostas, N.; Chryssolouris, G. E-Collaboration for Ship Repair Supply Chain Management. In Proceedings of the IEEE International Conference on Emerging Technologies and Factory Automation (ETFA), Catania, Italy, 19–22 September 2005; Volume 1, pp. 713–718. 120. Ahlers, R. Cooperative design by using intelligent electronic supplier catalogs in the maritime industry. J. Sh. Prod. 2004, 20, 193–199. 121. Mehdi, N.; Starly, B. Witness Box Protocol: Automatic machine identification and authentication in industry 4.0. Comput. Ind. 2020, 123, 103340. [CrossRef] 122. Chandriah, K.K.; Raghavendra, N.V. Architectural Framework for Industry 4.0 Compliance Supply Chain System for Automotive Industry. In Advances in Intelligent Systems and Computing; Springer: Berlin/Heidelberg, Germany, 2019; Volume 986, pp. 107–116. 123. Das, A.; Franciosa, P.; Prakash, P.K.S.; Ceglarek, D. Transfer function of assembly process with compliant non-ideal parts. In Proceedings of the Procedia CIRP; Elsevier: Amsterdam, The Netherlands, 2014; Volume 21, pp. 177–182. 124. Finke, G.R.; Schmitt, A.J.; Singh, M. Modeling and simulating supply chain schedule risk. In Proceedings of the Proceedings—Winter Simulation Conference, Baltimore, MD, USA, 5–8 December 2010; pp. 3472–3481. 125. Satterberg, J.; Bloyer, D. Boeing commercial airplane factory modeling part 2—Simulation modeling for decision support to “Right-Size” receiving and inspection. In Proceedings of the IIE Annual Conference and Exhibition 2004, Houston, TX, USA, 15–19 May 2004; pp. 3771–3821. 126. Li, G.; Lv, C.; Cheng, Y. Research on innovation of shipbuilding supply chain management based on complexity adaptive system. In Proceedings of the ICEIS 2011—13th International Conference on Enterprise Information Systems, Beijing, China, 8–11 June 2011; Volume 1, pp. 389–395. 127. Dev, A.K.; Fung, Z.K. Simulation of hull panel logistics improvement in a shipyard. In Proceedings of the Royal Institution of Naval Architects—19th International Conference on Computer Applications in Shipbuilding (ICCAS 2019), Rotterdam, The Netherlands, 24–26 September 2019; Volume 1, p. 2020. 128. References In Proceedings of the IFIP Advances in Information and Communication Technology, Seville, Spain, 10–12 July 2017; Volume 517, pp. 59–68. 107. Chiang, P.H.; Torng, C.C. Development of total logistics support management information system and its applications to the aerospace industry. Int. J. Ind. Syst. Eng. 2016, 23, 482–499. [CrossRef] 107. Chiang, P.H.; Torng, C.C. Development of total logistics support management information system and its applications to the aerospace industry. Int. J. Ind. Syst. Eng. 2016, 23, 482–499. [CrossRef] 108. Cherry, S. Who Goes There? Proc. IEEE Spectr. 2002, 39, 39–40. , S. Who Goes There? Proc. IEEE Spectr. 2002, 39, 39–4 108. Cherry, S. Who Goes There? Proc. IEEE Spectr. 2 109. Ren, N.; Liu, J.; Su, X.; Wang, P.; Yin, J. A research on the project of digital comprehensive capability platform for shipbuilding. In IFIP Advances in Information and Communication Technology; Springer: Berlin/Heidelberg, Germany, 2008; Volume 254, pp. 487–491. 109. Ren, N.; Liu, J.; Su, X.; Wang, P.; Yin, J. A research on the project of digital comprehensive capability platform for shipbuilding. In IFIP Advances in Information and Communication Technology; Springer: Berlin/Heidelberg, Germany, 2008; Volume 254, pp. 487–491. 110. Gorecki, S.; Possik, J.; Zacharewicz, G.; Ducq, Y.; Perry, N. A multicomponent distributed framework for smart production system modeling and simulation. Sustainability 2020, 12, 6969. [CrossRef] 110. Gorecki, S.; Possik, J.; Zacharewicz, G.; Ducq, Y.; Perry, N. A multicomponent distributed framework for smart production system modeling and simulation. Sustainability 2020, 12, 6969. [CrossRef] 21 of 23 21 of 23 Materials 2020, 13, 5625 111. Yang, J.; Lee, S.; Kang, Y.S.; Do Noh, S.; Choi, S.S.; Jung, B.R.; Lee, S.H.; Kang, J.T.; Lee, D.Y.; Kim, H.S. Integrated Platform and Digital Twin Application for Global Automotive Part Suppliers. In IFIP Advances in Information and Communication Technology; Springer: Berlin/Heidelberg, Germany, 2020; Volume 592, pp. 230–237. 112. Sokol, D.Z. Improving the supply chain by sharing intelligent technical data packages. SAE Int. J. Aerosp. 2010, 2, 83–86. [CrossRef] 113. Zhao, J.Z. JT as the foundation of collaboration in ship lifecycle: The first iso standard for lightweight 3D visualization. In Proceedings of the RINA, Royal Institution of Naval Architects—International Conference on Computer Applications in Shipbuilding (ICCAS 2013), Busan, Korea, 24–26 September 2013; Volume 3, pp. 1–8. 114. Swan, M. Blockchain for Business: Next-Generation Enterprise Artificial Intelligence Systems. Adv. Comput. 2018, 111, 121–162. [CrossRef] 115. Idris, M.R.; Prakash, P.S.; Abdullah, A. References Verma, A.; Devulapalli, J. Design of simulation tools for training programs in lean manufacturing. In Proceedings of the 2006 ASME International Mechanical Engineering Congress and Exposition, IMECE 2006—Mechanical Engineering Technology Department Heads, Chicago, IL, USA, 5–10 November 2006. 129. Taluru, D.R.; Allabanda, R.P.U. Application of data analytics in gas turbine engines. In Proceedings of the ASME 2019 Gas Turbine India Conference (GTINDIA 2019), Chennai, India, 5–6 December 2019; Volume 2. 130. Nikolopoulos, L.; Boulougouris, E. A methodology for the holistic, simulation driven ship design optimization under uncertainty. In Proceedings of the Marine Design XIII, Espoo, Finland, 10–14 June 2018; Volume 1, pp. 227–244. 22 of 23 Materials 2020, 13, 5625 131. Skobelev, P. Towards autonomous ai systems for resource management: Applications in industry and lessons learned. In Lecture Notes in Computer Science (Including Subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics); Springer: Berlin/Heidelberg, Germany, 2018; Volume 10978, pp. 12–25. 132. Queen, K.H. Six emerging aerospace capabilities to watch. Manuf. Eng. 2017, 159, 81–83. 133. Van Dyke Parunak, H.; Baker, A.D.; Clark, S.J. AARIA agent architecture: From manufacturing requirements to agent-based system design. Integr. Comput. Aided. Eng. 2001, 8, 45–58. [CrossRef] 134. Serfati, C.; Sauviat, C. Global supply chains and intangible assets in the automotive and aeronautical industries. Int. J. Automot. Technol. Manag. 2019, 19, 183–205. [CrossRef] 135. Dumitrascu, O.; Dumitrascu, M.; Dobrotˇa, D. Performance evaluation for a sustainable supply chain management system in the automotive industry using artificial intelligence. Processes 2020, 8, 1384. [CrossRef] 136. Schulze-Horn, I.; Hueren, S.; Scheffler, P.; Schiele, H. Artificial Intelligence in Purchasing: Facilitating Mechanism Design-based Negotiations. Appl. Artif. Intell. 2020, 34, 618–642. [CrossRef] 137. Linger, R. Behavior computation to validate aerospace software cyber security: A knowledge management process. In Proceedings of the Proceedings of the International Astronautical Congress, Washington, DC, USA, 21–25 October 2019; Volume 2019. 138. Frazier, P.D.; Gilmore, E.T.; Collins, I.J.; Chouikha, M.F. Novel counterfeit detection of integrated circuits via infrared analysis: A case study based on the Intel Cyclone II FPGAS. In Proceedings of the Proceedings—International Conference on Machine Learning and Cybernetics, Jeju, Korea, 10–13 July 2016; Volume 1, pp. 404–409. 139. Kennedy, J.; Holt, T.; Cheng, B. Automotive cybersecurity: Assessing a new platform for cybercrime and malicious hacking. J. Crime Justice 2019, 42, 632–645. [CrossRef] 140. Hutchins, M.J.; Bhinge, R.; Micali, M.K.; Robinson, S.L.; Sutherland, J.W.; Dornfeld, D. Framework for Identifying Cybersecurity Risks in Manufacturing. Procedia Manuf. References 2015, 1, 47–63. [CrossRef] 141. Warwick, G. Aerospace tackles barriers to use of cloud computing. In Proceedings of the Aviation Week and Space Technology, New York, NY, USA, 26 October–8 November 2010; Volume 172, p. 76. 142. Zeiler, J.; Fottner, J. Architectural Design for Special Load Carriers as Smart Objects in a Cloud-based Service System. In Proceedings of the 2019 IEEE 6th International Conference on Industrial Engineering and Applications (ICIEA 2019), Tokyo, Japan, 12–15 April 2019; pp. 644–652. 43. Smythe, S.A.; Thomas, B.M.; Jackson, M. Recycling of titanium alloy powders and swarf through continu extrusion (Conformtm) into affordable wire for additive manufacturing. Metals 2020, 10, 843. [CrossRe 144. Strong, D.; Kay, M.; Wakefield, T.; Sirichakwal, I.; Conner, B.; Manogharan, G. Rethinking reverse logistics: Role of additive manufacturing technology in metal remanufacturing. J. Manuf. Technol. Manag. 2020, 31, 124–144. [CrossRef] 145. Qiao, D.; Wang, B.; Cridland, M. Additive manufacturing for marine and offshore applications. In SNAME Maritime Convention; The Society of Naval Architects and Marine Engineers: Alexandria, VA, USA, 2020. 146. Szymczyk, P.; Smolina, I.; Rusi´nska, M.; Wo´zna, A.; Tomassetti, A.; Chlebus, E. Logistical aspects of transition from traditional to additive manufacturing. In Proceedings of the Advances in Intelligent Systems and Computing, Wroclaw, Poland, 17–18 September 2018; Volume 835, pp. 752–760. 147. Mahaboob Sheriff, K.M.; Kerbache, L. Implementation of additive manufacturing (Am) for automotive supply chain transformation in post covid-19 scenario-a barrier analysis. In Proceedings of the International Conference on Industrial Engineering and Operations Management, Detroit, MI, USA, 10–14 August 2020; p. 2020. 148. Eryilmaz, U.; Dijkman, R.; Van Jaarsveld, W.; Van Dis, W.; Alizadeh, K. Traceability blockchain prototype for regulated manufacturing industries. In Proceedings of the ACM International Conference Proceeding Series, Singapore, 8–10 July 2020; pp. 9–16. 149. Butean, A.; Pournaras, E.; Tara, A.; Turesson, H.; Ivkushkin, K. Dynamic consensus: Increasing blockchain adaptability to enterprise applications. In Advances in Intelligent Systems and Computing; Springer: Berlin/Heidelberg, Germany, 2020; Volume 1226, pp. 433–442. 150. Zhu, J.; Wu, M.; Liu, C. Research on the Application Mode of Blockchain Technology in the Field of Shipbuilding. In Proceedings of the Proceedings of 2020 IEEE International Conference on Artificial Intelligence and Computer Applications (ICAICA 2020), Dalian, China, 27–29 June 2020; pp. 34–37. 151. Dutta, P.; Choi, T.M.; Somani, S.; Butala, R. Blockchain technology in supply chain operations: Applications, challenges and research opportunities. Transp. Res. Part E Logist. Transp. Rev. 2020, 142, 102067. References [CrossRef] [PubMed] 23 of 23 Materials 2020, 13, 5625 152. Stroud, A.; Ertas, A. Complex multi-system integration problems associated with titanium metalworking and manufacture: System of systems aproach—Part I. Metals 2019, 9, 424. [CrossRef] 153. Silva-Martinez, J.; Schrage, D. Proposed variants for a collaborative aerospace lifecycle systems engineering master’s program. In Proceedings of the 52nd Aerospace Sciences Meeting, National Harbor, MD, USA, 13–17 January 2014; p. 2020. y p 154. Braglia, M.; Frosolini, M. An integrated approach to implement Project Management Information Systems within the Extended Enterprise. Int. J. Proj. Manag. 2014, 32, 18–29. [CrossRef] 155. Wang, X.; Le, X. Discussions about Supply Logistics in Automobile Industry of China. J. Phys. Conf. Ser. 2020, 1544, 2020. [CrossRef] 156. Bartlett, P.A.; Julien, D.M.; Baines, T.S. Improving supply chain performance through improved visibility. Int. J. Logist. Manag. 2007, 18, 294–313. [CrossRef] 157. Vilko, J.; Ritala, P.; Hallikas, J. Risk management abilities in multimodal maritime supply chains: Visibility and control perspectives. Accid. Anal. Prev. 2019, 123, 469–481. [CrossRef] 158. Fraser, I.J.; Müller, M.; Schwarzkopf, J. Transparency for multi-tier sustainable supply chain management: A case study of a multi-tier transparency approach for SSCM in the automotive industry. Sustainability 2020, 12, 1814. [CrossRef] 59. Keivanpour, S.; Ait Kadi, D.; Mascle, C. End of life aircrafts recovery and green supply chain (a concep framework for addressing opportunities and challenges). Manag. Res. Rev. 2015, 38, 1098–1124. [Cross 160. Sherafati, M.; Bashiri, M.; Tavakkoli-Moghaddam, R.; Pishvaee, M.S. Achieving sustainable development of supply chain by incorporating various carbon regulatory mechanisms. Transp. Res. Part D Transp. Environ. 2020, 81, 102253. [CrossRef] 161. Li, G.; Li, L.; Choi, T.M.; Sethi, S.P. Green supply chain management in Chinese firms: Innovative measures and the moderating role of quick response technology. J. Oper. Manag. 2020, 66, 958–988. [CrossRef] 162. Liboni, L.B.; Cezarino, L.O.; Jabbour, C.J.C.; Oliveira, B.G.; Stefanelli, N.O. Smart industry and the pathways to HRM 4.0: Implications for SCM. Supply Chain Manag. 2019, 24, 124–146. [CrossRef] 163. Thaib, D. Drivers of the green supply chain initiatives: Evidence from indonesian automotive industry. Uncertain Supply Chain Manag. 2020, 8, 105–116. [CrossRef] 64. Ahmed, S.M.; Karmaker, C.L.; Doss, D.A.; Khan, A.H. Modeling the barriers in managing closed loop sup chains of automotive industries in Bangladesh. Int. J. Supply Oper. Manag. 2020, 7, 76–92. [CrossRef] 65. Pop, A.B.; ¸Tî¸tu, A.M. Implementation of advanced product quality planning in the aerospace industry a to improve the quality management. Qual. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Access Success 2020, 21, 56–61. 166. Abollado, J.R.; Shehab, E. A Systems Approach for the Definition of Lean Workflows in Global Aerospace Manufacturing Companies. Procedia CIRP 2018, 70, 446–450. [CrossRef] 167. Lascu, E.; Lascu, F.D.; Stinga, F.; Severin, I. Process redesign to reduce stocks of obsolete parts in automotive industry. J. Gen. Manag. 2020, 21, 43–49. 168. Elhammouchi, C.; Abouabdellah, A. Proposal of professional referential evaluating the performance of downstream supply chain in Moroccan automotive industry. In Proceedings of the International Conference on Industrial Engineering and Operations Management, Paris, France, 26–27 July 2018; Volume 2018, pp. 716–722. 169. Turi, A.; Mocan, M.; Ivascu, L.; Goncalves, G. Managing success: The benefits of marketing effectiveness and strategic plan adaptation in automotive industry. In Proceedings of the 29th International Business Information Management Association Conference—Education Excellence and Innovation Management through Vision 2020: From Regional Development Sustainability to Global Economic Growth, Vienna, Austria, 3–4 May 2017; pp. 2047–2059. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 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/). © 2020 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/).
https://openalex.org/W2144531925
https://europepmc.org/articles/pmc4149311?pdf=render
English
null
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
2,014
cc-by
6,512
* 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, Yamatomo G, Nannya Y, Koehler R, Flohr T, Miller CW, Harbott J, Ludwig WD, Stanulla M, Schrappe M, Bartram CR, Koeffler HP: Molecular allelokaryotyping of pediatric acute lymphoblastic leukemias by high-resolution single nucleotide polymorphism oligonucleotide genomic microarray. Blood 2008, 111(2):776–84. This study attests to the fact that some patients with a myelodysplastic syndrome exhibiting a normal karyotype may have underlying genetic abnormalities detectable by chromosomal microarray and/or targeted mutation analyses. Further genomic and molecular studies on a 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 genomic abnormalities in acute myeloid leukemia/myelodysplastic syndrome with normal karyotype. Haematologica 2009, 94:213–223. 6. References Delhommeau F, Dupont S, Della Valle V, James C, Trannoy S, Massé A, Kosmider O, Le Couedic JP, Robert F, Alberdi A, Lécluse Y, Plo I, Dreyfus FJ, Marzac C, Casadevall N, Lacombe C, Romana SP, Dessen P, Soulier J, Viguié F, Fontenay M, Vainchenker W, Bernard OA: Mutation in TET2 in myeloid cancers. N Engl J Med 2009, 360(22):2289–301. 40. Wu PK, Hong SK, Veeranki S, Karkhanis M, Starenki D, Plaza JA, Park JI: A mortalin/HSPA9-mediated switch in tumor-suppressive signaling of Raf/MEK/extracellular signal-regulated kinase. Mol Cell Biol 2013, 33(20):4051–67. 25. Mohamedali A, Gäken J, Twine NA, Ingram W, Westwood N, Lea NC, Hayden J, Donaldson N, Aul C, Gattermann N, Giagounidis A, Germing U, List AF, Mufti GJ: Prevalence and prognostic significance of allelic imbalance by single-nucleotide polymorphism analysis in low-risk myelodysplastic syndromes. Blood 2007, 110(9):3365–73. 41. Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL: Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 1983, 305(5937):779–84. 42. Fearon ER, Vogelstein B, Feinberg AP: Somatic deletion and duplication of genes on chromosome 11 in Wilms’ tumours. Nature 1984, 309:176–8. 26. Gondek LP, Tiu R, O'Keefe CL, Sekeres MA, Thiel KS, Maciejewski JP: Chromosomal lesions and uniparental disomy detected by SNP arrays in MDS. Blood 2008, 111:1534–42. 43. Jankowska AM, Szpurka H, Tiu RV, Makishima H, Afable M, Huh J, O’Keefe CL, Ganetzky R, McDevitt MA, Maciejewski JP: Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms. Blood 2009, 113(25):6403–10. 27. Gorletta TA, Gasparini P, D’Elios MM, Trubia M, Pelicci PG, Di Fiore PP: Frequent loss of heterozygosity without loss of genetic material in acute myeloid leukemia with a normal karyotype. Genes Chromosomes Cancer 2005, 44:334–337. 44. Wang J, Ai X, Gale RP, Xu Z, Qin T, Fang L, Zhang H, Pan L, Hu N, Zhang Y, Xiao Z: TET2, ASXL1 and EZH2 mutations in Chinese with myelodysplastic syndromes. Leuk Res 2013, 37(3):305–11. 28. Raghavan M, Lillington DM, Skoulakis S, Debernardi S, Chaplin T, Foot NJ, Lister TA, Young BD: Genome-wide single nucleotide polymorphism analysis reveals frequent partial uniparental disomy due to somatic recombination in acute myeloid leukemias. Cancer Res 2005, 65:375–378. 45. References Molecular Cytogenetics 2014, 7:35 http://www.molecularcytogenetics.org/content/7/1/35 nucleotide polymorphism array karyotyping identifies acquired uniparental disomy and homozygous mutations, including novel missense substitutions of c-Cbl, in myeloid malignancies. Cancer Res 2008, 68(24):10349–57. candidates for a tumor suppressor gene in malignant myeloid disorders on 5q31. Blood 2000, 95(7):2372–7. 36. Kask M1, Pruunsild P, Timmusk T: Bidirectional transcription from human LRRTM2/CTNNA1 and LRRTM1/CTNNA2 gene loci leads to expression of N-terminally truncated CTNNA1 and CTNNA2 isoforms. Biochem Biophys Res Commun 2011, 411(1):56–61. 21. Ernst T, Chase AJ, Score J, Hidalgo-Curtis CE, Bryant C, Jones AV, Waghorn K, Zoi K, Ross FM, Reiter A, Hochhaus A, Drexler HG, Duncombe A, Cervantes F, Oscier D, Boultwood J, Grand FH, Cross NC: Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet 2010, 42(8):722–6. 37. Liu TX, Becker MW, Jelinek J, Wu WS, Deng M, Mikhalkevich N, Hsu K, Bloomfield CD, Stone RM, DeAngelo DJ, Galinsky IA, Issa JP, Clarke MF, Look AT: Chromosome 5q deletion and epigenetic suppression of the gene encoding alpha-catenin (CTNNA1) in myeloid cell transformation. Nat Med 2007, 13(1):78–83. 22. Langemeijer SM, Kuiper RP, Berends M, Knops R, Aslanyan MG, Massop M, Stevens-Linders E, van Hoogen P, van Kessel AG, Raymakers RA, Kamping EJ, Verhoef GE, Verburgh E, Hagemeijer A, Vandenberghe P, de Witte T, van der Reijden BA, Jansen JH: Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat Genet 2009, 41(7):838–42. 38. Ye Y, McDevitt MA, Guo M, Zhang W, Galm O, Gore SD, Karp JE, Maciejewski JP, Kowalski J, Tsai HL, Gondek LP, Tsai HC, Wang X, Hooker C, Smith BD, Carraway HE, Herman JG: Progressive chromatin repression and promoter methylation of CTNNA1 associated with advanced myeloid malignancies. Cancer Res 2009, 69(21):8482–90. 23. Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tönnissen ER, van der Heijden A, Scheele TN, Vandenberghe P, de Witte T, van der Reijden BA, Jansen JH: Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet 2010, 42(8):665–7. 39. Xie H, Hu Z, Chyna B, Horrigan SK, Westbrook CA: Human mortalin (HSPA9): a candidate for the myeloid leukemia tumor suppressor gene on 5q31. Leukemia 2000, 14(12):2128–34. 24. References Jerez A1, Sugimoto Y, Makishima H, Verma A, Jankowska AM, Przychodzen B, Visconte V, Tiu RV, O’Keefe CL, Mohamedali AM, Kulasekararaj AG, Pellagatti A, McGraw K, Muramatsu H, Moliterno AR, Sekeres MA, McDevitt MA, Kojima S, List A, Boultwood J, Mufti GJ, Maciejewski JP: Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic pathogenesis. Blood 2012, 119(25):6109–17. 29. 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. 46. Khan SN, Jankowska AM, Mahfouz R, Dunbar AJ, Sugimoto Y, Hosono N, Hu Z, Cheriyath V, Vatolin S, Przychodzen B, Reu FJ, Saunthararajah Y, O’Keefe C, Sekeres MA, List AF, Moliterno AR, McDevitt MA, Maciejewski JP, Makishima H: Multiple mechanisms deregulate EZH2 and histone H3 lysine 27 epigenetic changes in myeloid malignancies. Leukemia 2013, 27(6):1301–9. 30. Gondek LP, Dunbar AJ, Szpurka H, McDevitt MA, Maciejewski JP: SNP array karyotyping allows for the detection of uniparental disomy and cryptic chromosomal abnormalities in MDS/MPD-U and MPD. PLoS One 2007, 2(11):e122. 31. Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA, Walker H, Wheatley K, Bowen DT, Burnett AK, Goldstone AH, Linch DC: The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: Analysis of 854 patients from the United Kingdom medical research council. AML 10 and 12 trials. Blood 2001, 98:1752–1759. 47. Dicker F, Haferlach C, Sundermann J, Wendland N, Weiss T, Kern W, Haferlach T, Schnittger S: Mutation analysis for RUNX1, MLL-PTD, FLT3-ITD, NPM1 and NRAS in 269 patients with MDS or secondary AML. Leukemia 2010, 24:1528–1532. 48. Schnittger S, Dicker F, Kern W, Wendland N, Sundermann J, Alpermann T, Alpermann T, Haferlach C, Haferlach T: RUNX1 mutations are frequent in de novo AML with noncomplex karyotype and confer an unfavorable prognosis. Blood 2011, 117:2348–2357. 32. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, La Starza R, Diverio D, Colombo E, Santucci A, Bigerna B, Pacini R, Pucciarini A, Liso A, Vignetti M, Fazi P, Meani N, Pettirossi V, Saglio G, Mandelli F, Lo-Coco F, Pelicci PG, Martelli MF: Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005, 352:254–266. 49. References 1. Mullighan CG, Goorha S, Radtke I, Miller CB, Coustan-Smith E, Dalton JD, Girtman K, Mathew S, Ma J, Pounds SB, Su X, Pui CH, Relling MV, Evans WE, Shurtleff SA, Downing JR: Genome-wide analysis of genetic alterations in acute lymphoblastic leukemia. Nature 2007, 446:758–764. 1. Mullighan CG, Goorha S, Radtke I, Miller CB, Coustan-Smith E, Dalton JD, Girtman K, Mathew S, Ma J, Pounds SB, Su X, Pui CH, Relling MV, Evans WE, Shurtleff SA, Downing JR: Genome-wide analysis of genetic alterations in acute lymphoblastic leukemia. Nature 2007, 446:758–764. 18. Grand FH, Hidalgo-Curtis CE, Ernst T, Zoi K, Zoi C, McGuire C, Kreil S, Jones A, Score J, Metzgeroth G, Oscier D, Hall A, Brandts C, Serve H, Reiter A, Chase AJ, Cross NC: Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms. Blood 2009 113(24):6182–92 y 2. Suela J, Alvarez S, Cifuentes F, Largo C, Ferreira BI, Blesa D, Ardanaz M, Garcia R, Marquez JA, Odero MD, Calasanz MJ, Cigudosa JC: DNA profiling analysis of 100 consecutive de novo acute myeloid leukemia cases reveals patterns of genomic instability that affect all cytogenetic risk groups. Leukemia 2007, 21:1224–1231. 2. Suela J, Alvarez S, Cifuentes F, Largo C, Ferreira BI, Blesa D, Ardanaz M, Garcia R, Marquez JA, Odero MD, Calasanz MJ, Cigudosa JC: DNA profiling analysis of 100 consecutive de novo acute myeloid leukemia cases reveals patterns of genomic instability that affect all cytogenetic risk groups. Leukemia 2007, 21:1224–1231. 19. Sanada M, Suzuki T, Shih LY, Otsu M, Kato M, Yamazaki S, Tamura A, Honda H, Sakata-Yanagimoto M, Kumano K, Oda H, Yamagata T, Takita J, Gotoh N, Nakazaki K, Kawamata N, Onodera M, Nobuyoshi M, Hayashi Y, Harada H, Kurokawa M, Chiba S, Mori H, Ozawa K, Omine M, Hirai H, Nakauchi H, Koeffler HP, Ogawa S: Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms. Nature 2009, 460(7257):904–8. 3. Tyyba kinoja A, Elonen E, Piippo K, Porkka K, Knuutila S: Oligonucleotide array-CGH reveals cryptic gene copy number alterations in karyotypically normal acute myeloid leukemia. Leukemia 2007, 21:571–574. 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 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 Page 8 of 9 Hemmat et al. 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 52. Boultwood J, Perry J, Zaman R, Fernandez-Santamaria C, Littlewood T, Kusec R, Pellagatti A, Wang L, Clark RE, Wainscoat JS: High-density single nucleotide polymorphism array analysis and ASXL1 gene mutation screening in chronic myeloid leukemia during disease progression. Leukemia 2010, 24(6):1139–45. 53. Pérez B, Kosmider O, Cassinat B, Renneville A, Lachenaud J, Kaltenbach S, Bertrand Y, Baruchel A, Chomienne C, Fontenay M, Preudhomme C, Cavé H: Genetic typing of CBL, ASXL1, RUNX1, TET2 and JAK2 in juvenile myelomonocytic leukaemia reveals a genetic profile distinct from chronic myelomonocytic leukaemia. Br J Haematol 2010, 151(5):460–8. 54. Sugimoto Y, Muramatsu H, Makishima H, Prince C, Jankowska AM, Yoshida N, Xu Y, Nishio N, Hama A, Yagasaki H, Takahashi Y, Kato K, Manabe A, Kojima S, Maciejewski JP: Spectrum of molecular defects in juvenile myelomonocytic leukaemia includes ASXL1 mutations. Br J Haematol 2010, 150(1):83–7. 54. Sugimoto Y, Muramatsu H, Makishima H, Prince C, Jankowska AM, Yoshida N, Xu Y, Nishio N, Hama A, Yagasaki H, Takahashi Y, Kato K, Manabe A, Kojima S, Maciejewski JP: Spectrum of molecular defects in juvenile myelomonocytic leukaemia includes ASXL1 mutations. Br J Haematol 2010, 150(1):83–7. 55. Kosmider O, Gelsi-Boyer V, Cheok M, Grabar S, Della-Valle V, Picard F, Viguié F, Quesnel B, Beyne-Rauzy O, Solary E, Vey N, Hunault-Berger M, Fenaux P, Mansat-De Mas V, Delabesse E, Guardiola P, Lacombe C, Vainchenker W, Preudhomme C, Dreyfus F, Bernard OA, Birnbaum D, Fontenay M, Groupe Francophone des Myélodysplasies: TET2 mutation is an independent favorable prognostic factor in myelodysplastic syndromes (MDSs). Blood 2009, 114(15):3285–91. 56. Thol F, Friesen I, Damm F, Yun H, Weissinger EM, Krauter J, Wagner K, Chaturvedi A, Sharma A, Wichmann M, Göhring G, Schumann C, Bug G, Ottmann O, Hofmann WK, Schlegelberger B, Heuser M, Ganser A: Prognostic significance of ASXL1 mutations in patients with myelodysplastic syndromes. J Clin Oncol 2011, 29(18):2499–506. 56. Thol F, Friesen I, Damm F, Yun H, Weissinger EM, Krauter J, Wagner K, Chaturvedi A, Sharma A, Wichmann M, Göhring G, Schumann C, Bug G, Ottmann O, Hofmann WK, Schlegelberger B, Heuser M, Ganser A: Prognostic significance of ASXL1 mutations in patients with myelodysplastic syndromes. J Clin Oncol 2011, 29(18):2499–506. References Gaidzik VI, Bullinger L, Schlenk RF, Zimmermann AS, Rock J, Paschka P, Corbacioglu A, Krauter J, Schlegelberger B, Ganser A, Späth D, Kündgen A, Schmidt-Wolf IG, Götze K, Nachbaur D, Pfreundschuh M, Horst HA, Döhner H, Döhner K: RUNX1 mutations in acute myeloid leukemia: results from a comprehensive genetic and clinical analysis from the AML study group. J Clin Oncol 2011, 29:1364–1372. 33. Baldus CD, Mrozek K, Marcucci G, Bloomfield CD: Clinical outcome of de novo acute myeloid leukaemia patients with normal cytogenetics is affected by molecular genetic alterations: a concise review. Br J Haematol 2007, 137:387–400. 50. Gelsi-Boyer V, Trouplin V, Adélaïde J, Bonansea J, Cervera N, Carbuccia N, Lagarde A, Prebet T, Nezri M, Sainty D, Olschwang S, Xerri L, Chaffanet M, Mozziconacci MJ, Vey N, Birnbaum D: Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br J Haematol 2009, 145(6):788–800. 34. Mead AJ, Linch DC, Hills RK, Wheatley K, Burnett AK, Gale RE: FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia. Blood 2007, 110:1262–1270. 51. chou WC, Hou HA, Chen CY, Tang JL, Yao M, Tsay W, Ko BS, Wu SJ, Huang SY, Hsu SC, Chen YC, Huang YN, Chang YC, Lee FY, Liu MC, Liu CW, Tseng MH, Huang CF, Tien HF: Distinct clinical and biologic characteristics in adult acute myeloid leukemia bearing the isocitrate dehydrogenase 1 mutation. Blood 2010, 115(14):2749–54. 35. Horrigan SK, Arbieva ZH, Xie HY, Kravarusic J, Fulton NC, Naik H, Le TT, Westbrook CA: Delineation of a minimal interval and identification of 9 Page 9 of 9 Page 9 of 9 Hemmat et al. Molecular Cytogenetics 2014, 7:35 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 and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W2807283266
https://europepmc.org/articles/pmc6008786?pdf=render
English
null
Newer Oral Anticoagulants in the Treatment of Acute Portal Vein Thrombosis in Patients with and without Cirrhosis
International journal of hepatology
2,018
cc-by
7,028
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 (rivaroxaban, apixaban, and dabigatran) in patients without cirrhosis was 25% higher than patients with cirrhosis [10]. In another study, involving 20 patients with VTE, including 12 patients with splanchnic vein thrombosis, 75% were treated 7 International Journal of Hepatology International Journal of Hepatology International Journal of Hepatology 8 Use in Acute Portal Vein Thrombosis Unrelated to Cirrhosis,” Gastroenterology Research, vol. 10, no. 2, pp. 141–143, 2017. [6] M. Senzolo, T. M. Sartori, V. Rossetto et al., “Prospective evaluation of anticoagulation and transjugular intrahepatic por- tosistemic shunt for the management of portal vein thrombosis in cirrhosis,” Liver International, vol. 32, no. 6, pp. 919–927, 2012. Use in Acute Portal Vein Thrombosis Unrelated to Cirrhosis,” Gastroenterology Research, vol. 10, no. 2, pp. 141–143, 2017. [22] M. Martinez, A. Tandra, and R. Vuppalanchi, “Treatment of acute portal vein thrombosis by nontraditional anticoagula- tion,” Hepatology, vol. 60, no. 1, pp. 425-426, 2014. [7] E. Villa, C. Camm`a, M. Marietta et al., “Enoxaparin prevents portal vein thrombosis and liver decompensation in patients with advanced cirrhosis,” Gastroenterology, vol. 143, no. 5, pp. 1253–1260, 2012. [23] S. Pannach, J. Babatz, and J. Beyer-Westendorf, “Successful treatment of acute portal vein thrombosis with rivaroxaban,” Thrombosis and Haemostasis, vol. 110, no. 4, pp. 626-627, 2013. [8] N. M. Intagliata, Z. H. Henry, H. Maitland et al., “Direct Oral Anticoagulants in Cirrhosis Patients Pose Similar Risks of Bleeding When Compared to Traditional Anticoagulation,” Digestive Diseases and Sciences, vol. 61, no. 6, pp. 1721–1727, 2016. [24] W. Potze, F. Arshad, J. Adelmeijer et al., “Routine coagulation assays underestimate levels of antithrombin-dependent drugs but not of direct anticoagulant drugs in plasma from patients with cirrhosis,” British Journal of Haematology, vol. 163, no. 5, pp. 666–673, 2013. [9] J. Hum, J. J. Shatzel, J. H. Jou, and T. G. Deloughery, “The efficacy and safety of direct oral anticoagulants vs traditional anticoagulants in cirrhosis,” European Journal of Haematology, vol. 98, no. 4, pp. 393–397, 2017. [25] M. A. Smythe, J. Fanikos, M. P. Gulseth et al., “Rivaroxaban: Practical considerations for ensuring safety and efficacy,” Phar- macotherapy, vol. 33, no. 11, pp. 1223–1245, 2013. [10] A. De Gottardi, J. Trebicka, C. Klinger et al., “Antithrombotic treatment with direct-acting oral anticoagulants in patients with splanchnic vein thrombosis and cirrhosis,” Liver International, vol. 37, no. 5, pp. 694–699, 2017. [26] A. M. Morrill, D. Ge, and K. C. Willett, “Dosing of Target- Specific Oral Anticoagulants in Special Populations,” Annals of Pharmacotherapy, vol. 49, no. 9, pp. 1031–1045, 2015. [27] S. M. Bates and J. S. Ginsberg, “Clinical practice. Treatment of deep-vein thrombosis,” The New England Journal of Medicine, vol. 351, no. 3, pp. 268–277, 2004. [11] K. Lenz, B. Dieplinger, R. International Journal of Hepatology Buder, P. Piringer, M. Rauch, and M. Voglmayr, “Successful treatment of partial portal vein thrombosis (PVT) with low dose rivaroxaban,” Zeitschrift f¨ur Gastroenterologie, vol. 52, no. 10, pp. 1175–1177, 2014. [28] E. Liakoni, A. E. R¨atz Bravo, and S. Kr¨ahenb¨uhl, “Hepatotoxicity of New Oral Anticoagulants (NOACs),” Drug Safety, vol. 38, no. 8, pp. 711–720, 2015. [12] H. Yang, S. R. Kim, and M. J. Song, “Recurrent acute portal vein thrombosis in liver cirrhosis treated by rivaroxaban,” Clinical and Molecular Hepatology, vol. 22, no. 4, pp. 499–502, 2016. [29] D. Caldeira, M. Barra, A. T. Santos et al., “Risk of drug-induced liver injury with the new oral anticoagulants: Systematic review and meta-analysis,” Heart, vol. 100, no. 7, pp. 550–556, 2014.f [13] L. D. DeLeve, D.-C. Valla, and G. Garcia-Tsao, “Vascular disorders of the liver,” Hepatology, vol. 49, no. 5, pp. 1729–1764, 2009. [30] J. Graff and S. Harder, “Anticoagulant therapy with the oral direct factor Xa inhibitors rivaroxaban, apixaban and edoxaban and the thrombin inhibitor dabigatran etexilate in patients with hepatic impairment,” Clinical Pharmacokinetics, vol. 52, no. 4, pp. 243–254, 2013. [14] B. Condat, F. Pessione, S. Hillaire et al., “Current outcome of portal vein thrombosis in adults: risk and benefit of anticoagu- lant therapy,” Gastroenterology, vol. 120, no. 2, pp. 490–497, 2001. [31] W. Potze, J. Adelmeijer, and T. Lisman, “Decreased in vitro anticoagulant potency of Rivaroxaban and Apixaban in plasma from patients with cirrhosis,” Hepatology, vol. 61, no. 4, pp. 1435- 1436, 2015. [15] W. Ageno, N. Riva, S. Schulman et al., “Long-term clinical out- comes of splanchnic vein thrombosis results of an international registry,” JAMA Internal Medicine, vol. 175, no. 9, pp. 1474–1480, 2015. [32] M. A. Zocco, E. di Stasio, R. de Cristofaro et al., “Thrombotic risk factors in patients with liver cirrhosis: correlation with MELD scoring system and portal vein thrombosis develop- ment,” Journal of Hepatology, vol. 51, no. 4, pp. 682–689, 2009. [16] W. Ageno, A. S. Gallus, A. Wittkowsky, M. Crowther, E. M. Hylek, and G. Palareti, “Oral anticoagulant therapy: antithrom- botic therapy and prevention of thrombosis, 9th ed: American College of Chest Physicians evidence-based clinical practice guidelines,” CHEST, vol. 141, no. 2, supplement, pp. e44S–e88S, 2012. [33] Xarelto (rivaroxaban) [package insert], Janssen Pharmaceuticals Inc. Titusville, http://www.xareltohcp.com/shared/product/ prescribing-information.pdf. [17] K. I. Rodr´ıguez-Castro, R. J. Porte, E. Nadal, G. Germani, P. Burra, and M. References There are some limitations to the studies included in this review. First, it is significant heterogeneity which makes comparison between studies difficult. There are differences in baseline patient characteristics, the indications for anti- coagulation, the anticoagulant agents used, the doses given, and the primary endpoints. It is difficult to standardize a review when such parameters do not align. The second major limitation is the quality of the studies available. None of the included studies were randomized controlled trials, and, barring one study, all were retrospective. The study sizes were small. These were not controlled studies as the anticoagulation prescribing (initiation, choice of the agents, discontinuation, and transitioning to and from traditional anticoagulants due to intolerance or adverse effects) was not under the investigators’ control. Many studies did not have a control group. Additionally, patients with CPT C cirrhosis were largely excluded. Patients with mesenteric ischemia or renal impairment were also excluded from these studies limiting our insight into management of these subgroups with NOACs. [1] L. Amitrano, M. A. Guardascione, V. Brancaccio et al., “Risk factors and clinical presentation of portal vein thrombosis in patients with liver cirrhosis,” Journal of Hepatology, vol. 40, no. 5, pp. 736–741, 2004. [2] X. Qi, V. de Stefano, H. Li, J. Dai, X. Guo, and D. Fan, “Anticoagulation for the treatment of portal vein thrombosis in liver cirrhosis: a systematic review and meta-analysis of observational studies,” European Journal of Internal Medicine, vol. 26, no. 1, pp. 23–29, 2015. [3] L. Loffredo, D. Pastori, A. Farcomeni, and F. Violi, “Effects of Anticoagulants in Patients With Cirrhosis and Portal Vein Thrombosis: A Systematic Review and Meta-analysis,” Gas- troenterology, vol. 153, no. 2, pp. 480–487.e1, 2017. [4] H. Chen, F. Turon, V. Hern´andez-Gea et al., “Nontumoral portal vein thrombosis in patients awaiting liver transplantation,” Liver Transplantation, vol. 22, no. 3, pp. 352–365, 2016. [5] M. G. Delgado, S. Seijo, and I. Yepes, “Efficacy and safety of anticoagulation on patients with cirrhosis and portal vein thrombosis,” Clinical Gastroenterology and Hepatology, vol. 10, no. 7, pp. 776–783, 2012. International Journal of Hepatology 9 of coagulation assays and reversal of anticoagulant activity,” Thrombosis & Haemostasis, vol. 103, no. 6, pp. 1116–1127, 2010. [40] S. J. Connolly, M. D. Ezekowitz, S. Yusuf et al., “Dabigatran versus warfarin in patients with atrial fibrillation,” The New England Journal of Medicine, vol. 361, no. 12, pp. 1139–1151, 2009. [41] J. Stangier, B. I. Eriksson, O. E. Dahl et al., “Pharmacokinetic profile of the oral direct thrombin inhibitor dabigatran etexilate in healthy volunteers and patients undergoing total hip replace- ment,” Clinical Pharmacology and Therapeutics, vol. 45, no. 5, pp. 555–563, 2005. [42] H. Tran, J. Joseph, L. Young et al., “New oral anticoagulants: A practical guide on prescription, laboratory testing and peri- procedural/bleeding management,” Internal Medicine Journal, vol. 44, no. 6, pp. 525–536, 2014. [43] J. W. Eikelboom, D. J. Quinlan, J. Hirsh, S. J. Connolly, and J. I. Weitz, “Laboratory monitoring of non-Vitamin K antagonist oral anticoagulant use in patients with atrial fibrillation: A review,” JAMA Cardiology, vol. 2, no. 5, pp. 566–574, 2017.f [44] I. L. Holster, V. E. Valkhoff, E. J. Kuipers, and E. T. T. L. Tjwa, “New oral anticoagulants increase risk for gastrointestinal bleeding: a systematic review and meta-analysis,” Gastroenterol- ogy, vol. 145, no. 1, pp. 105.e15–112.e15, 2013. [45] C. Francoz, J. Belghiti, V. Vilgrain et al., “Splanchnic vein thrombosis in candidates for liver transplantation: usefulness of screening and anticoagulation,” Gut, vol. 54, no. 5, pp. 691–697, 2005. [46] L. Amitrano, M. A. Guardascione, A. Menchise et al., “Safety and efficacy of anticoagulation therapy with low molecular weight heparin for portal vein thrombosis in patients with liver cirrhosis,” Journal of Clinical Gastroenterology, vol. 44, no. 6, pp. 448–451, 2010. [47] C. D. Seaman, K. J. Smith, and M. V. Ragni, “Cost-effectiveness of rivaroxaban versus warfarin anticoagulation for the preven- tion of recurrent venous thromboembolism: A U.S. perspec- tive,” Thrombosis Research, vol. 132, no. 6, pp. 647–651, 2013. [48] D. Kubitza, A. Roth, M. Becka et al., “Effect of hepatic impair- ment on the pharmacokinetics and pharmacodynamics of a single dose of rivaroxaban—an oral, direct Factor Xa inhibitor,” British Journal of Clinical Pharmacology, vol. 76, no. 1, pp. 89–98, 2013. [49] J. Mendell, L. Johnson, and S. Chen, “An open-label, phase 1 study to evaluate the effects of hepatic impairment on edoxaban pharmacokinetics and pharmacodynamics,” Clinical Pharmacology and Therapeutics, vol. 55, no. 12, pp. 1395–1405, 2015. [50] M. Tornkvist, J. G. Smith, and A. International Journal of Hepatology Senzolo, “Management of nonneoplastic portal vein thrombosis in the setting of liver transplantation: a systematic review,” Transplantation, vol. 94, no. 11, pp. 1145–1153, 2012. [34] Eliquis(apixaban) [package insert], Bristol-Myers Squibb, New York, NY, USA, http://packageinserts.bms.com/pi/ pi eliquis.pdf. [35] Savaysa (edoxaban) [package insert], Daiichi Sankyo, Tokyo, Japan, http://www.accessdata.fda.gov/drugsatfda docs/label/ 2015/206316lbl.pdf. [18] S. Seijo and J. C. Garcia-Pagan, “Anticoagulation in cirrhosis: Ready ... set ... wait!,” Hepatology, vol. 58, no. 3, pp. 1175-1176, 2013. [36] T. A. Dewald and R. C. Becker, “The pharmacology of novel oral anticoagulants,” Journal of Thrombosis and Thrombolysis, vol. 37, no. 2, pp. 217–233, 2014. [19] D. T. Janczak, M. K. Mimier, R. D. McBane et al., “Rivaroxaban and Apixaban for Initial Treatment of Acute Venous Throm- boembolism of Atypical Location,” Mayo Clinic Proceedings, vol. 93, no. 1, pp. 40–47, 2018. [37] D. A. Parasrampuria, T. Kanamaru, A. Connor et al., “Evalua- tion of regional gastrointestinal absorption of edoxaban using the enterion capsule,” Clinical Pharmacology and Therapeutics, vol. 55, no. 11, pp. 1286–1292, 2015. [20] X. Qi, E. M. Yoshida, N. Mendez-Sanchez, and X. Guo, “Rivaroxaban recanalized occlusive superior mesenteric vein thrombosis, but increased the risk of bleeding in a cirrhotic patient,” Liver International, vol. 37, no. 10, pp. 1574-1575, 2017. [38] Pradaxa (dabigatran) [package insert], Boehringer Ingelheim Pharmaceuticals, Inc, 2015. [39] J. van Ryn, J. Stangier, S. Haertter et al., “Dabigatran etexilate—a novel, reversible, oral direct thrombin inhibitor: interpretation [21] F. Nery, D. Valadares, S. Morais, M. T. Gomes, and A. De Got- tardi, “Efficacy and Safety of Direct-Acting Oral Anticoagulants International Journal of Hepatology International Journal of Hepatology Labaf, “Current evidence of oral anticoagulant reversal: A systematic review,” Thrombosis Research, vol. 162, pp. 22–31, 2018. [51] T. Y. Hu, V. R. Vaidya, and S. J. Asirvatham, “Reversing anticoag- ulant effects of novel oral anticoagulants: Role of ciraparantag, andexanetalfa, and idarucizumab,” Vascular Health and Risk Management, vol. 12, pp. 35–44, 2016.
https://openalex.org/W3098846484
https://www.ams.org/proc/2011-139-05/S0002-9939-2010-10632-9/S0002-9939-2010-10632-9.pdf
English
null
Mertens’ theorem for toral automorphisms
Proceedings of the American Mathematical Society
2,011
public-domain
3,086
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) ( ) [3] G. Everest and T. Ward. Heights of polynomials and entropy in algebraic dynamics. Springer- Verlag, London, 1999. MR1700272 (2000e:11087) ( ) [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
https://openalex.org/W2994930907
https://europepmc.org/articles/pmc6950032?pdf=render
English
null
Characterizing Unsafe Sexual Behavior among Factory Workers in the Context of Rapid Industrialization in Northern Vietnam
International journal of environmental research and public health/International journal of environmental research and public health
2,019
cc-by
6,584
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., Q.N.N., C.A.L., C.S.H.H., and R.C.M.H. References HIV risk behaviours among contracted and non-contracted male migrant workers in India: Potential role of 18. Saggurti, N.; Verma, R.K.; Jain, A.; RamaRao, S.; Kumar, K.A.; Subbiah, A.; Modugu, H.R.; Halli, S.; Bharat, S. HIV risk behaviours among contracted and non-contracted male migrant workers in India: Potential role of labour contractors and contractual systems in HIV prevention. AIDS 2008, 22, S127–S136. [CrossRef] 19. Li, X.; Zhang, L.; Stanton, B.; Fang, X.; Xiong, Q.; Lin, D. HIV/AIDS-related sexual risk behaviors among rural residents in China: Potential role of rural-to-urban migration. AIDS Educ. Prev. 2007, 19, 396–407. [CrossRef] 19. Li, X.; Zhang, L.; Stanton, B.; Fang, X.; Xiong, Q.; Lin, D. HIV/AIDS-related sexual risk behaviors among rural residents in China: Potential role of rural-to-urban migration. AIDS Educ. Prev. 2007, 19, 396–407. [CrossRef] 20. Rojanapithayakorn, W. The 100% condom use programme in Asia. Reprod. Health Matters 2006, 14, 41–52. [C R f] 20. Rojanapithayakorn, W. The 100% condom use programme in Asia. Reprod. Health Matters 2006, 14, 41–52. [CrossRef] 21. Hong, S.A.; Thepthien, B.-o. HIV risk-related sexual behavior by cohabiting partner status among factory workers: Results from the 2015 Bangkok behavioral surveillance survey (BSS). Cogent Soc. Sci. 2017, 3, 1364070. [CrossRef] 22. Hu, Z.; Liu, H.; Li, X.; Stanton, B.; Chen, X. HIV-related sexual behaviour among migrants and non-migrants in a rural area of China: Role of rural-to-urban migration. Public Health 2006, 120, 339–345. [CrossRef] [PubMed] 23. Ishtiaque, A.; Ullah, M.S. The Influence of Factors of Migration on the Migration Status of Rural-Urban Migrants in Dhaka, Bangladesh. Hum. Geogr. 2013, 7, 45–52. [CrossRef] 24. Yang, H.; Li, X.; Stanton, B.; Fang, X.; Lin, D.; Mao, R.; Liu, H.; Chen, X.; Severson, R. Workplace and HIV-related sexual behaviours and perceptions among female migrant workers. AIDS Care 2005, 17, 819–833. [CrossRef] 25. Wang, B.; Li, X.; Stanton, B.; Fang, X.; Liang, G.; Liu, H.; Lin, D.; Yang, H. Gender differences in HIV-related perceptions, sexual risk behaviors, and history of sexually transmitted diseases among Chinese migrants visiting public sexually transmitted disease clinics. AIDS Patient Care STDS 2007, 21, 57–68. [CrossRef] 26. Do, M.; Fu, H. Is women’s self-efficacy in negotiating sexual decisionmaking associated with condom use in marital relationships in Vietnam? Stud. Fam. Plann. 2011, 42, 273–282. [CrossRef] 27. Bui, T.C.; Markham, C.M.; Ross, M.W.; Williams, M.L.; Beasley, R.P.; Tran, L.T.; Nguyen, H.T.; Le, T.N. References HIV-Risk Behavior Among the Male Migrant Factory Workers in a North Indian City. Indian J. Community Med. Off. Publ. Indian Assoc. Prev. Soc. Med. 2015, 40, 108–115. [CrossRef] 8. Chamratrithirong, A.; Ford, K.; Punpuing, S.; Prasartkul, P. A workplace intervention program and the increase in HIV knowledge, perceived accessibility and use of condoms among young factory workers in Thailand. SAHARA J. 2017, 14, 132–139. [CrossRef] 9. Mucci, N.; Traversini, V.; Giorgi, G.; Garzaro, G.; Fiz Perez, J.; Campagna, M.; Rapisarda, V.; Tommasi, E.; Montalti, M.; Arcangeli, G. Migrant workers and physical health: An umbrella review. Sustainability 2019, 11, 232. [CrossRef] 10. Puri, M.; Cleland, J. Sexual behavior and perceived risk of hiv/aids among young migrant factory workers in nepal. J. Adolesc. Health Off. Publ. Soc. Adolesc. Med. 2006, 38, 237–246. [CrossRef] 11. Dai, W.; Gao, J.; Gong, J.; Xia, X.; Yang, H.; Shen, Y.; Gu, J.; Wang, T.; Liu, Y.; Zhou, J.; et al. Sexual behavior of migrant workers in Shanghai, China. BMC Public Health 2015, 15, 1067. [CrossRef] 12. Mondal, M.N.; Hoque, M.N.; Khan Chowdhury, M.R.; Moni, S.; Howard, J.; Rahman Choudhury, S. Risky Sexual Behaviors and HIV Vulnerability of Male Migrant Workers in Rajshahi City, Bangladesh. Epidemiol. Open Access 2014, 4, 160. 13. Nguyen, V.H.; Dunne, M.P.; Debattista, J.; Hien Nguyen, T.; Minh An Dao, T. Social Contexts of Risk Behaviors for HIV Among Male, Unskilled, Unregistered Laborers in Urban Vietnam. Qual. Health Res. 2011, 22, 871–879. [CrossRef] [PubMed] 14. Tran, A.N.; Nørlund, I. Globalization, industrialization, and labor markets in Vietnam. J. Asia Pac. Econ. 2015, 20, 143–163. [CrossRef] 15. Bradley, K.A.; DeBenedetti, A.F.; Volk, R.J.; Williams, E.C.; Frank, D.; Kivlahan, D.R. AUDIT-C as a brief screen for alcohol misuse in primary care. Alcohol. Clin. Exp. Res. 2007, 31, 1208–1217. [CrossRef] [PubMed] Int. J. Environ. Res. Public Health 2019, 16, 5085 10 of 11 10 of 11 16. Tran, B.X.; Nguyen, H.L.T.; Le, Q.N.H.; Mai, H.T.; Ngo, C.; Hoang, C.D.; Nguyen, H.H.; Le, H.Q.; Van Nguyen, H.; Le, H.T.; et al. Alcohol and tobacco use among methadone maintenance patients in Vietnamese rural mountainside areas. Addict. Behav. Rep. 2018, 7, 19–25. [CrossRef] [PubMed] 7. Tran, B.X.; Ohinmaa, A.; Nguyen, L.T. Quality of life profile and psychometric properties of the EQ-5D-5 HIV/AIDS patients. Health Qual. Outcomes 2012, 10, 132. [CrossRef] 18. Saggurti, N.; Verma, R.K.; Jain, A.; RamaRao, S.; Kumar, K.A.; Subbiah, A.; Modugu, H.R.; Halli, S.; Bharat, S. References Perceived gender inequality, sexual communication self-efficacy, and sexual behaviour among female undergraduate students in the Mekong Delta of Vietnam. Sex Health 2012, 9, 314–322. [CrossRef] 28. Tran, T.D.H.; Tuan, D.K.; Anh, N.D.; Le, T.K.A.; Bui, T.T.H. Premarital sex, contraceptive use among unmarried women migrant workers in industrial parks in Vietnam, 2015. Health Care Women Int. 2018, 39, 377–388. [CrossRef] 29. Wolffers, I.; Fernandez, I.; Verghis, S.; Vink, M. Sexual behavior and vulnerability of migrant workers for HIV infection. Cult. Health Sex. 2002, 4, 459–473. [CrossRef] 30. Tiruneh, K.; Wasie, B.; Gonzalez, H. Sexual behavior and vulnerability to HIV infection among seasonal migrant laborers in Metema district, northwest Ethiopia: A cross-sectional study. BMC Public Health 2015, 15, 122. [CrossRef] [ ] 31. Weine, S.M.; Kashuba, A.B. Labor migration and HIV risk: A systematic review of the literature. AIDS Behav. 2012, 16, 1605–1621. [CrossRef] 32. Chen, Y.; Li, X.; Zhang, C.; Hong, Y.; Zhou, Y.; Liu, W. Alcohol use and sexual risks: Use of the Alcohol Use Disorders Identification Test (AUDIT) among female sex workers in China. Health Care Women Int. 2013, 34, 122–138. [CrossRef] [PubMed] 33. Singh, S.K.; Schensul, J.J.; Gupta, K.; Maharana, B.; Kremelberg, D.; Berg, M. Determinants of alcohol use, risky sexual behavior and sexual health problems among men in low income communities of Mumbai, India. AIDS Behav. 2010, 14, S48–S60. [CrossRef] [PubMed] 34. Cooper, M.L. Alcohol use and risky sexual behavior among college students and youth: Evaluating the evidence. J. Stud. Alcohol Suppl. 2002, 14, 101–117. [CrossRef] [PubMed] 35. Fortenberry, J.D.; Orr, D.P.; Katz, B.P.; Brizendine, E.J.; Blythe, M.J. Sex under the influence. A diary self-report study of substance use and sexual behavior among adolescent women. Sex. Transm. Dis. 1997, 24, 313–319. [CrossRef] [PubMed] Int. J. Environ. Res. Public Health 2019, 16, 5085 11 of 11 36. Li, Q.; Li, X.; Stanton, B. Alcohol use among female sex workers and male clients: An integrative review of global literature. Alcohol Alcohol. 2010, 45, 188–199. [CrossRef] 37. Tran, B.X.; Nguyen, L.H.; Nguyen, C.T.; Phan, H.T.; Latkin, C.A. Alcohol abuse increases the risk of HIV infection and diminishes health status of clients attending HIV testing services in Vietnam. Harm. Reduct. J. 2016, 13, 6. [CrossRef] 38. Carey, K.B.; Senn, T.E.; Walsh, J.L.; Scott-Sheldon, L.A.; Carey, M.P. Alcohol Use Predicts Number of Sexual Partners for Female but not Male STI Clinic Patients. AIDS Behav. 2016, 20, S52–S59. [CrossRef] 39. © 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/). References Khan, M.R.; Kaufman, J.S.; Pence, B.W.; Gaynes, B.N.; Adimora, A.A.; Weir, S.S.; Miller, W.C. Depression, sexually transmitted infection, and sexual risk behavior among young adults in the United States. Arch. Pediatr. Adolesc. Med. 2009, 163, 644–652. [CrossRef] 40. Rao, U. Links between depression and substance abuse in adolescents: Neurobiological mechanisms. Am. J. Prev. Med. 2006, 31, S161–S174. [CrossRef] 41. Ngo, A.D.; Ha, T.H.; Rule, J.; Dang, C.V. Peer-based education and the integration of HIV and Sexual and Reproductive Health services for young people in Vietnam: evidence from a project evaluation. PLoS ONE 2013, 8, e80951. [CrossRef] © 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/).
https://openalex.org/W4321248200
https://journals.iucr.org/e/issues/2023/03/00/jq2025/jq2025.pdf
English
null
Synthesis, crystal structure and thermal properties of di-μ-iodido-bis[bis(2-chloropyrazine-κ<i>N</i>)copper(I)]
Acta crystallographica. Section E, Crystallographic communications
2,023
cc-by
4,869
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
https://openalex.org/W3210221540
https://www.researchsquare.com/article/rs-900422/latest.pdf
English
null
The Exotic Materia Medica of Chinese Medicine: A Survey of Its Resources and Medical Information in 1029 BC–1999 AD
Research Square (Research Square)
2,021
cc-by
4,563
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
https://openalex.org/W4322504123
https://escholarship.org/content/qt7rg6q8nt/qt7rg6q8nt.pdf?t=rxqqhs
English
null
Intradiscal treatment of the cartilage endplate for improving solute transport and disc nutrition
Frontiers in bioengineering and biotechnology
2,023
cc-by
8,917
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 applications of collagenase (metalloproteases): A review. Asian Pac. J. Trop. Biomed. 6, 975–981. doi:10.1016/j.apjtb.2016.07.017 Lotz, J. C., Fields, A. J., and Liebenberg, E. C. (2013). The role of the vertebral end plate in low back pain. Glob. Spine J. 3, 153–163. doi:10.1055/s-0033-1347298 Maroudas, A., Stockwell, R. A., Nachemson, A., and Urban, A. J. (1975). Factors involved in the nutrition of the human lumbar intervertebral disc: Cellularity and diffusion of glucose in vitro. J. Anat. 120, 113–130. Antoniou, J., Goudsouzian, N. M., Heathfield, T. F., Winterbottom, N., Steffen, T., Poole, A. R., et al. (1996). The human lumbar endplate. Spine (Phila. Pa. 1976) 21, 1153–1161. doi:10.1097/00007632-199605150-00006 Matta, A., Karim, M. Z., Isenman, D. E., and Erwin, W. M. (2017). Molecular therapy for degenerative disc disease: Clues from secretome analysis of the notochordal cell-rich nucleus pulposus. Sci. Rep. 7, 45623. doi:10.1038/srep45623 Bae, W. C., and Masuda, K. (2011). Emerging technologies for molecular therapy for intervertebral disk degeneration. Orthopedic Clinics of North America. 42, 585, doi:10. 1016/j.ocl.2011.07.004 Moore, R. J. (2006). “The vertebral endplate: Disc degeneration, disc regeneration,” in European spine journal (Springer, Germany). doi:10.1007/s00586-006-0170-4 Benneker, L. M., Heini, P. F., Alini, M., Anderson, S. E., and Ito, K. (2005). 2004 Young investigator award winner: Vertebral endplate marrow contact channel occlusions and intervertebral disc degeneration. Spine (Phila. Pa. 1976) 30, 167–173. doi:10.1097/01.brs.0000150833.93248.09 Nachemson, A., Lewin, T., Maroudas, A., and Freeman, M. A. R. (1970). In vitro diffusion of DYE through the end-plates and the annulus fibrosus of human lumbar inter-vertebral discs. Acta Orthop. 41, 589–607. doi:10.3109/17453677008991550 Binch, A. L. A., Fitzgerald, J. C., Growney, E. A., and Barry, F. (2021). Cell-based strategies for IVD repair: Clinical progress and translational obstacles. Nat. Rev. Rheumatol. 17 (3), 158–175. doi:10.1038/s41584-020-00568-w Nachemson, A. M. D. (1966). The load on lumbar disks in different positions of the body. Clin. Orthop. Relat. Res. March 45, 107–122. doi:10.1097/00003086-196600450- 00014 Bonnheim, N. B., Wang, L., Lazar, A. A., Zhou, J., Chachad, R., Sollmann, N., et al. (2022). The contributions of cartilage endplate composition and vertebral bone marrow fat to intervertebral disc degeneration in patients with chronic low back pain. Eur. Spine J. 31, 1866–1872. doi:10.1007/s00586-022-07206-x Nagase, H., and Kashiwagi, M. (2003). Aggrecanases and cartilage matrix degradation. Arthritis Res. Ther. 5, 94–103. doi:10.1186/ar630 Nosikova, Y. S., Santerre, J. P., Grynpas, M., Gibson, G., and Kandel, R. A. (2012). References Rep. 4, 151–160. doi:10.1007/s40610-018-0105-y Urban, J. P., Holm, S., Maroudas, A., and Nachemson, A. (1977). Nutrition of the intervertebral disk. An in vivo study of solute transport. Clin. Orthop. Relat. Res., 101–114. Fields, A. J., Lee, G. L., and Keaveny, T. M. (2010). Mechanisms of initial endplate failure in the human vertebral body. J. Biomechanics 43, 3126–3131. doi:10.1016/j. jbiomech.2010.08.002 Fields, A. J., Rodriguez, D., Gary, K. N., Liebenberg, E. C., and Lotz, J. C. (2014). Influence of biochemical composition on endplate cartilage tensile properties in the human lumbar spine. J. Orthop. Res. 32, 245–252. doi:10.1002/jor.22516 Urban, J. P. G., Smith, S., and Fairbank, J. C. T. (2004). Nutrition of the intervertebral disc. Spine (Phila. Pa. 1976) 29, 2700–2709. Vassilaki, M., and Hurwitz Dc, E. L. (2014). Insights in public health: Perspectives on pain in the low back and neck: Global burden, epidemiology, and management. Hawai’i J. Med. public health a J. Asia Pac. Med. Public Health 73 (4), 122–126. Gawri, R., Antoniou, J., Ouellet, J., Awwad, W., Steffen, T., Roughley, P., et al. (2013). Best paper NASS 2013: Link-N can stimulate proteoglycan synthesis in the degenerated human intervertebral discs. Eur. Cells Mater. 26, 107–119. doi:10.22203/eCM.v026a08 Velikonja, N. K., Urban, J., Frohlich, M., Neidlinger-Wilke, C., Kletsas, D., Potocar, U., et al. (2014). “Cell sources for nucleus pulposus regeneration,” in European spine journal: Official publication of the European spine society, the European spinal deformity society, and the European section of the cervical spine research society Vol Suppl 3, S364–74. doi:10.1007/s00586-013-3106-9 Grant, M., Epure, L., Bokhari, R., Roughle, P., Antoniou, J., and Mwale, F. (2016). Human cartilaginous endplate degeneration is induced by calcium and the extracellular calcium-sensing receptor in the intervertebral disc. Eur. Cells Mater 32, 137–151. doi:10. 22203/eCM.v032a09 Haralson, R. H., Iii, and Zuckerman, J. D. (2009). Prevalence, health care expenditures, and orthopedic surgery workforce for musculoskeletal conditions. JAMA 302, 1586–1587. doi:10.1001/jama.2009.1489 Wang, L., Han, M., Wong, J., Zheng, P., Lazar, A. A., Krug, R., et al. (2021). Evaluation of human cartilage endplate composition using MRI: Spatial variation, association with adjacent disc degeneration, and in vivo repeatability. J. Orthop. 39, 1470–1478. doi:10. 1002/jor.24787 Horner,H.A.,and Urban,J.P.(2001).2001volvo awardwinnerinbasic sciencestudies:Effect of nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral disc. Spine (Phila Pa 1976) 26 (23), 2543–2549. doi:10.1097/00007632-200112010-00006 Wilke, H. J., Neef, P., Caimi, M., Hoogland, T., and Claes, L. E. (1999). Ethics statement The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1111356/ full#supplementary-material This study utilized bovine coccygeal discs that were obtained from a local abattoir and human cadaveric lumbar discs that were 08 Frontiers in Bioengineering and Biotechnology Frontiers in Bioengineering and Biotechnology frontiersin.org 10.3389/fbioe.2023.1111356 Habib et al. References Characterization of the annulus fibrosus-vertebral body interface: Identification of new structural features. J. Anat. 221, 577–589. doi:10.1111/j.1469-7580.2012.01537.x Chou, D., Samartzis, D., Bellabarba, C., Patel, A., Luk, K. D. K., Kisser, J. M. S., et al. (2011). Degenerative magnetic resonance imaging changes in patients with chronic low back pain: A systematic review. Spine (Phila Pa 1976). 36, S43, doi:10.1097/BRS.0b013e31822ef700 Ohshima, H., Tsuji, H., Hirano, N., Ishihara, H., Katoh, Y., and Yamada, H. (1989). Water diffusion pathway, swelling pressure, and biomechanical properties of the intervertebral disc during compression load. Spine (Phila. Pa. 1976) 14, 1234–1244. DeLucca, J. F., Cortes, D. H., Jacobs, N. T., Vresilovic, E. J., Duncan, R. L., and Elliott, D. M. (2016). Human cartilage endplate permeability varies with degeneration and intervertebral disc site. J. Biomechanics 49, 550–557. doi:10.1016/j.jbiomech.2016. 01.007 Roberts, S., Menage, J., Duance, V., Wotton, S., and Ayad, S. 1991. Volvo Award in basic sciences. Collagen types around the cells of the intervertebral disc and cartilage end plate: An immunolocalization study. Spine 16 (9), 1030–8. Dolor, A., Sampson, S. L., Lazar, A. A., Lotz, J. C., Szoka, F. C., and Fields, A. J. (2019). Matrix modification for enhancing the transport properties of the human cartilage endplate to improve disc nutrition. PLoS ONE 14, e0215218. doi:10.1371/journal.pone. 0215218 Roberts, S., Urban, J. P., Evans, H., and Eisenstein, S. M. (1996). Transport properties of the human cartilage endplate in relation to its composition and calcification. Spine 21 (4), 415–420. doi:10.1097/00007632-199602150-00003 Farndale, R. W., Buttle, D. J., and Barrett, A. J. (1986). Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta 883, 173–177. doi:10.1016/0304-4165(86)90306-5 Rodriguez, A. G., Slichter, C. K., Acosta, F. L., Rodriguez-Soto, A. E., Burghardt, A. J., Majumdar, S., et al. (2011). Human disc nucleus properties and vertebral endplate permeability. Spine (Phila Pa 1976) 36, 512–520. doi:10.1097/BRS.0b013e3181f72b94 Sampson, S. L., Sylvia, M., and Fields, A. J. (2019). Effects of dynamic loading on solute transport through the human cartilage endplate. J. Biomechanics 83, 273–279. doi:10.1016/j.jbiomech.2018.12.004 Fernandez-Moure, J., Moore, C. A., Kim, K., Karim, A., Smith, K., Barbosa, Z., et al. (2018). Novel therapeutic strategies for degenerative disc disease: Review of cell biology and intervertebral disc cell therapy. SAGE Open Med. 6, 205031211876167. doi:10.1177/ 2050312118761674 Schollmeier, G., Lahr-Eigen, R., and Lewandrowski, K. U. (1976). Observations on fiber-forming collagens in the anulus fibrosus. Spine (Phila Pa 25 (21), 2736–2741. doi:10.1097/00007632-200011010-00004 Fields,A.J.,Ballatori,A.,Liebenberg,E.C.,andLotz,J.C.(2018).Contributionoftheendplates to disc degeneration. Curr. Mol. Biol. Frontiers in Bioengineering and Biotechnology References New in vivo measurements of pressures in the intervertebral disc in daily life. Spine (Phila Pa 1976) 24 (8), 755–762. doi:10.1097/00007632-199904150-00005 Huang, Y. C., Urban, J. P. G., and Luk, K. D. K. (2014). Intervertebral disc regeneration: Do nutrients lead the way? Nat. Rev. Rheumatol. 10, 561–566. doi:10. 1038/nrrheum.2014.91 Wong, J., Sampson, S. L., Bell-Briones, H., Ouyang, A., Lazar, A. A., Lotz, J. C., et al. (2019). Nutrient supply and nucleus pulposus cell function: Effects of the transport properties of the cartilage endplate and potential implications for intradiscal biologic therapy. Osteoarthr. Cartil. 27, 956–964. doi:10.1016/j.joca. 2019.01.013 Jeon, O., Shin, J. Y., Marks, R., Hopkins, M., Kim, T. H., Park, H. H., et al. (2017). Highly elastic and tough interpenetrating polymer network-structured hybrid hydrogels for cyclic mechanical loading-enhanced tissue engineering. Chem. Mater. 29, 8425–8432. doi:10.1021/acs.chemmater.7b02995 Woods, B. I., Vo, N., Sowa, G., and Kang, J. D. (2011). Gene therapy for intervertebral disk degeneration. Orthop. Clin. North Am. 42 (4), 563–74. doi:10.1016/j.ocl.2011. 07.002 Jiang, C. C., Hsieh, C. H., Liao, C. J., Chang, W. H., Liao, W. J., Tsai-Wu, J. J., et al. (2020). Collagenase treatment of cartilaginous matrix promotes fusion of adjacent cartilage. Regen. Ther. 15, 97–102. doi:10.1016/j.reth.2020.05.006 Zhu, Q., Gao, X., Temple, H. T., Brown, M. D., and Gu, W. (2016). Simulation of biological therapies for degenerated intervertebral discs. J. Orthop. Res. 34 (4), 699–708. doi:10.1002/jor.23061 Ju, D. G., Kanim, L. E., and Bae, H. W. (2022). Is there clinical improvement associated with intradiscal therapies? A comparison across randomized controlled studies. Global Spine J. 12 (5), 756–764. doi:10.1177/2192568220963058 Frontiers in Bioengineering and Biotechnology 09 frontiersin.org
https://openalex.org/W3124652729
https://skeletalmusclejournal.biomedcentral.com/counter/pdf/10.1186/s13395-021-00261-w
English
null
A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment
Skeletal muscle
2,021
cc-by
9,156
© 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 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. Benedetti et al. Skeletal Muscle (2021) 11:7 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 Clin Investig. 2014;124:4257–65. 33. 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 Clin Investig. 2014;124:4257–65. 9. Tedesco FS, Dellavalle A, Diaz-Manera J, Messina G, Cossu G. Repairing skeletal muscle: Regenerative potential of skeletal muscle stem cells. J Clin Investig. 2010;120:11–9. 34. Sonna L, Fujita J, Gaffin SL, Craig M. Molecular Biology of Thermoregulation Invited Review: Effects of heat and cold stress on mammalian gene expression. J Appl Physiol. 2002;92:1725–42. 34. Sonna L, Fujita J, Gaffin SL, Craig M. Molecular Biology of Thermoregulation Invited Review: Effects of heat and cold stress on mammalian gene expression. J Appl Physiol. 2002;92:1725–42. 10. Keire P, Shearer A, Shefer G, Yablonka-Reuveni Z. Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol. 2013;946:431–68. 35. Judson RN, Quarta M, Oudhoff MJ, Soliman H, Yi L, Chang CK, et al. Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential. Cell Stem Cell. 2018;22:177–90. 11. Danoviz ME, Yablonka-Reuveni Z. Skeletal muscle satellite cells: Background and methods for isolation and analysis in a primary culture system. Methods Mol Biol. 2012;798:21–52. 36. Ito T, Sawada R, Fujiwara Y, Seyama Y, Tsuchiya T. FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF- β2. Biochem Biophys Res Commun. 2007;359:108–14. 12. Jonah D, Lee BCS, Lisa M, Larkin KWV. Isolation and Purification of Satellite Cells for Skeletal Muscle Tissue Engineering. J Regen Med. 2015;3:117. 13. Rinaldi F, Perlingeiro RCR. Stem cells for skeletal muscle regeneration: Therapeutic potential and roadblocks. Transl Res. 2014. Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Page 12 of 12 Page 12 of 12 Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle (2021) 11:7 Benedetti et al. Skeletal Muscle (2021) 11:7 Received: 21 December 2020 Accepted: 18 February 2021 Received: 21 December 2020 Accepted: 18 February 2021 Received: 21 December 2020 Accepted: 18 February 2021 27. Juliano RL, Gagalang E. The adhesion of Chinese hamster cells. I. Effects of temperature, metabolic inhibitors and proteolytic dissection of cell surface macromolecules. J Cell Physiol. 1977;92:209–20. The authors declare that they have no competing interests. The authors declare that they have no competing interests. 3. Benedetti A, Fiore PF, Madaro L, Lozanoska-Ochser B, Bouché M. T 23. Benedetti A, Fiore PF, Madaro L, Lozanoska-Ochser B, Bouché M. Targeting pkcθ promotes satellite cell self-renewal. Int J Mol Sci. 2020;21:2419. Competing interests 22. Blanco-Bose WE, Yao CC, Kramer RH, Blau HM. Purification of mouse primary myoblasts based on α7 integrin expression. Exp Cell Res. 2001;265:212–20. Author details 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 muscle injury. Int J Mol Sci. 2020;21:932. 25. Rizzo G, di Maggio R, Benedetti A, Morroni J, Bouche M, Lozanoska-Ochser B. Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair. JCI Insight. 2020;5:e130807. 3Department of Orthopaedics and Traumatology, Policlinico Umberto I, Rome, Italy. 3Department of Orthopaedics and Traumatology, Policlinico Umberto I, Rome, Italy. 26. Lozanoska-Ochser B, Benedetti A, Rizzo G, Marrocco V, di Maggio R, Fiore P, et al. Targeting early PKCθ-dependent T-cell infiltration of dystrophic muscle reduces disease severity in a mouse model of muscular dystrophy. J Pathol. 2018;244:323–333. References 1. Chang NC, Rudnicki MA. Satellite Cells: The Architects of Skeletal Muscle. Curr Top Dev Biol. 2014;107:161–81. 28. Rico F, Chu C, Abdulreda MH, Qin Y, Moy VT. Temperature modulation of integrin-mediated cell adhesion. Biophys J. 2010;99:1387–96. 2. Wang YX, Dumont NA, Rudnicki MA. Muscle stem cells at a glance. J Cell Sci. 2014;127:4543–8. 2. Wang YX, Dumont NA, Rudnicki MA. Muscle stem cells at a glance. J Cell Sci. 2014;127:4543–8. 29. Machida S, Spangenburg EE, Booth FW. Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages. Cell Prolif. 2004;37:267–77. 3. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. 1961;9:493–5. 3. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. 1961;9:493–5. 4. Wang YX, Rudnicki MA. Satellite cells, the engines of muscle repair. Nat Rev Mol Cell Biol. 2012;13:127–33. 4. Wang YX, Rudnicki MA. Satellite cells, the engines of muscle repair. Nat Rev Mol Cell Biol. 2012;13:127–33. 30. Tierney MT, Sacco A. Satellite Cell Heterogeneity in S Homeostasis. Trends Cell Biol. 2016;26:434–444. 0. Tierney MT, Sacco A. Satellite Cell Heterogeneity in Skeletal Musc Homeostasis. Trends Cell Biol. 2016;26:434–444. 5. Evano B, Tajbakhsh S. Skeletal muscle stem cells in comfort and stress. npj Regen Med. 2018;3:24. 5. Evano B, Tajbakhsh S. Skeletal muscle stem cells in comfort and stress. npj Regen Med. 2018;3:24. 31. Ono Y, Masuda S, Nam HS, Benezra R, Miyagoe-Suzuki Y, Takeda S. Slow- dividing satellite cells retain long-term self-renewal ability in adult muscle. J Cell Sci. 2012;125:1309–17. 6. Feige P, Rudnicki MA. Muscle stem cells. Curr Biol. 2018;28:581–98. 7. Sambasivan R, Yao R, Kissenpfennig A, van Wittenberghe L, Paldi A, Gayraud-Morel B, et al. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development. 2011;138:3647–56. 7. Sambasivan R, Yao R, Kissenpfennig A, van Wittenberghe L, Paldi A, Gayraud-Morel B, et al. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development. 2011;138:3647–56. 32. Charville GW, Cheung TH, Yoo B, Santos PJ, Lee GK, Shrager JB, et al. Ex vivo expansion and in vivo self-renewal of human muscle stem cells. Stem Cell Reports. 2015;5:621–32. 8. Relaix F, Zammit PS. Satellite cells are essential for skeletal muscle regeneration: The cell on the edge returns centre stage. Development (Cambridge). 2012;139:2845–56. 8. Relaix F, Zammit PS. Satellite cells are essential for skeletal muscle regeneration: The cell on the edge returns centre stage. Development (Cambridge). 2012;139:2845–56. 33. Publisher’s Note 14. Gharaibeh B, Lu A, Tebbets J, Zheng B, Feduska J, Crisan M, et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc. 2008;163:409–17. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 15. Yoshioka K, Kitajima Y, Okazaki N, Chiba K, Yonekura A, Ono Y. A Modified Pre-plating Method for High-Yield and High-Purity Muscle Stem Cell Isolation From Human/Mouse Skeletal Muscle Tissues. Front Cell Dev Biol. 2020;8:793. 16. Pasut A, Oleynik P, Rudnicki MA. Isolation of muscle stem cells by fluorescence activated cell sorting cytometry. Methods Mol Biol. 2012;798: 53–64. 17. Liu L, Cheung TH, Charville GW, Rando TA. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat Protoc. 2015;10:1612–24. 18. Chapman MR, Balakrishnan KR, Li J, Conboy MJ, Huang H, Mohanty SK, et al. Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity. Integr Biol (United Kingdom). 2013;5:692–702. 19. Fukada SI, Higuchi S, Segawa M, Koda KI, Yamamoto Y, Tsujikawa K, et al. Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody. Exp Cell Res. 2004;296:245–55. 20. Sherwood RI, Christensen JL, Conboy IM, Conboy MJ, Rando TA, Weissman IL, et al. Isolation of adult mouse myogenic progenitors: Functional heterogeneity of cells within and engrafting skeletal muscle. Cell. 2004;119: 543–54. 21. Montarras D, Morgan J, Collins C. Direct Isolation of Satellite Cells for Skeletal Muscle Regeneration. Mol Cell Biol. 2005;309:2064–7.
https://openalex.org/W2657697978
https://www.int-res.com/articles/esep2017/17/e017p051.pdf
English
null
The maladies of enlightenment science
Ethics in science and environmental politics
2,017
cc-by
9,798
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 the hit parades of pop music. LITERATURE CITED Houghton Mifflin, Boston, MA Jackson M (2008) Distracted: the erosion of attention and the coming dark age. Prometheus Books, Amherst, NY Collins H, Pinch T (1993) The golem: what everyone should know about science. Cambridge University Press, Cam- bridge Katz J (1997) Birth of a digital nation. Wired 5 (1 April) www. wired.com/1997/04/netizen-3/ Dawkins R (1976) The selfish gene. Oxford University Press, Oxford Kearns CE, Schmidt LA, Glantz SA (2016) Sugar industry and coronary heart disease research: a historical analysis of internal industry. JAMA Intern Med 176: 1680−1685 Dawkins R (1996) The blind watchmaker: why the evidence of evolution reveals a universe without design. WW Nor- ton & Company, New York, NY Klein N (2000) No logo. Vintage Canada, Toronto Krimsky S (2003) Science in the private interest: Has the lure of profits corrupted biomedical research? Rowman & Lit- tlefield Publishers, New York, NY p y Eco E (1994) How to travel with a salmon and other essays. Harcourt, Orlando, FL Ernst E (2010) Homeopathy: What does the ‘best’ evidence tell us? Med J Aust 192: 458−460 Kuhn TS (1962) The structure of scientific revolutions. Uni- versity of Chicago Press, Chicago, IL Kuntz M (2012) The postmodern assault on science. If all truths are equal, who cares what science has to say? EMBO Rep 13: 885−889 Fanelli D (2012) Negative results are disappearing from most disciplines and countries. Scientometrics 90: 891−904 Feyerabend P (1988) Against method. Outline of an anar- chistic theory of knowledge. Verso, London Lakatos I, Feyerabend P (1999) For and against method. Uni- versity of Chicago Press, Chicago, IL Foucart S (2016) La discrète influence de Monsanto. Le Monde Science et Techno, 12.07.2016 Lave R, Mirowski P, Randalls S (2010) Introduction: STS and neoliberal science. Soc Stud Sci 40: 659−675 Fromm E (1964) The heart of man: its genius for good and evil. Harper & Row, New York, NY Lessig L (2004) Free culture: how big media uses technology and the law to lock down culture and control creativity. Penguin Press, New York, NY Funtowicz S, Ravetz JR (1993) Science for the post-normal age. Futures 25: 739−755 g Fuster Morell M (2015) Digital commons and free culture. Editorial Icaria, Barcelona Loehle C (1990) A guide to increased creativity in research —Inspiration or perspiration? Bioscience 40: 123−129 Long PO (2001) Openness, secrecy, authorship: technical arts and the culture of knowledge from antiquity to the renaissance. LITERATURE CITED p y Gray J (2009) False dawn: the delusions of global capitalism. Granta Books, New York, NY Agin D (2007) Junk science: how politicians, corporations, and other hucksters betray us. Macmillan Publishers, New York, NY Gray J (2014) The silence of animals. Penguin Books, London Anon (2005) The influence of the pharmaceutical industry, Vol 1. United Kingdom House of Commons Health Com- mittee Stationary Office, London Gross PR, Levitt N, Lewis MW (1996) The flight from science and reason. New York Academy of Sciences, New York, NY mittee Stationary Office, London Guazzo S (1925) The civile conversation of M. Steeven Guazzo. The first three books translated by George Pet- tie […] and the fourth by Barth. Young […] With an intro- duction by Sir Edward Sullivan. Tudor translations, Series 2, Vols. 7,8. Constable and Company, London Bauerlein M (2009) The dumbest generation: how the digital age stupefies young Americans and jeopardizes our future. The Penguin Group, New York, NY Bauwens M (2005) The political economy of peer produc- tion. www.ctheory.net/articles.aspx?id=499 Bes-Rastrollo M, Schulze MB, Ruiz-Canela M, Martinez- Gonzalez MA (2013) Financial conflicts of interest and reporting bias regarding the association between sugar- sweetened beverages and weight gain: a systematic review of systematic reviews. PLOS MED 10: e1001578 Hamilton WD (1995) Narrow roads of gene land, Vol 1: evo- lution of social behaviour. WH Freeman & Company, New York, NY Hey T (2006) Foreword. In: C Hine (ed) New infrastructures for knowledge production. Information Science Publish- ing, London, p vi−vii Biagioli M (2006) Galileo’s instruments of credit: telescopes, images, secrecy. University of Chicago Press, Chicago, IL Hobbes T (1668) Behemoth, or the long parliament. Simp- kin, Marshall, & Company, London Bok D (2003) Universities in the market place: the commer- cialization of higher education. Princeton University Press, Princeton, NJ Hollings K (2008) Welcome to Mars. Politics, pop culture and weird science in 1950s America. North Atlantic Books, Berkeley, CA Borges JL (1962) Labyrinths. New Directions Publishing, New York, NY Horton R (2004a) The Dawn of McScience. New York Rev Books 51: 7−9 Horton R (2004b) MMR science & fiction: exploring the vac- cine crisis. Granta Books, London Carey A (1997) Taking the risk out of democracy: corporate propaganda versus freedom and liberty. University of Illinois Press, Urbana, IL Huxley A (1932) Brave new world. Chatto & Windus, London Carson R (1962) Silent spring. DISCUSSION The need to accede to market demands is fulfilled by semantic tricks such as assertive sen- tence titles. The metaphysicians of Tlön in Jorge Luis Borges’ Labyrinths (Borges 1962) seek neither truth nor plausibility, but to astonish (‘Los metafísicos de Tlön no buscan la verdad ni siquiera la verosimilitud: buscan el asombro.’). Assertive titles ‘provide not only the product but the advertisement for it’ (Rosner 1990), and are frequently accompanied by insubstan- tial texts. Besides, anyone who offers certainty is cer- tainly wrong. A century ago, Veblen (1918, p. 9) wrote: ‘It is always possible, of course, that this pre-eminence of intellectual enterprise in the civilization of the West- ern peoples is a transient episode; that it may eventu- ally … perhaps even precipitately, with the next im - pending turn in the fortunes of this civilization … again be relegated to a secondary place in the scheme of things and become only an instrumentality in the service of some dominant aim or impulse, such as a vainglorious patriotism, or dynastic politics, or the breeding of a commercial aristocracy’. This analysis has been brought up to date by Bok (2003), Krimsky (2003), Mirowski (2011) and others. If scien- tists turn away from or shun the traditional ethos without adopting another equally fertile, science itself will languish and degenerate into a mere instrumentality, as Veblen suggested, of gold and iron, money and power. Distinctions between science, technology, and business management are increasingly confounded. Knowledge which formerly belonged to a universal commons is now commodified. As other Commons have been in the past, knowledge production is now a commercial enterprise. Productivity is measured by numbers of publications, by patents, and by biblio- metric indices. These measures effect a new kind of selection pressure on scientists minds, and the prod- uct is recognizably different from that of traditional science (Lynch 2014), and has a distinct aura. It dresses differently too. In our new increasingly online communication channels, the scientific con- tent sometimes seems to take a back seat to the medium, like a ‘juicy piece of meat carried by the Wyatt: Maladies of enlightenment science 61 Gould SJ (1987) An urchin in the storm: essays about books and ideas. WW Norton & Company, New York, NY LITERATURE CITED LITERATURE CITED John Hopkins University Press, London Gardner M (1952) Fads and fallacies in the name of science. GP Putnam’s Sons, New York, NY Gibbons M, Limoges C, Nowotny H, Schwartzman S, Scott P, Trow M (1994) The new production of knowledge: the dynamics of science and research in contemporary soci- eties. Sage Publications, London Lynch K (2014) New managerialism, neoliberalism and ranking. Ethics Sci Environ Polit 13: 141−153 Marx K (1846) The German ideology. Cited in: Tucker RC (ed) (1972) The Marx-Engels Reader. WW Norton & Company, New York, NY Goldacre B (2008) Bad science. Fourth Estate, London Gombrich EH (1979) Ideals & idols. A collection of essays on values and their place in humanity. Phaidon, London Gombrich EH (1979) Ideals & idols. A collection of essays on McHenry L (2010) Of sophists and spin-doctors: industry- sponsored ghostwriting and the crisis of academic medi- cine. Mens Sana Monogr 8: 129−145 values and their place in humanity. Phaidon, London Gould SJ (1981) The mismeasure of man. WW Norton & Company, New York, NY Ethics Sci Environ Polit 17: 51–62, 2017 62 Ravetz JR (1996) Scientific knowledge and its social prob- lems. Transaction Publishers, London McLuhan M (1962) Gutenberg galaxy: the making of typo- graphic man. University of Toronto Press, Toronto Resnik DB (2006) Openness versus secrecy in scientific research. Episteme 2: 135−147 Medawar P (1967) The art of the soluble. Oxford University Press, Oxford Medawar P (1977) Unnatural science. New York Rev Books 24: 13 Ritzer G (1998) The McDonaldization thesis: explorations and extensions. Sage Publications, London Ritzer G (2011) The McDonaldization of society, 6th edn. Sage Publications, London Medawar P (1986) Memoirs of a thinking radish. Oxford University Press, Oxford y Merton R (1942) A note on science and democracy. J Legal Polit Sociol 1: 115−126 Rosner JL (1990) Reflections of science as a product. Nature 345: 108 Russo L (1996) La rivoluzione dementicata. Feltrinelli, Milan Michaels D (2008) Doubt is their product: how industry’s assault on science threatens your health. Oxford Univer- sity Press, Oxford Schlain L (1998) The alphabet versus the goddess. The conflict between word and image. Penguin Books, London Mirowski P (2011) Science-mart: privatizing American science. Harvard University Press, Harvard, MA Shneiderman B (2008) Science 2.0. Science 319: 1349−1350 Shneiderman B (2016) The new ABCs of research: achieving breakthrough collaborations. LITERATURE CITED Oxford University Press, Oxford Moskovkin VM, Serkina OV (2016) Is sustainable develop- ment of scientific systems possible in the neo-liberal agenda? Ethics Sci Environ Polit 16: 1−9 Smaldino PE, McElreath R (2016) The natural selection of bad science. R Soc Open Sci 3: 160384 Moynihan R, Henry D (2006) The fight against disease mon- gering: generating knowledge for action. PLOS MED 3: e191 Smithson M (1989) Ignorance and uncertainty: emerging paradigms. Springer-Verlag, New York, NY Nentwich M (2003) Cyberscience: research in the age of the internet. Österreichische Akademie der Wissenschaften, Vienna Sprat T (1667) The history of the Royal Society of London, for the improving of natural knowledge. J Martyn, London Nietzsche F (1962) Philosophy in the tragic age of the Greeks. Regnery Publishing, Washington, DC Starr P (1982) The social transformation of American medi- cine. Basic Books, London Nosek BA, Spies JR, Motyl M (2012) Scientific utopia: II. Restructuring incentives and practices to promote truth over publishability. Perspect Psychol Sci 7: 615−631 Stocking SH, Holstein LW (2009) Manufacturing doubt: jour- nalists’ roles and the construction of ignorance in a scien- tific controversy. Public Underst Sci 18: 23−42 Nowotny H, Scott P, Gibbons M (2003) ‘Mode 2’ revisited: the new production of knowledge. Minerva 41: 179−194 Storetvedt KM (1997) Our evolving planet: earth history in a new perspective. Alma Mater Vorlag, Bergen Oreskes N (1999) The rejection of continental drift: theory and method in American earth science. Oxford Univer- sity Press, Oxford Tuchman G (2009) Wannabe U: inside the corporate univer- sity. University of Chicago Press, Chicago, IL Van Valen L (1973) A new evolutionary law. Evol Theory 1: 1−30 Oreskes N, Conway EM (2012) Merchants of doubt. Blooms- bury Press, London Pigliucci M (2002) Denying evolution: creationism, scien- tism, and the nature of science. Sinauer Associates, Sun- derland, MA Veblen T (1918) The higher learning in America. BW Huebsch, New York, NY Waddington CH (1948) The scientific attitude, 2nd edn. Pen- guin Books, London Polanyi M (1958) Personal knowledge: towards a post-criti- cal philosophy. University of Chicago Press, Chicago, IL Watermeyer R, Olssen M (2016) ‘Excellence’ and exclusion: the individual costs of institutional competitiveness. Minerva 54: 201−218 cal philosophy. University of Chicago Press, Chicago, IL Polanyi M (1974) Personal knowledge: towards a post-criti- cal philosophy. University of Chicago Press, Chicago, IL Popper KR (1959) The logic of scientific discovery. Submitted: February 8, 2017; Accepted: June 18, 2017 Proofs received from author(s): September 21, 2017 Editorial responsibility: Konstantinos Stergiou, Thessaloniki, Greece LITERATURE CITED Hutchin- son & Company, London Polanyi M (1974) Personal knowledge: towards a post-criti- cal philosophy. University of Chicago Press, Chicago, IL Polanyi M (1974) Personal knowledge: towards a post criti cal philosophy. University of Chicago Press, Chicago, IL Popper KR (1959) The logic of scientific discovery. Hutchin- son & Company, London Popper KR (1959) The logic of scientific discovery. Hutchin- son & Company, London Watson J (1968) The double helix. Weidenfeld & Nicolson, New York, NY Wouters PF (1996) Cyberscience. Kennis en Methode 20: 155−186 Pringle JR (2013) An enduring enthusiasm for academic sci- ence, but with concerns. Mol Biol Cell 24: 3281−3284 Proctor RN (1995) Cancer wars: how politics shapes what we know and don’t know about cancer. Basic Books, New York, NY Ziman J (1994) Prometheus bound: science in a dynamic steady state. Cambridge University Press, Cambridge Proctor RN (1995) Cancer wars: how politics shapes what we know and don’t know about cancer. Basic Books, New York, NY Ziman J (2000) Real science: What it is and what it means. Cambridge University Press, Cambridge Ramanathan VD (2002) A case of numerophilia. BMJ 325: 8 Editorial responsibility: Konstantinos Stergiou, Thessaloniki, Greece
https://openalex.org/W4395467360
https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-024-10312-3
English
null
Genome composition and GC content influence loci distribution in reduced representation genomic studies
BMC genomics
2,024
cc-by
10,591
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 performed statistical analyses. CP conducted the genomic category analyses. References Wang J, Qin J, Sun P, Ma X, Yu J, Li Y, et al. Polyploidy Index and its implications for the evolution of polyploids. Front Genet. 2019;10:807. 13. Hidalgo O, Pellicer J, Christenhusz M, Schneider H, Leitch AR, Leitch IJ. Is there an Upper limit to genome size? Trends Plant Sci. 2017;22:567–73. 39. Kress WJ, Soltis DE, Kersey PJ, Wegrzyn JL, Leebens-Mack JH, Gostel MR et al. Green plant genomes: What we know in an era of rapidly expanding oppor­ tunities. Proc Natl Acad Sci U S A. 2022;119. 14. Platt RN 2nd, Vandewege MW, Ray DA. Mammalian transposable elements d th i i t l ti Ch R 2018 26 25 43 14. Platt RN 2nd, Vandewege MW, Ray DA. Mammalian transposable elements and their impacts on genome evolution. Chromosome Res. 2018;26:25–43.i 40. Wendel JF, Jackson SA, Meyers BC, Wing RA. Evolution of plant genome architecture. Genome Biol. 2016;17:37.i 15. Wu C, Lu J. Diversification of transposable elements in Arthropods and its impact on genome evolution. Genes. 2019;10. 41. Ågren JA, Wright SI. Selfish genetic elements and plant genome size evolu­ tion. Trends Plant Sci. 2015;20:195–6. 16. Li Z, Tiley GP, Galuska SR, Reardon CR, Kidder TI, Rundell RJ, et al. Multiple large-scale gene and genome duplications during the evolution of hexa­ pods. Proc Natl Acad Sci U S A. 2018;115:4713–8. 42. Grau-Bové X, Ruiz-Trillo I, Irimia M. Origin of exon skipping-rich transcrip­ tomes in animals driven by evolution of gene architecture. Genome Biol. 2018;19:135. 17. Heckenhauer J, Frandsen PB, Sproul JS, Li Z, Paule J, Larracuente AM et al. Genome size evolution in the diverse insect order Trichoptera. Gigascience. 2022;11. 43. Sacerdot C, Louis A, Bon C, Berthelot C, Roest Crollius H. Chromosome evolution at the origin of the ancestral vertebrate genome. Genome Biol. 2018;19:166. 18. Amit M, Donyo M, Hollander D, Goren A, Kim E, Gelfman S, et al. Differential GC content between exons and introns establishes distinct strategies of splice-site recognition. Cell Rep. 2012;1:543–56. 44. Kapusta A, Suh A, Feschotte C. Dynamics of genome size evolution in birds and mammals. Proc Natl Acad Sci U S A. 2017;114:E1460–9.f 19. Haerty W, Ponting CP. Unexpected selection to retain high GC content and splicing enhancers within exons of multiexonic lncRNA loci. RNA. 2015;21:333–46. 45. Naville M, Henriet S, Warren I, Sumic S, Reeve M, Volff J-N, et al. References Massive changes of genome size driven by expansions of non-autonomous transpos able elements. Curr Biol. 2019;29:1161–e86. 20. Francis WR, Wörheide G. Similar ratios of introns to intergenic sequence across animal genomes. Genome Biol Evol. 2017;9:1582–98. 46. Elkon R, Agami R. Characterization of noncoding regulatory DNA in the human genome. Nat Biotechnol. 2017;35:732–46. 47. Borys SM, Younger ST. Identification of functional regulatory elements in the human genome using pooled CRISPR screens. BMC Genomics. 2020;21:107. 21. Zhu L, Zhang Y, Zhang W, Yang S, Chen J-Q, Tian D. Patterns of exon-intron architecture variation of genes in eukaryotic genomes. BMC Genomics. 2009;10:47. 48. Chueca LJ, Schell T, Pfenninger M. De novo genome assembly of the land snail Candidula unifasciata (Mollusca: Gastropoda). G3. 2021;11. 22. Rhie A, McCarthy SA, Fedrigo O, Damas J, Formenti G, Koren S, et al. Towards complete and error-free genome assemblies of all vertebrate species. Nature. 2021;592:737–46. 49. Belser C, Istace B, Denis E, Dubarry M, Baurens F-C, Falentin C, et al. Chromo­ some-scale assemblies of plant genomes using nanopore long reads and optical maps. Nat Plants. 2018;4:879–87. 23. Barbanti A, Torrado H, Macpherson E, Bargelloni L, Franch R, Carreras C et al. Helping decision making for reliable and cost-effective 2b-RAD sequencing and genotyping analyses in non-model species. Mol Ecol Resour. 2020;20. 50. Feng S, Stiller J, Deng Y, Armstrong J, Fang Q, Reeve AH, et al. Dense sam­ pling of bird diversity increases power of comparative genomics. Nature. 2020;587:252–7. 24. Marshall JJT, Halford SE. The type IIB restriction endonucleases. Biochem Soc Trans. 2010;38:410–6. 51. Chen Z, Omori Y, Koren S, Shirokiya T, Kuroda T, Miyamoto A, et al. De novo assembly of the goldfish (Carassius auratus) genome and the evolution of genes after whole-genome duplication. Sci Adv. 2019;5:eaav0547. 25. Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJM, Birol I. ABySS: a paral­ lel assembler for short read sequence data. Genome Res. 2009;19:1117–23. 52. Berthelot C, Brunet F, Chalopin D, Juanchich A, Bernard M, Noël B, et al. The rainbow trout genome provides novel insights into evolution after whole- genome duplication in vertebrates. Nat Commun. 2014;5:3657. 26. Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM. BUSCO Update: Novel and Streamlined Workflows along with broader and deeper phyloge­ netic Coverage for Scoring of Eukaryotic, Prokaryotic, and viral genomes. Mol Biol Evol. 2021;38:4647–54. 53. Glémin S, Clément Y, David J, Ressayre A. References lme4: linear mixed-effects models using S4 classes. http model and non-model species. PLoS ONE. 2012;7:e37135. 7. Davey JW, Blaxter ML. RADSeq: next-generation population genetics. Brief Funct Genomics. 2010;9:416–23. 33. Fox J, Weisberg S, Adler D, Bates D, Baud-Bovy G, Ellison S, et al. Package car. Volume 16. Vienna: R Foundation for Statistical Computing; 2012. 8. Galià-Camps C, Carreras C, Turon X, Pascual M. The impact of ada 8. Galià-Camps C, Carreras C, Turon X, Pascual M. The impact of adaptor selec­ tion on genotyping in 2b-RAD studies. Front Mar Sci. 2022;9. 34. Zhang D. rsq: R-squared and related measures. R package version. 2018. tion on genotyping in 2b-RAD studies. Front Mar Sci. 2022;9. y 9. Wang S, Meyer E, McKay JK, Matz MV. 2b-RAD: a simple and flexible method f id t i N t M th d 2012 9 808 10 9. Wang S, Meyer E, McKay JK, Matz MV. 2b-RAD: a simple and flexible method for genome-wide genotyping. Nat Methods. 2012;9:808–10. 9. Wang S, Meyer E, McKay JK, Matz MV. 2b-RAD: a simple and f 35. Lenth R, Singmann H, Love J, Buerkner P, Herve M. Emmeans: estimated marginal means. R package version 1.4. 4. Am Stat. 2020. for genome-wide genotyping. Nat Methods. 2012;9:808–10 marginal means. R package version 1.4. 4. Am Stat. 2020. g g y g 10. López A, Carreras C, Pascual M, Pegueroles C. Evaluating restriction enzyme 10. López A, Carreras C, Pascual M, Pegueroles C. Evaluating restriction enzyme selection for reduced representation sequencing in conservation genomics. Mol Ecol Resour. 2023. 10. López A, Carreras C, Pascual M, Pegueroles C. Evaluating rest 36. Qiao X, Li Q, Yin H, Qi K, Li L, Wang R, et al. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol. 2019;20:38. selection for reduced representation sequencing in conservation genomics. Mol Ecol Resour. 2023. 11. Hotaling S, Kelley JL, Frandsen PB. Toward a genome sequence for every animal: where are we now? Proc Natl Acad Sci U S A. 2021;118. 37. Li Z, McKibben MTW, Finch GS, Blischak PD, Sutherland BL, Barker MS. Pat­ terns and processes of diploidization in land plants. Annu Rev Plant Biol. 2021;72:387–410. 12. Campbell CR, Poelstra JW, Yoder AD. What is Speciation Genomics? The roles of ecology, gene flow, and genomic architecture in the formation of species. Biol J Linn Soc Lond. 2018;124:561–83. 38. References GC content evolution in coding regions of angiosperm genomes: a unifying hypothesis. Trends Genet. 2014;30:263–70. 27. Kumar S, Stecher G, Suleski M, Hedges SB. TimeTree: A Resource for timelines, timetrees, and divergence Times. Mol Biol Evol. 2017;34:1812–9.l 28. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26:841–2. genomic features. Bioinformatics. 2010;26:841–2. References 1. Formenti G, Theissinger K, Fernandes C, Bista I, Bombarely A, Bleidorn C, et al. The era of reference genomes in conservation genomics. Trends Ecol Evol. 2022;37:197–202. 2. Lewin HA, Richards S, Lieberman Aiden E, Allende ML, Archibald JM, Bálint M et al. The Earth BioGenome Project 2020: Starting the clock. Proc Natl Acad Sci U S A. 2022;119. 3. Manuzzi A, Zane L, Muñoz-Merida A, Griffiths AM, Veríssimo A. Population genomics and phylogeography of a benthic coastal shark (Scyliorhinus canicula) using 2b-RAD single nucleotide polymorphisms. Biol J Linn Soc Lond. 2019;126:289–303. 4. Guo C, Ma P-F, Yang G-Q, Ye X-Y, Guo Y, Liu J-X, et al. Parallel ddRAD and genome skimming analyses reveal a Radiative and Reticulate Evolutionary History of the Temperate bamboos. Syst Biol. 2021;70:756–73. 5. Torrado H, Carreras C, Raventos N, Macpherson E, Pascual M. Individual-based population genomics reveal different drivers of adaptation in sympatric fish. Sci Rep. 2020;10:12683. 1. Formenti G, Theissinger K, Fernandes C, Bista I, Bombarely A, Bleidorn C, et al. The era of reference genomes in conservation genomics. Trends Ecol Evol. 2022;37:197–202. 2. Lewin HA, Richards S, Lieberman Aiden E, Allende ML, Archibald JM, Bálint M et al. The Earth BioGenome Project 2020: Starting the clock. Proc Natl Acad Sci U S A. 2022;119.fi 3. Manuzzi A, Zane L, Muñoz-Merida A, Griffiths AM, Veríssimo A. Population genomics and phylogeography of a benthic coastal shark (Scyliorhinus canicula) using 2b-RAD single nucleotide polymorphisms. Biol J Linn Soc Lond. 2019;126:289–303. 4. Guo C, Ma P-F, Yang G-Q, Ye X-Y, Guo Y, Liu J-X, et al. Parallel ddRAD and genome skimming analyses reveal a Radiative and Reticulate Evolutionary History of the Temperate bamboos. Syst Biol. 2021;70:756–73. 5. Torrado H, Carreras C, Raventos N, Macpherson E, Pascual M. Individual-based population genomics reveal different drivers of adaptation in sympatric fish. Sci Rep. 2020;10:12683. Page 12 of 12 Galià-Camps et al. BMC Genomics (2024) 2 Galià-Camps et al. BMC Genomics (2024) 25:410 6. Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE. Double digest RAD­ seq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. PLoS ONE. 2012;7:e37135. 31. Wickham H, Chang W, Wickham MH. Package ggplot2. Create Elegant data Visualisations Using Gramm Graphics Version. 2016;2:1–189. Visualisations Using Gramm Graphics Version. 2016;2:1–189. g 32. Bates D. lme4: linear mixed-effects models using S4 classes. http://cran.r- project.org/package=lme4. 2010. 32. Bates D. Publisher’s Note 29. Seetharam AS, Stuart GW. Whole genome phylogeny for 21 Drosophila spe­ cies using predicted 2b-RAD fragments. PeerJ. 2013;1:e226. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 30. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.
https://openalex.org/W3127844953
http://umu.diva-portal.org/smash/get/diva2:1525840/FULLTEXT02
English
null
Abstract argumentation and the rational man
Journal of logic and computation
2,021
cc-by
26,661
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 g p p y 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 AF = ({a}, {}), AF′ = ({a, b, c}, {(a, b), (b, c), (c, a)}), AF ⪯N AF′.) [1] L. Amgoud and C. Cayrol. Inferring from inconsistency in preference-based argumentation frameworks. Journal of Automated Reasoning, 29, 125–169, 2002. Acknowledgements This work was partially supported by the Wallenberg AI, Autonomous Systems and Software Program (WASP) funded by the Knut and Alice Wallenberg Foundation. We thank Dov Gabbay, as well as the anonymous reviewers, for valuable feedback and discussions. [2] P. Baroni, M. Caminada and M. Giacomin. Abstract argumentation frameworks and their semantics. In Handbook of Formal Argumentation, P. Baroni, D. Gabbay, G. Massimiliano and L. van der Torre, eds, pp. 159–236. College Publications, 2018. [1] L. Amgoud and C. Cayrol. Inferring from inconsistency in preference-based argumentation frameworks. Journal of Automated Reasoning, 29, 125–169, 2002. [2] P. Baroni, M. Caminada and M. Giacomin. Abstract argumentation frameworks and their semantics. In Handbook of Formal Argumentation, P. Baroni, D. Gabbay, G. Massimiliano and L. van der Torre, eds, pp. 159–236. College Publications, 2018. References [1] L. Amgoud and C. Cayrol. Inferring from inconsistency in preference-based argumentation frameworks. Journal of Automated Reasoning, 29, 125–169, 2002. [2] P. Baroni, M. Caminada and M. Giacomin. Abstract argumentation frameworks and their semantics. In Handbook of Formal Argumentation, P. Baroni, D. Gabbay, G. Massimiliano and L. van der Torre, eds, pp. 159–236. College Publications, 2018. 692 Abstract argumentation and the rational man [3] P. Baroni, D. M. Gabbay and M. Giacomin. Introduction to the special issue on loops in argumentation. Journal of Logic and Computation, 26, 1051–1053. [4] P. Baroni and M. Giacomin. On principle-based evaluation of extension-based argumentation semantics. Artificial Intelligence, 171, 675–700, 2007. Argumentation in Artificial Intelli- gence. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 [5] P. Baroni, M. Giacomin and G. Guida. Scc-recursiveness: a general schema for argumentation semantics. Artificial Intelligence, 168, 162–210, 2005. [6] I. Bateman, A. Munro, B. Rhodes, C. Starmer and R. Sugden. A test of the theory of reference- dependent preferences. The Quarterly Journal of Economics, 112, 479–505, 1997. [7] R. Baumann. What does it take to enforce an argument? Minimal change in abstract argumentation. In Proceedings of the 20th European Conference on Artificial Intelligence (NLD, 2012), ECAI’12, pp. 127–132. IOS Press, 2012. ( ) pp [8] R. Baumann. On the nature of argumentation semantics: existence and uniqueness, expressibil- ity, and replaceability. Journal of Applied Logics, 4, 2779–2886, 2017. [9] R. Baumann and G. Brewka. Expanding argumentation frameworks: enforcing and monotonic- ity results. COMMA, 10, 75–86, 2010. [10] R. Baumann, G. Brewka and M. Ulbricht. Revisiting the foundations of abstract argumenta- tion—semantics based on weak admissibility and weak defense. In The Thirty-Fourth AAAI Conference on Artificial Intelligence, AAAI 2020, The Thirty-Second Innovative Applications of Artificial Intelligence Conference, IAAI 2020, The Tenth AAAI Symposium on Educational Advances in Artificial Intelligence, EAAI 2020, New York, NY, USA, February 7–12, 2020, pp. 2742–2749, 2020. [11] R. Baumann and S. Woltran. The role of self-attacking arguments in characterizations of equivalence notions. Journal of Logic and Computation, 26, 1293–1313, Aug 2016. [12] T. J. Bench-Capon. Persuasion in practical argument using value-based argumentation frame- works. Journal of Logic and Computation, 13, 429–448, 2003. [13] S. Benferhat, D. Dubois and H. Prade. Nonmonotonic reasoning, conditional objects and possibility theory. Artificial Intelligence, 92, 259–276, 1997. [14] M. Caminada. Comparing two unique extension semantics for formal argumentation: ideal and eager. References In Proceedings of the 19th Belgian-Dutch Conference on Artificial Intelligence (BNAIC 2007), pp. 81–87. Utrecht University Press, 2007. [15] M. Caminada. Rationality postulates: applying argumentation theory for non-monotonic reasoning. Journal of Applied Logics, 4, 2707–2734, 2017. [16] M. W. Caminada, W. A. Carnielli and P. E. Dunne. Semi-stable semantics. Journal of Logic and Computation, 22, 1207–1254, 2012. [17] K. ˇCyras and F. Toni. Non-monotonic inference properties for assumption-based argumenta- tion. In Theory and Applications of Formal Argumentation (Cham, 2015), E. Black, S. Modgil and N. Oren, eds, pp. 92–111. Springer International Publishing, 2015. [18] B. A. Davey and H. A. Priestly. Introduction to Lattices and Order, 2nd edn. Cambridge University Press, 2002. [19] V. Dignum, M. Baldoni, C. Baroglio, M. Caon, R. Chatila, L. Dennis, G. Génova, G. Haim, M. S. Kließ, M. Lopez-Sanchez, R. Micalizio, J. Pavón, M. Slavkovik, M. Smakman, M. van Steenbergen, S. Tedeschi, L. van der Torre, S. Villata and T. de Wildt. Ethics by design: Necessity or curse? In Proceedings of the 2018 AAAI/ACM Conference on AI, Ethics, and Society, pp. 60–66. AIES ‘18, ACM, New York, NY, USA, 2018. 693 Abstract argumentation and the rational man [20] S. Doutre and J. Mengin. On sceptical versus credulous acceptance for abstract argument systems. In Logics in Artificial Intelligence, J. J. Alferes and J. Leite, eds, pp. 462–473, Berlin, Heidelberg, Springer Berlin Heidelberg, 2004. Downloaded from https://academic.oup.com/logcom/article/31/2/654/6128412 by Umea University Library user on 16 March 2021 [21] J. R. Doyle, D. J. O’Connor, G. M. Reynolds and P. A. Bottomley. The robustness of the asymmetrically dominated effect: Buying frames, phantom alternatives, and in-store purchases. Psychology & Marketing, 16, 225–243, 1999. [22] P. M. Dung. On the acceptability of arguments and its fundamental role in nonmonotonic reasoning, logic programming and n-person games. Artificial intelligence, 77, 321–357, 1995. [ ] . . u g. O t e acceptab ty o a gu e ts a d ts u da e ta o e o o oto c reasoning, logic programming and n-person games. Artificial intelligence, 77, 321–357, 1995. [23] P. M. Dung, P. Mancarella and F. Toni. Computing ideal sceptical argumentation. Artificial Intelligence, 171, 642–674, 2007. [23] P. M. Dung, P. Mancarella and F. Toni. Computing ideal sceptical argumentation. Artificial Intelligence, 171, 642–674, 2007. [24] W. Dvoˇrák and S. A. Gaggl. Stage semantics and the SCC-recursive schema for argumentation semantics. References Journal of Logic and Computation 26, 4, 1149–1202, 02 2014. [25] D. Gabbay. The handling of loops in argumentation networks. Journal of Logic and Computa- tion 26, 4, 1065–1147, 01 2014. [26] D. M. Gabbay. Theoretical foundations for non-monotonic reasoning in expert systems. In Logics and Models of Concurrent Systems, K. R. Apt, ed, pp. 439–457. Berlin, Heidelberg, Springer Berlin Heidelberg, 1985. [27] D. Kahneman. Maps of bounded rationality: Psychology for behavioral economics. American economic review, 93, 1449–1475, 2003. [28] T. Kampik and D. Gabbay. Towards diarg: An argumentation-based dialogue reasoning engine. In SAFA@ COMMA, 2020. [29] H. Li, N. Oren and T. J. Norman. Probabilistic argumentation frameworks. In International Workshop on Theorie and Applications of Formal Argumentation, pp. 1–16. Springer, 2011. [30] M. Lippi and P. Torroni. Argumentation mining: State of the art and emerging trends. ACM Trans. Internet Technol. 16, 2, 10:1–10:25, Mar. 2016. [31] J. F. Nash. Equilibrium points in n-person games. Proceedings of the National Academy of Sciences, 36, 48–49, 1950. [32] J. C. Nieves and R. Confalonieri. A possibilistic argumentation decision making framework with default reasoning. Fundamenta Informaticae, 113, 41–61, 2011. [33] I. Rahwan, M. Cebrian, N. Obradovich, J. Bongard, J.-F. Bonnefon, C. Breazeal, J. W. Crandall, N. A. Christakis, I. D. Couzin, M. O. Jackson, N. R. Jennings, E. Kamar, I. M. Kloumann, H. Larochelle, D. Lazer, R. McElreath, A. Mislove, D. C. Parkes, A. Pentland, M. E. Roberts, A. Shariff, J. B. Tenenbaum and M. P. Wellman. Machine behaviour. Nature, 568, 477–486, 2019. , , , , , , , Shariff, J. B. Tenenbaum and M. P. Wellman. Machine behaviour. Nature, 568, 477–486, 2019. [34] A. Rubinstein. Modeling Bounded Rationality. MIT press, 1998. [34] A. Rubinstein. Modeling Bounded Rationality. MIT press, 1998. [35] S. J. Russell and P. Norvig. Artificial Intelligence: A Modern Approach. Pearson Education Limited, Malaysia, 2016. [36] L. Schröder, D. Pattinson and D. Hausmann. Optimal tableaux for conditional logics with cautious monotonicity. In Proceedings of the 2010 Conference on ECAI 2010: 19th European Conference on Artificial Intelligence (NLD, 2010), pp. 707–712. IOS Press, 2010. [37] A. Sen. Utility: Ideas and terminology. Economics and Philosophy, 7, 277–283, 1991 [38] H. A. Simon. A behavioral model of rational choice. The Quarterly Journal of Economics, 69, 99–118, 1955. [39] A. Tversky, P. Slovic and D. Kahneman. The causes of preference reversal. The American Economic Review, 80, 204–217, 1990. [40] L. References van der Torre and S. Vesic. The principle-based approach to abstract argumentation semantics. IfCoLog Journal of Logics and Their Applications, 4, 8, 2017. 694 Abstract argumentation and the rational man [41] B. Verheij. Two approaches to dialectical argumentation: admissible sets and argumentation stages. In Proceedings of the Eighth Dutch Conference on Artificial Intelligence (NAIC 1996), vol. 96, pp. 357–368, 1996. [42] J. Von Neumann, O. Morgenstern and H. W. Kuhn. Theory of Games and Economic Behavior (Commemorative Edition). Princeton University Press, 2007. [43] Y. Wu and M. Caminada. A labelling-based justification status of arguments. Studies in Logic, 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
https://openalex.org/W4361236675
https://figshare.com/ndownloader/files/39861714
English
null
Supplemental Figures S1-S4 from Modeling Alveolar Soft Part Sarcoma Unveils Novel Mechanisms of Metastasis
null
2,023
cc-by
426
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
https://openalex.org/W3138774926
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 the first time, that eye and mouse parameters behave similarly, Frontiers in Psychology | www.frontiersin.org REFERENCES Griffiths, L., and Chen, Z. (2007). “Investigating the differences in web browsing behaviour of chinese and european users using mouse tracking,” in Usability and Internationalization. HCI and Culture, Vol. 4559 (Berlin; Heidelberg: Springer Berlin Heidelberg), 502–512. doi: 10.1007/978-3-540-73287-7_59 Anderson, N. C., Ort, E., Kruijne, W., Meeter, M., and Donk, M. (2015). It depends on when you look at it: Salience influences eye movements in natural scene viewing and search early in time. J. Vis. 15:9. doi: 10.1167/15.5.9 Guo, Q., and Agichtein, E. (2010). “Towards predicting web searcher gaze position from mouse movements,” in Proceedings of the 28th of the International Conference Extended Abstracts on Human Factors in Computing Systems - CHI EA ’10 (Atlanta, GA: ACM Press), 3601. doi: 10.1145/1753846.1754025 l A h S S d ( ) Th f Antal, M., and Egyed-Zsigmond, E. (2019). Intrusion detection using mouse dynamics. IET Biometr. 8, 285–294. doi: 10.1049/iet-bmt.2018.5126 Boi, P., Fenu, G., Spano, L. D., and Vargiu, V. (2016). Reconstructing user’s attention on the web through mouse movements and perception-based content identification. ACM Trans. Appl. Percept. 13, 1–21. doi: 10.1145/2912124 Helo, A., Pannasch, S., Sirri, L., and Rämä, P. (2014). The maturation of eye movement behavior: scene viewing characteristics in children and adults. Vis. Res. 103, 83–91. doi: 10.1016/j.visres.2014.08.006 Brady, K., Cho, S. J., Narasimham, G., Fisher, D., and Goodwin, A. (2018). “Is scrolling disrupting while reading?” in Proceedings of the 13th International Conference of the Learning Sciences (London), 8. Henderson, J. M. (2003). Human gaze control during real-world scene perception. Trends Cogn. Sci. 7, 498–504. doi: 10.1016/j.tics.2003.09.006 Braganza, C., Marriott, K., Moulder, P., Wybrow, M., and Dwyer, T. (2009). “Scrolling behaviour with single- and multi-column layout,” in Proceedings of the 18th international conference on World wide web - WWW ’09 (Madrid: ACM Press), 831–840. doi: 10.1145/1526709.1526821 Henderson, J. M., and Hollingworth, A. (1998). “Eye movements during scene viewing: an overview,” in Eye Guidance in Reading and Scene Perception, ed G. Underwood (Amsterdam: Elsevier), 269–293. doi: 10.1016/B978-008043361-5/50013-4 Bruyer, R., Abdi, H., and Benoit, J. (1987). Stimulus versus face recognition in laterally displayed stimuli. Am. J. Psychol. 100, 117–121. doi: 10.2307/1422645 Henderson, J. M. and Hollingworth, A. (1999). High-level scene perception. Annu. Rev. Psychol. 50, 243–271. doi: 10.1146/annurev.psych.50.1.243 Buscher, G., Cutrell, E., and Morris, M. R. (2009). “What do you see when you’re surfing? REFERENCES Using eye tracking to predict salient regions of web pages,” in Proceedings of the SIGCHI Conference on Human Factors in Computing Systems - CHI ’09 (Boston, MA: Association for Computing Machinery), 21–30. doi: 10.1145/1518701.1518705 Holmqvist, K., Nyström, M., Andersson, R., Dewhurst, R., Halszka, J., and van de Weijer, J. (2011). Eye Tracking: A Comprehensive Guide to Methods and Measures. Oxford: Oxford University Press. Huang, J., White, R., and Buscher, G. (2012). “User see, user point: gaze and cursor alignment in web search,” in Proceedings of the 2012 ACM annual conference on Human Factors in Computing Systems - CHI ’12 (Austin, TX: ACM Press), 1341–1350. doi: 10.1145/2207676.2208591 Buswell, G. T. (1935). How People Look at Pictures: A Study of the Psychology and Perception in Art. Chicago, IL: University of Chicago Press. Itti, L., and Borji, A. (2015). Computational models: bottom-up and top-down aspects. arXiv:1510.07748 [cs]. doi: 10.1093/oxfordhb/9780199675111.013.026 Chen, M. C., Anderson, J. R., and Sohn, M. H. (2001). “What can a mouse cursor tell us more? Correlation of eye/mouse movements on web browsing,” in CHI ’01 Extended Abstracts on Human Factors in Computing Systems, CHI EA ’01 (New York, NY: Association for Computing Machinery), 281–282. doi: 10.1145/634067.634234 Krejtz, K., Duchowski, A., Krejtz, I., Szarkowska, A., and Kopacz, A. (2016). Discerning ambient/focal attention with coefficient K. ACM Trans. Appl. Percept. 13, 1–20. doi: 10.1145/2896452 Kumar, M., Winograd, T., and Paepcke, A. (2007). “Gaze-enhanced scrolling techniques,” in CHI’07 Extended Abstracts on Human Factors in Computing Systems (San Jose, CA), 2531–2536. doi: 10.1145/1240866.1241036 Cronin, D. A., Hall, E. H., Goold, J. E., Hayes, T. R., and Henderson, J. M. (2020). Eye movements in real-world scene photographs: general characteristics and 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 temporal dynamic of ambient and focal modes,” in Proceedings of the 11th ACM Symposium on Eye Tracking Research & Applications - ETRA ’19 (Denver, CO: ACM Press), 1–4. doi: 10.1145/3314111.3323075 Ferreira, S., Arroyo, E., Tarrago, R., and Blat, J. (2010). Applying Mouse Tracking to Investigate Patterns of Mouse Movements in Web Forms. Universitat Pompeu Fabra. Fessenden, T. (2017). Horizontal attention leans left. Milisavljevic, A., Hamard, K., Petermann, C., Gosselin, B., Doré-Mazars, K., and Mancas, M. (2018). “Eye and mouse coordination during task: from behaviour to prediction,” in Proceedings of the 13th International Joint Conference on Computer Vision, Imaging and Computer Graphics Theory and Applications (Funchal: SCITEPRESS - Science and Technology Publications), 86–93. doi: 10.5220/0006618800860093 Fu, E. Y., Kwok, T. C., Wu, E. Y., Leong, H. V., Ngai, G., and Chan, S. C. (2017). “Your mouse reveals your next activity: towards predicting user intention from mouse interaction,” in 2017 IEEE 41st Annual Computer Software and Applications Conference (COMPSAC) (Turin: IEEE), 869–874. doi: 10.1109/COMPSAC.2017.270 Gamboa, H., and Fred, A. (2004). “A behavioral biometric system based on human- computer interaction,” in Biometric Technology for Human Identification, Vol. 5404 (Orlando, FL: International Society for Optics and Photonics), 381–392. doi: 10.1117/12.542625 Moher, J., and Song, J.-H. (2019). A comparison of simple movement behaviors across three different devices. Attent. Percept. Psychophys. 81, 2558–2569. doi: 10.3758/s13414-019-01856-8 Mueller, F., and Lockerd, A. (2001). “Cheese: tracking mouse movement activity on websites, a tool for user modeling,” in CHI’01 Extended Goodale, M. A., and Milner, A. D. (1992). Separate visual pathways for perception and action. Trends Neurosci. 15, 20–25. doi: 10.1016/0166-2236(92)90344-8 March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 15 Similarities and Differences Between Eye and Mouse Milisavljevic et al. Abstracts on Human Factors in Computing Systems (Seattle, WA), 279–280. doi: 10.1145/634067.634233 Still, J. D., and Masciocchi, C. M. (2010). “A saliency model predicts fixations in web interfaces,” in Proceedings of the 5th International Workshop on Model-Driven Development of Advanced User Interactions, Vol. 617 (Atlanta, GA), 25–28. Abstracts on Human Factors in Computing Systems (Seattle, WA), 279–280. REFERENCES doi: 10.1145/634067.634233 Navalpakkam, V., Jentzsch, L., Sayres, R., Ravi, S., Ahmed, A., and Smola, A. (2013). “Measurement and modeling of eye-mouse behavior in the presence of nonlinear page layouts,” in Proceedings of the 22nd International Conference on World Wide Web - WWW ’13 (Rio de Janeiro: ACM Press), 953–964. doi: 10.1145/2488388.2488471 Stone, K. D., and Gonzalez, C. L. R. (2015). The contributions of vision and haptics to reaching and grasping. Front. Psychol. 6:1403. doi: 10.3389/fpsyg.2015.01403 Tatler, B. W., and Vincent, B. T. (2008). Systematic tendencies in scene viewing. J. Eye Mov. Res. 2, 1–18. doi: 10.16910/jemr.2.2.5 Nielsen, J. (2006). F-Shaped Pattern for Reading Web Content. Available online at: https://www.nngroup.com/articles/f-shaped-pattern-reading-web- content-discovered/ Theeuwes, J., and Failing, M. (2020). Attentional Selection: Top-Down, Bottom- Up and History-Based Biases. Elements in Perception. Cambridge: Cambridge University Press. doi: 10.1017/9781108891288 Nielsen, J. (2010). Horizontal Attention Leans Left (Early Research). Available online at: https://www.nngroup.com/articles/horizontal-attention-original- research/ Torralba, A., Oliva, A., Castelhano, M. S., and Henderson, J. M. (2006). Contextual guidance of eye movements and attention in real-world scenes: the role of global features in object search. Psychol. Rev. 113:766. doi: 10.1037/0033-295X.113.4.766 Pan, B., Hembrooke, H. A., Gay, G. K., Granka, L. A., Feusner, M. K., and Newman, J. K. (2004). “The determinants of web page viewing behavior: an eye-tracking study,” in Proceedings of the 2004 Symposium on Eye Tracking Research & Applications (San Antonio, TX), 147–154. doi: 10.1145/968363.968391 Tullis, T. S. (2007). “Older adults and the web: lessons learned from eye-tracking,” in Universal Acess in Human Computer Interaction. Coping with Diversity, Vol. 4554 (Berlin; Heidelberg: Springer Berlin Heidelberg), 1030–1039. doi: 10.1007/978-3-540-73279-2_115 Pannasch, S., Helmert, J. R., Roth, K., Herbold, A.-K., and Walter, H. (2008). Visual fixation durations and saccade amplitudes: shifting relationship in a variety of conditions. J. Eye Mov. Res. 2. doi: 10.16910/jemr.2.2.4 Tzafilkou, K., and Protogeros, N. (2018). Mouse behavioral patterns and keystroke dynamics in end-user development: what can they tell us about users’ behavioral attributes? Comput. Hum. Behav. 83, 288–305. doi: 10.1016/j.chb.2018.02.012 Pannasch, S., and Velichkovsky, B. M. (2009). Distractor effect and saccade amplitudes: further evidence on different modes of processing in free exploration of visual images. Vis. Cogn. 17, 1109–1131. doi: 10.1080/13506280902764422 Unema, P. J. A., Pannasch, S., Joos, M., and Velichkovsky, B. M. (2005). Time course of information processing during scene perception: the relationship between saccade amplitude and fixation duration. Vis. Cogn. 12, 473–494. doi: 10.1080/13506280444000409 Reeder, R., and Maxion, R. (2006). REFERENCES “User interface defect detection by hesitation analysis,” in International Conference on Dependable Systems and Networks (DSN’06) (Philadelphia, PA: IEEE), 61–72. doi: 10.1109/DSN.2006.71 Rheem, H., Verma, V., and Becker, D. V. (2018). Use of mouse-tracking method to measure cognitive load. Proc. Hum. Fact. Ergon. Soc. Annu. Meet. 62, 1982–1986. doi: 10.1177/1541931218621449 Velichkovsky, B. M., Rothert, A., Kopf, M., Dornhöfer, S. M., and Joos, M. (2002). Towards an express-diagnostics for level of processing and hazard perception. Transport. Res. Part F Traff. Psychol. Behav. 5, 145–156. doi: 10.1016/S1369-8478(02)00013-X Rodden, K., and Fu, X. (2007). “Exploring how mouse movements relate to eye movements on web search results pages,” in 30th Annual International ACM SIGIR Conference (Amsterdam), 29–32. Yarbus, A. L. (1967). Eye Movements and Vision. New York, NY: Plenum. doi: 10.1007/978-1-4899-5379-7 Rodden, K., Fu, X., Aula, A., and Spiro, I. (2008). “Eye-mouse coordination patterns on web search results pages,” in Proceeding of the Twenty-Sixth Annual CHI Conference Extended Abstracts on Human Factors in Computing Systems - CHI ’08 (Florence: ACM Press), 2997–3002. doi: 10.1145/1358628.1358797 Zheng, N., Paloski, A., and Wang, H. (2011). “An efficient user verification system via mouse movements,” in Proceedings of the 18th ACM Conference on Computer and Communications Security - CCS ’11 (Chicago, IL: ACM Press), 139–150. doi: 10.1145/2046707.2046725 Roth, S. P., Tuch, A. N., Mekler, E. D., Bargas-Avila, J. A., and Opwis, K. (2013). Location matters, especially for non-salient features-an eye-tracking study on 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 South Africa, ETSA ’13 (New York, NY: Association for Computing Machinery), 24–31. doi: 10.1145/2509315.2509319 March 2021 | Volume 12 | Article 554595 Frontiers in Psychology | www.frontiersin.org 16
https://openalex.org/W3024990838
https://www.nature.com/articles/s41598-020-68263-0.pdf
English
null
Gait asymmetry, and bilateral coordination of gait during a six-minute walk test in persons with multiple sclerosis
bioRxiv (Cold Spring Harbor Laboratory)
2,020
cc-by
9,899
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 gi ( , ) p η 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 η g p p y 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 y 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 g p 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 y g 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 g 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 y 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​:// doi.org/10.1016/j.apmr.2007.08.142 (2008).hlf g j p 13. Lewek, M. D., Osborn, A. J. & Wutzke, C. J. The influence of mechanically and physiologically imposed stiff-knee gait patterns on the energy cost of walking. Arch. Phys. Med. Rehabil. 93, 123–128. https​://doi.org/10.1016/j.apmr.2011.08.019 (2012). 4. Bot, J. et al. Spinal cord abnormalities in recently diagnosed MS patients added value of spinal MRI examination. Neurology 62 226–233 (2004). 5. Ikuta, F. & Zimmerman, H. Distribution of plaques in 70 autopsy cases of multiple sclerosis in the United States. Neurology 26 26–28 (1976).f 6. Naismith, R. T. et al. Spinal cord tract diffusion tensor imaging reveals disability substrate in demyelinating disease. Neurology 80 2201–2209. https​://doi.org/10.1212/WNL.0b013​e3182​96e8f​1 (2013). p g 7. Reich, D. S. et al. Multiparametric magnetic resonance imaging analysis of the corticospinal tract in multiple sclerosis. Neuroimage 38, 271–279 (2007).f ( ) 8. von Meyenburg, J. et al. Spinal cord diffusion-tensor imaging and motor-evoked potentials in multiple sclerosis patients: Micro- structural and functional asymmetry. Radiology 267, 869–879 (2013). y y gy 9. Krasovsky, T. & Levin, M. F. Review: Toward a better understanding of coordination in healthy and poststroke gait. Neurorehabil Neural. Repair 24, 213–224. https​://doi.org/10.1177/15459​68309​34850​9 (2010). 19. Krasovsky, T. & Levin, M. F. Review: Toward a better understanding of coordination in h Neural. Repair 24, 213–224. https​://doi.org/10.1177/15459​68309​34850​9 (2010). p p g 20. Dietz, V. Spinal cord pattern generators for locomotion. Clin. Neurophysiol. 114, 1379–1389 (2003). 1. Guertin, P. A. Central pattern generator for locomotion: Anatomical, physiological, and pathophysiological considerations. Front Neurol. 3, 183 (2013). 2. O’Connor, S. M. & Donelan, J. M. Fast visual prediction and slow optimization of preferred walking speed. J. Neurophysiol. 107(9) 2549–2559 (2012).ff 3. Plotnik, M., Giladi, N. & Hausdorff, J. M. A new measure for quantifying the bilateral coordination of human gait: Effects of aging and Parkinson’s disease. Exp. Brain Res. 181, 561–570. https​://doi.org/10.1007/s0022​1-007-0955-7 (2007). p p g 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.gaitp​ost.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 1. Motl, R. W. & Learmonth, Y. C. Neurological disability and its association with walking impairment in multiple sclerosis: Brief review. Neurodegen. Dis. Manag. 4, 491–500 (2014). review. Neurodegen. Dis. Manag. 4, 491–500 (2014). 2 Givon U Zeilig G & Achiron A Gait analysis in multiple sclerosis: Characterization of temporal spatial parameters using g g 2. Givon, U., Zeilig, G. & Achiron, A. Gait analysis in multiple sclerosis: Characterization of temporal–spatial parameters using GAITRite functional ambulation system. Gait Posture 29, 138–142 (2009).ff Givon, U., Zeilig, G. & Achiron, A. Gait analysis in multiple scle y 3. Socie, M. J., Motl, R. W., Pula, J. H., Sandroff, B. M. & Sosnoff, J. J. Gait variability and disability in multiple sclerosis. Gait Posture 38, 51–55 (2013).f 4. Socie, M. J., Motl, R. W. & Sosnoff, J. J. Examination of spatiotemporal gait parameters during the 6-min walk in individuals with multiple sclerosis. Int. J. Rehabil. Res. 37, 311–316. https​://doi.org/10.1097/MRR.00000​00000​00007​4 (2014). f multiple sclerosis. Int. J. Rehabil. Res. 37, 311–316. https​://doi.o g 5. Goldman, M. D., Marrie, R. A. & Cohen, J. A. Evaluation of the six-minute walk in multiple sclerosis subjects and healthy controls Multiple Sclerosis J. 14, 383–390 (2008). p 6. Phan-Ba, R. et al. Motor fatigue measurement by distance-induced slow down of walking speed in multiple sclerosis. PLoS ONE 7, e34744. https​://doi.org/10.1371/journ​al.pone.00347​44 (2012). p g j p 7. Kalron, A. Gait variability across the disability spectrum in people with multiple sclerosis. J. Neurol. Sci. 361, 1–6. https​://doi org/10.1016/j.jns.2015.12.012 (2016).hl g j j 8. Kalron, A. & Frid, L. The, “butterfly diagram”: A gait marker for neurological and cerebellar impairment in people with multiple sclerosis. J. Neurol. Sci. 358, 92–100. https​://doi.org/10.1016/j.jns.2015.08.028 (2015).i 9. Kalron, A. Association between gait variability, falls and mobility in people with multiple sclerosis: A specific observation on the EDSS 4.0–4.5 level. NeuroRehabilitation 40, 579–585. https​://doi.org/10.3233/NRE-17144​5 (2017).lt g 10. Hsu, A.-L., Tang, P.-F. & Jan, M.-H. Analysis of impairments influencing gait velocity and asymmetry of hemiplegic patients after mild to moderate stroke. Arch. Phys. Med. Rehabil. 84, 1185–1193 (2003).h y ( ) 11. Lin, P.-Y., Yang, Y.-R., Cheng, S.-J. & Wang, R.-Y. The relation between ankle impairments and gait velocity and symmet people with stroke. Arch. Phys. Med. Rehabil. 87, 562–568. https​://doi.org/10.1016/j.apmr.2005.12.042 (2006). p p y p g j p 2. Patterson, K. K. et al. Gait asymmetry in community-ambulating stroke survivors. Arch. Phys. Med. Rehabil. 89, 304–310. Data availability Th d d The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ BMC Neurol. 13, 37 (2013).fh Learmonth, Y. C., Motl, R. W., Sandroff, B. M., Pula, J. H. & Cada 7. Learmonth, Y. C., Motl, R. W., Sandroff, B. M., Pula, J. H. & Cadavid, D. Validation of patient determined disease steps (PDDS) scale scores in persons with multiple sclerosis. BMC Neurol. 13, 37 (2013).fh 48. Plotnik, M. & Hausdorff, J. M. The role of gait rhythmicity and bilateral coordination of stepping in the pathophysiology of freezing of gait in Parkinson’s disease. Movement Disord. Off. J. Movement Disord. Soc. 23, S444–S450 (2008). g ff 49. DeLuca, G., Ebers, G. & Esiri, M. Axonal loss in multiple sclerosis: A pathological survey of the corticospinal and sensory tracts. Brain 127, 1009–1018 (2004). 0. Newland, P. et al. Exploring the feasibility and acceptability of sensor monitoring of gait and falls in the homes of persons with multiple sclerosis. Gait Posture 49, 277–282 (2016). p 51. Shammas, L. et al. Home-based system for physical activity monitoring in patients with multiple sclerosis (Pilot study). Biomed. Eng. Online 13, 10 (2014). 52. van den Heuvel, L. et al. Actigraphy detects greater intra-individual variability during gait in non-manifesting LRRK2 mutation carriers. J. Parkinson’s Disease 8, 131–139 (2018).f 3. Fasano, A. et al. Split-belt locomotion in Parkinson’s disease links asymmetry, dyscoordination and sequence effect. Gait Posture 48, 6–12 (2016). 54. Reisman, D. S., McLean, H., Keller, J., Danks, K. A. & Bastian, A. J. Repeated split-belt treadmill training improves poststroke step length asymmetry. Neurorehabil. Neural. Repair. 27, 460–468 (2013). 55. Ricciardi, L. et al. Working on asymmetry in Parkinson’s disease: Randomized, controlled pilot study. Neurol. Sciences. 36, 1337– 1343 (2015). d l ll l l l & ( ) 56. Matsuda, P. N. et al. Falls in multiple sclerosis. PM&R 3, 624–632 (2011). Acknowledgements g We thank Ms. Natalie Karlibach and Ms. Tamar Azrad from the Center of Advanced Technologies in Rehabilita- 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, Behavioral, and Performance Unit). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. Competing interests p g RTN has received honorarium for speaking/consulting from Alexion, Alkermes, Biogen, Celgene, EMD Serono, Genentech, Genzyme, Novartis, Alexion, TG Therapeutics, Viela Bio. All other authors declare no competing interests. Author contributions M.P., J.M.W., G.A., and R.T.N. designed and performed the study. AG and MP did the data analysis, interpreted the data, prepared the figures and drafted the main manuscript text. J.M.W. and R.T.N. substantively revised the manuscript. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 38. Kiss, R. M. Effect of walking speed and severity of hip osteoarthritis on gait variability. J. Electromyogr. Kinesiol. 20, 1044–1051 (2010). ( ) 9. Schniepp, R. et al. Locomotion speed determines gait variability in cerebellar ataxia and vestibular failure. Mov. Disord. 27, 125–131 (2012).ff ( ) 0. Plotnik, M., Bartsch, R. P., Zeev, A., Giladi, N. & Hausdorff, J. M. Effects of walking speed on asymmetry and bilateral coordination of gait. Gait Posture 38, 864–869 (2013). 1. Pajek, M. B. et al. Six-minute walk test in renal failure patients: representative results, performance analysis and perceived dyspnea predictors. PLoS ONE 11, e0150414 (2016).f predictors. PLoS ONE 11, e0150414 (2016). 42. Mirelman, A. et al. Effects of aging on arm swing during gait: the role of gait speed and dual tasking. PLoS ONE 10, e0136043 (2015). 2. Mirelman, A. et al. Effects of aging on arm swing during gait: the role of gait speed and dual tasking. PLoS ONE 10, e0136043 (2015).f ( ) 43. Yogev, G., Plotnik, M., Peretz, C., Giladi, N. & Hausdorff, J. M. Gait asymmetry in patients with Parkinson’s disease and elderly fallers: When does the bilateral coordination of gait require attention?. Exp. Brain Res. 177, 336–346. https​://doi.org/10.1007/s0022​ 1-006-0676-3 (2007). 44. Plotnik, M., Giladi, N., Balash, Y., Peretz, C. & Hausdorff, J. M. Is freezing of gait in Parkinson’s disease related to asymm motor function?. Ann. Neurol. Off. J. Am. Neurol. Assoc. Child Neurol. Soc. 57, 656–663 (2005).f 4. Plotnik, M., Giladi, N., Balash, Y., Peretz, C. & Hausdorff, J. M. Is freezing of gait in Parkinsons disease rel motor function?. Ann. Neurol. Off. J. Am. Neurol. Assoc. Child Neurol. Soc. 57, 656–663 (2005).f f . Socie, M. J. & Sosnoff, J. J. Gait variability and multiple sclerosis f & Sosnoff, J. J. Gait variability and multiple sclerosis. Multiple Scl f 46. Kaipust, J. P., Huisinga, J. M., Filipi, M. & Stergiou, N. Gait variability measures reveal differences between multiple scle patients and healthy controls. Mot. Control 16(2), 229–244 (2012).f p y ( ), ( ) 7. Learmonth, Y. C., Motl, R. W., Sandroff, B. M., Pula, J. H. & Cadavid, D. Validation of patient determined disease steps (PDDS) l h l l l C l ( ) y 47. Learmonth, Y. C., Motl, R. W., Sandroff, B. M., Pula, J. H. & Cadavid, D. Validation of pati scale scores in persons with multiple sclerosis. References 266(12), 3022–3030 (2019). p 7. Butland, R., Pang, J., Gross, E., Woodcock, A. & Geddes, D. Two-, six-, and 12-minute walking tests in respiratory disease. Br. Med J. (Clin. Res. Ed.) 284, 1607 (1982). 28. Cutter, G. R. et al. Development of a multiple sclerosis functional composite as a clinical trial outcome measure. Brain 122(Pt 5), 871–882 (1999).h 29. Hobart, J. C., Riazi, A., Lamping, D. L., Fitzpatrick, R. & Thompson, A. J. Measuring the impact of MS on walking ability: the 12-Item MS Walking Scale (MSWS-12). Neurology 60, 31–36 (2003).hii g gy 0. Powell, L. E. & Myers, A. M. The Activities-specific Balance Confidence (ABC) Scale. J. Gerontol. A Biol. Sci. Med. Sci. 50A, M28-34 (1995).i 31. Meijer, R. et al. Markedly impaired bilateral coordination of gait in post-stroke patients: Is this deficit distinct from asymmetry? A cohort study. J. Neuroeng. Rehabil. 8, 23. https​://doi.org/10.1186/1743-0003-8-23 (2011).f y g p g 32. Plotnik, M., Giladi, N. & Hausdorff, J. M. Bilateral coordination of walking and freezing of gait in Parkinson’s disease. Eur. J. Neurosci. 27, 1999–2006 (2008). ( ) 3. Nilsagård, Y., Gunnarsson, L.-G. & Denison, E. Self-perceived limitations of gait in persons with multiple sclerosis. Adv. Physiother 9, 136–143 (2007).f 34. Nasirzade, A., Sadeghi, H., Mokhtarinia, H. R. & Rahimi, A. A review of selected factors affecting gait symmetry. Phys. Treat. Spec. Phys. Ther. J. 7, 3–12 (2017). yh 35. Gimmon, Y. et al. Gait coordination deteriorates in independent old–old adults. J. Aging Phys. Activity 26, 382–389 (2018). H S H l Q l f h b l l d d l b d 35. Gimmon, Y. et al. Gait coordination deteriorates in independent old–old adults. J. Aging Phys. Activity 26, 382–389 (2018). 36. Han, S. H. et al. Quantitative analysis of the bilateral coordination and gait asymmetry using inertial measurement unit-based gait analysis. PLoS ONE 14 (2019). 36. Han, S. H. et al. Quantitative analysis of the bilateral coordination and gait asymmetry using inertial measurement unit-based analysis. PLoS ONE 14 (2019).f 37. Kang, H. G. & Dingwell, J. B. Separating the effects of age and walking speed on gait variability. Gait Posture 27(4), 572–577 (2008). Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. Additional information Supplementary information is available for this paper at https​://doi.org/10.1038/s4159​8-020-68263 Correspondence and requests for materials should be addressed to M.P. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0 m/scientificreports/ Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. © Th A th ( ) 2020 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. © The Author(s) 2020 Scientific Reports | (2020) 10:12382 | https://doi.org/10.1038/s41598-020-68263-0
https://openalex.org/W2914132714
https://www.frontiersin.org/articles/10.3389/fneur.2019.00097/pdf
English
null
Dynamic Trajectory of Long-Term Cognitive Improvement Up to 10 Years in Young Community-Dwelling Stroke Survivors: A Cohort Study
Frontiers in neurology
2,019
cc-by
7,213
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 are independent risk factors for cognitive decline: a population-based study. Stroke (2000) 31:2431–6. 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) 314:41–51. doi: 10.1001/jama.2015.6968 7. Dik MG, Deeg DJ, Bouter LM, Corder EH, Kok A, Jonker C. Stroke and apolipoprotein E epsilon4 are independent risk factors for cognitive decline: a population-based study. Stroke (2000) 31:2431–6. doi: 10.1161/01.STR.31.10.2431 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 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 REFERENCES 1. O’Brien JT, Erkinjuntti T, Reisberg B, Roman G, Sawada T, Pantoni L, et al. Vascular cognitive impairment. Lancet Neurol. (2003) 2:89–98. doi: 10.1016/S1474-4422(03)00305-3 2. Feigin VL, Krishnamurthi RV, Parmar P, Norrving B, Mensah GA, Bennett DA, et al. Update on the Global Burden of Ischemic and Hemorrhagic Stroke in 1990–2013: the GBD 2013 study. Neuroepidemiology (2015) 45:161–76. doi: 10.1159/000441085 2. Feigin VL, Krishnamurthi RV, Parmar P, Norrving B, Mensah GA, Bennett DA, et al. Update on the Global Burden of Ischemic and Hemorrhagic Stroke in 1990–2013: the GBD 2013 study. Neuroepidemiology (2015) 45:161–76. doi: 10.1159/000441085 3. Schaapsmeerders P, Maaijwee NA, van Dijk EJ, Rutten-Jacobs LC, Arntz RM, Schoonderwaldt HC, et al. Long-term cognitive impairment after first-ever ischemic stroke in young adults. Stroke (2013) 44:1621–8. doi: 10.1161/STROKEAHA.111.0 00792 3. Schaapsmeerders P, Maaijwee NA, van Dijk EJ, Rutten-Jacobs LC, Arntz RM, Schoonderwaldt HC, et al. Long-term cognitive impairment after first-ever ischemic stroke in young adults. Stroke (2013) 44:1621–8. doi: 10.1161/STROKEAHA.111.0 00792 February 2019 | Volume 10 | Article 97 Frontiers in Neurology | www.frontiersin.org 9 Cognitive Improvement Among Stroke Survivors Elgh and Hu 21. Knoflach M, Matosevic B, Rucker M, Furtner M, Mair A, Wille G, et al. Functional recovery after ischemic stroke–a matter of age: data from the Austrian Stroke Unit Registry. Neurology (2012) 78:279–85. doi: 10.1212/WNL.0b013e31824367ab 8. Suzuki M, Sugimura Y, Yamada S, Omori Y, Miyamoto M, Yamamoto J. Predicting recovery of cognitive function soon after stroke: differential modeling of logarithmic and linear regression. PLoS ONE (2013) 8: e53488. doi: 10.1371/journal.pone.0053488 22. Baddeley A. Working memory: looking back and looking forward. Nat Rev Neurosci. (2003) 4:829–39. doi: 10.1038/nrn1201 9. Leeds L, Meara RJ, Woods R, Hobson JP. A comparison of the new executive functioning domains of the CAMCOG-R with existing tests of executive function in elderly stroke survivors. Age Ageing (2001) 30:251–4. doi: 10.1093/ageing/30.3.251 23. Kessels RP, Eikelboom WS, Schaapsmeerders P, Maaijwee NA, Arntz RM, van Dijk EJ, et al. Effect of formal education on vascular cognitive impairment after stroke: a meta-analysis and study in Young-stroke patients. J Int Neuropsychol Soc. (2017) 23:223–38. doi: 10.1017/S1355617716001016 10. Wagle J, Farner L, Flekkoy K, Wyller TB, Sandvik L, Eiklid KL, et al. Cognitive impairment and the role of the ApoE epsilon4-allele after stroke– a 13 months follow-up study. Int J Geriatr Psychiatry (2010) 25:833–42. doi: 10.1002/gps.2425 24. Teasell R, Salter K, Cotoi A, Iliescu A, Donais J. Perceptual Disorders (2018). REFERENCES Available online at: www.ebrsr.com/sites/default/files/v18-SREBR- CH13-NET.pdf 11. Middleton LE, Lam B, Fahmi H, Black SE, McIlroy WE, Stuss DT, et al. Frequency of domain-specific cognitive impairment in sub-acute and chronic stroke. Neuro Rehabilitation (2014) 34:305–12. doi: 10.3233/NRE-1 31030 25. Klingberg T. Training and plasticity of working memory. Trends Cogn Sci. (2010) 14:317–24. doi: 10.1016/j.tics.2010.05.002 26. Murphy TH, Corbett D. Plasticity during stroke recovery: from synapse to behaviour. Nat Rev Neurosci. (2009) 10:861–72. doi: 10.1038/nrn2735 12. Tang EY, Amiesimaka O, Harrison SL, Green E, Price C, Robinson L, et al. Longitudinal effect of stroke on cognition: a systematic review. J Am Heart Assoc. (2018) 7:e006443. doi: 10.1161/JAHA.117.006443 27. Eckert M, Keren NI, Roberts D, Calhoun V, Harris K. Age-related changes in processing speed: unique contributions of cerebellar and prefrontal cortex. Front Hum Neurosci. (2010) 4:10. doi: 10.3389/neuro.09.010.2010 13. Tulsky D, Zhu J, Ledbetter MF. WAIS-III/WMS-III Technical Manual. San Antonio, TX: Psychological Corporation (1997). 28. Hedden T, Gabrieli J. Insights into the ageing mind: a view from cognitive neuroscience. Nat Rev Neurosci. (2004) 5:87–96. doi: 10.1038/ nrn1323 14. Weschler D. WAIS-IV: Weschler Adult Intelligence Scale. 4th ed. Manual del 1, Swedish Version. Stockholm: NCS Pearson, Inc. (2010). 15. Meyers JE, Meyers KR. Rey Complex Figure Test and Recognition Trial. Odessa: Psychological Assessment Resources, Inc. (1995). 29. Bull R, Espy KA, Wiebe SA. Short-term memory, working memory, and executive functioning in preschoolers: longitudinal predictors of mathematical achievement at age 7 years. Dev Neuropsychol. (2008) 33:205– 28. doi: 10.1080/87565640801982312 16. Claeson LE, Esbjörnsson E, Tännérus BM, Wahlbin M. Claeson-Dahls Test för Inlärning och Minne: Manual. Stockholm: Hogrefe Psykologiförlaget AB (1998). 17. Gauthier L, Dehaut F, Joanette Y. The bells test: a quantitative and qualitative test for visual neglect. Int J Clin Neuropsychol. (1989) 11:49–54. 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. 18. Delis DC, Kaplan E, Kramer JH. D-KEFS: Examiner’s Manual. San Antonio, 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 reproduction is permitted which does not comply with these terms. 20. Tombaugh TN. Trail Making Test A and B: normative data stratified by age and education. Arch Clin Neuropsychol. (2004) 19:203–14. doi: 10.1016/S0887-6177(03)00039-8 February 2019 | Volume 10 | Article 97 Frontiers in Neurology | www.frontiersin.org 10
https://openalex.org/W2920456096
https://vestnik.tsuab.ru/jour/article/download/566/524
Russian
null
PRODUCTION OF FOAMED HEAT INSULATION MATERIALS FROM COPPER ORE MILL TAILINGS
Vestnik Tomskogo gosudarstvennogo arhitekturno-stroitelʹnogo universiteta
2,019
cc-by
4,332
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 Пас. 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 Пас, 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 Пас 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 // J 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
https://openalex.org/W2024504202
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380016/pdf/pone.0039379.pdf
English
null
Interaction of Caveolin-1 with Ku70 Inhibits Bax-Mediated Apoptosis
PloS one
2,012
cc-by
11,637
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 dystrophy. Trends Mol Med 7: 435–441. 7. Parton RG, Richards AA (2003) Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic 4: 724–738. 8. Razani B, Schlegel A, Lisanti MP (2000) Caveolin proteins in signaling, oncogenic transformation and muscular dystrophy. J Cell Sci 113: 2103–2109. 9. Williams TM, Lisanti MP (2004) The Caveolin genes: from cell biology to medicine. Ann Med 36: 584–595. 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. 11. Parat MO (2009) The biology of caveolae: achievements and perspectives. Int Rev Cell Mol Biol 273: 117–162. 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. 9. Williams TM, Lisanti MP (2004) The Caveolin genes: from cell biology to medicine. Ann Med 36: 584–595. 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. 7. Parton RG, Richards AA (2003) Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic 4: 724–738. 8. Razani B, Schlegel A, Lisanti MP (2000) Caveolin proteins in signaling, oncogenic transformation and muscular dystrophy. J Cell Sci 113: 2103–2109. 6. Galbiati F, Razani B, Lisanti MP (2001) Caveolae and caveolin-3 in muscular dystrophy. Trends Mol Med 7: 435–441. 6. Galbiati F, Razani B, Lisanti MP (2001) Caveolae and caveolin-3 in muscular dystrophy. Trends Mol Med 7: 435–441. Caveolin-1, a Novel Ku70-Interacting Protein 12. Volonte D, Galbiati F (2009) Inhibition of thioredoxin reductase 1 by caveolin 1 promotes stress-induced premature senescence. EMBO Rep 10: 1334–1340. 26. Gomez JA, Gama V, Yoshida T, Sun W, Hayes P, et al. (2007) Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides. Biochem Soc Trans 35: 797–801. 13. Volonte D, Kahkonen B, Shapiro S, Di Y, Galbiati F (2009) Caveolin-1 expression is required for the development of pulmonary emphysema through activation of the ATM-p53-p21 pathway. J Biol Chem 284: 5462–5466. 27. Yoshida T, Tomioka I, Nagahara T, Holyst T, Sawada M, et al. (2004) Bax- inhibiting peptide derived from mouse and rat Ku70. Biochem Biophys Res Commun 321: 961–966. 14. Couet J, Li S, Okamoto T, Ikezu T, Lisanti MP (1997) Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J Biol Chem 272: 6525–6533. 28. Hwang PM, Bunz F, Yu J, Rago C, Chan TA, et al. (2001) Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells. Nat Med 7: 1111–1117. 29. Nechushtan A, Smith CL, Hsu YT, Youle RJ (1999) Conformation of the Bax C-terminus regulates subcellular location and cell death. Embo J 18: 2330–2341. 15. Jagannadham MV, Sharadadevi A, Nagaraj R (2002) Effects of deleting a tripeptide sequence observed in muscular dystrophy patients on the conforma- tion of synthetic peptides corresponding to the scaffolding domain of caveolin-3. Biochem Biophys Res Commun 298: 203–206. 30. Suzuki M, Youle RJ, Tjandra N (2000) Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103: 645–654. 16. Greenlee RT, Hill-Harmon MB, Murray T, Thun M (2001) Cancer statistics, 2001. CA Cancer J Clin 51: 15–36. 31. Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B (2000) Role of BAX in the apoptotic response to anticancer agents. Science 290: 989–992. 17. Ries LA, Wingo PA, Miller DS, Howe HL, Weir HK, et al. (2000) The annual report to the nation on the status of cancer, 1973–1997, with a special section on colorectal cancer. Cancer 88: 2398–2424. 32. Hasenjager A, Gillissen B, Muller A, Normand G, Hemmati PG, et al. (2004) Smac induces cytochrome c release and apoptosis independently from Bax/Bcl- x(L) in a strictly caspase-3-dependent manner in human carcinoma cells. Oncogene 23: 4523–4535. 18. Fine SW, Lisanti MP, Galbiati F, Li M (2001) Elevated expression of caveolin-1 in adenocarcinoma of the colon. Caveolin-1, a Novel Ku70-Interacting Protein Am J Clin Pathol 115: 719–724. g 33. Tessner TG, Muhale F, Riehl TE, Anant S, Stenson WF (2004) Prostaglandin E2 reduces radiation-induced epithelial apoptosis through a mechanism involving AKT activation and bax translocation. J Clin Invest 114: 1676–1685. J 19. Kim HA, Kim KH, Lee RA (2006) Expression of caveolin-1 is correlated with Akt-1 in colorectal cancer tissues. Exp Mol Pathol 80: 165–170. Akt-1 in colorectal cancer tissues. Exp Mol Pathol 80: 165–170. p 20. Patlolla JM, Swamy MV, Raju J, Rao CV (2004) Overexpression of caveolin-1 in experimental colon adenocarcinomas and human colon cancer cell lines. Oncol Rep 11: 957–963. 34. Brito RA, Medgyesy D, Zukowski TH, Royce ME, Ravandi-Kashani F, et al. (1999) Fluoropyrimidines: a critical evaluation. Oncology 57 Suppl 1: 2–8. ( ) py gy pp 35. Selga E, Morales C, Noe V, Peinado MA, Ciudad CJ (2008) Role of caveolin 1, E-cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells. BMC Med Genomics 1: 35. p 21. Featherstone C, Jackson SP (1999) Ku, a DNA repair protein with multiple cellular functions? Mutat Res 434: 3–15. 22. Kim SH, Kim D, Han JS, Jeong CS, Chung BS, et al. (1999) Ku autoantigen affects the susceptibility to anticancer drugs. Cancer Res 59: 4012–4017. 36. Tencer L, Burgermeister E, Ebert MP, Liscovitch M (2008) Rosiglitazone induces caveolin-1 by PPARgamma-dependent and PPRE-independent mech- anisms: the role of EGF receptor signaling and its effect on cancer cell drug resistance. Anticancer Res 28: 895–906. 23. Ouyang H, Nussenzweig A, Kurimasa A, Soares VC, Li X, et al. (1997) Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination In vivo. J Exp Med 186: 921–929. 37. Goetz JG, Lajoie P, Wiseman SM, Nabi IR (2008) Caveolin-1 in tumor progression: the good, the bad and the ugly. Cancer Metastasis Rev 27: 715– 735. 24. Chechlacz M, Vemuri MC, Naegele JR (2001) Role of DNA-dependent protein kinase in neuronal survival. J Neurochem 78: 141–154. 38. Zhu H, Yue J, Pan Z, Wu H, Cheng Y, et al. (2010) Involvement of Caveolin-1 in repair of DNA damage through both homologous recombination and non- homologous end joining. PLoS One 5: e12055. 25. Cohen HY, Lavu S, Bitterman KJ, Hekking B, Imahiyerobo TA, et al. (2004) Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax- mediated apoptosis. Mol Cell 13: 627–638. References 8. Razani B, Schlegel A, Lisanti MP (2000) Caveolin proteins in signaling, 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
https://openalex.org/W2056782937
https://leicester.figshare.com/articles/journal_contribution/OFD1_and_flotillins_are_integral_components_of_a_ciliary_signaling_protein_complex_organized_by_polycystins_in_renal_epithelia_and_odontoblasts_/10133054/1/files/18261872.pdf
English
null
OFD1 and Flotillins Are Integral Components of a Ciliary Signaling Protein Complex Organized by Polycystins in Renal Epithelia and Odontoblasts
PloS one
2,014
cc-by
12,643
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 Tikkanen [47]. GFP-OFD1 encoding full-length OFD1 fused in September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 11 References 1. Feather SA, Winyard PJ, Dodd S, Woolf AS (1997) Oral-facial-digital syndrome type 1 is another dominant polycystic kidney disease: clinical, radiological and histopathological features of a new kindred. Nephrol Dial Transplant 12: 1354– 1361. 20. Roitbak T, Surviladze Z, Tikkanen R, Wandinger-Ness A (2005) A polycystin multiprotein complex constitutes a cholesterol-containing signalling microdo- main in human kidney epithelia. Biochem J 392: 29–38. y p 21. Boucher CA, Ward HH, Case RL, Thurston KS, Li X, et al. (2011) Receptor protein tyrosine phosphatases are novel components of a polycystin complex. Biochim Biophys Acta 1812: 1225–1238. 2. Jerman S, Wandinger-Ness A, Ward HH (2013) Polycystins and the Pathophysiology of ADPKD. In: Bacallao RL, and Gattone II., V.H., editor. Polycystic Kidney Disease 1st edition electronic book. UK: Future Science Group. 22. Amaddii M, Meister M, Banning A, Tomasovic A, Mooz J, et al. (2012) Flotillin- 1/reggie-2 protein plays dual role in activation of receptor-tyrosine kinase/ mitogen-activated protein kinase signaling. J Biol Chem 287: 7265–7278. 3. Romio L, Fry AM, Winyard PJ, Malcolm S, Woolf AS, et al. (2004) OFD1 is a centrosomal/basal body protein expressed during mesenchymal-epithelial transition in human nephrogenesis. J Am Soc Nephrol 15: 2556–2568. 23. Qian F, Boletta A, Bhunia AK, Xu H, Liu L, et al. (2002) Cleavage of polycystin-1 requires the receptor for egg jelly domain and is disrupted by human autosomal-dominant polycystic kidney disease 1-associated mutations. Proc Natl Acad Sci U S A 99: 16981–16986. 4. Thivichon-Prince B, Couble ML, Giamarchi A, Delmas P, Franco B, et al. (2009) Primary cilia of odontoblasts: possible role in molar morphogenesis. J Dent Res 88: 910–915. 24. Merrick D, Bertuccio CA, Chapin HC, Lal M, Chauvet V, et al. (2013) Polycystin-1 cleavage and the regulation of transcriptional pathways. Pediatr Nephrol. 5. Giorgio G, Alfieri M, Prattichizzo C, Zullo A, Cairo S, et al. (2007) Functional characterization of the OFD1 protein reveals a nuclear localization and physical interaction with subunits of a chromatin remodeling complex. Mol Biol Cell 18: 4397–4404. p 25. Vieira OV, Gaus K, Verkade P, Fullekrug J, Vaz WL, et al. (2006) FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells. Proc Natl Acad Sci U S A 103: 18556–18561. 6. Ward HH, Brown-Glaberman U, Wang J, Morita Y, Alper SL, et al. (2011) A conserved signal and GTPase complex are required for the ciliary transport of polycystin-1. References Mol Biol Cell 22: 3289–3305. 26. Kang RS, Folsch H (2009) An old dog learns new tricks: novel functions of the exocyst complex in polarized epithelia in animals. F1000 Biol Rep 1: 83. 7. Ma R, Li WP, Rundle D, Kong J, Akbarali HI, et al. (2005) PKD2 functions as an epidermal growth factor-activated plasma membrane channel. Mol Cell Biol 25: 8285–8298. 27. Goetz SC, Anderson KV (2010) The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet 11: 331–344. 28. Van Adelsberg J, Chamberlain S, D’Agati V (1997) Polycystin expression is temporally and spatially regulated during renal development. Am J Physiol 272: F602–609. 8. Zullo A, Iaconis D, Barra A, Cantone A, Messaddeq N, et al. (2010) Kidney- specific inactivation of Ofd1 leads to renal cystic disease associated with upregulation of the mTOR pathway. Hum Mol Genet 19: 2792–2803. 29. Geng L, Burrow CR, Li HP, Wilson PD (2000) Modification of the composition of polycystin-1 multiprotein complexes by calcium and tyrosine phosphorylation. Biochim Biophys Acta 1535: 21–35. 9. Ohazama A, Haycraft CJ, Seppala M, Blackburn J, Ghafoor S, et al. (2009) Primary cilia regulate Shh activity in the control of molar tooth number. Development 136: 897–903. p y 30. Thymiakou E, Episkopou V (2011) Detection of signaling effector-complexes downstream of bmp4 using PLA, a proximity ligation assay. J Vis Exp. 10. Yoder BK, Tousson A, Millican L, Wu JH, Bugg CE Jr., et al. (2002) Polaris, a protein disrupted in orpk mutant mice, is required for assembly of renal cilium. Am J Physiol Renal Physiol 282: F541–552. 31. Xu C, Rossetti S, Jiang L, Harris PC, Brown-Glaberman U, et al. (2007) Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in the PKD1 gene exhibit defective ciliary polycystin localization and loss of flow- induced Ca2+ signaling. Am J Physiol Renal Physiol 292: F930–945. 11. Yoder BK, Hou X, Guay-Woodford LM (2002) The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J Am Soc Nephrol 13: 2508–2516. 32. Herbert BS, Grimes BR, Xu WM, Werner M, Ward C, et al. (2013) A telomerase immortalized human proximal tubule cell line with a truncation mutation (Q4004X) in polycystin-1. PLoS One 8: e55191. p 12. Singla V, Romaguera-Ros M, Garcia-Verdugo JM, Reiter JF (2010) Ofd1, a human disease gene, regulates the length and distal structure of centrioles. Dev Cell 18: 410–424. 33. References Lopes CA, Prosser SL, Romio L, Hirst RA, O’Callaghan C, et al. (2011) Centriolar satellites are assembly points for proteins implicated in human ciliopathies, including oral-facial-digital syndrome 1. J Cell Sci 124: 600–612. 13. Tran PV, Talbott GC, Turbe-Doan A, Jacobs DT, Schonfeld MP, et al. (2014) Downregulating Hedgehog Signaling Reduces Renal Cystogenic Potential of Mouse Models. J Am Soc Nephrol. 34. Tang P, Hou H, Zhang L, Lan X, Mao Z, et al. (2014) Autophagy reduces neuronal damage and promotes locomotor recovery via inhibition of apoptosis after spinal cord injury in rats. Mol Neurobiol 49: 276–287. 14. Nait Lechguer A, Couble ML, Labert N, Kuchler-Bopp S, Keller L, et al. (2011) Cell differentiation and matrix organization in engineered teeth. J Dent Res 90: 583–589. 35. Tanos BE, Yang HJ, Soni R, Wang WJ, Macaluso FP, et al. (2013) Centriole distal appendages promote membrane docking, leading to cilia initiation. Genes Dev 27: 163–168. 15. Miettinen PJ, Chin JR, Shum L, Slavkin HC, Shuler CF, et al. (1999) Epidermal growth factor receptor function is necessary for normal craniofacial development and palate closure. Nat Genet 22: 69–73. 36. Joo K, Kim CG, Lee MS, Moon HY, Lee SH, et al. (2013) CCDC41 is required for ciliary vesicle docking to the mother centriole. Proc Natl Acad Sci U S A 110: 5987–5992. 16. Otto EA, Ramaswami G, Janssen S, Chaki M, Allen SJ, et al. (2011) Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy. J Med Genet 48: 105–116. 37. Kim J, Krishnaswami SR, Gleeson JG (2008) CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium. Hum Mol Genet 17: 3796–3805. p g g q g gy J 17. Suda N, Kitahara Y, Ohyama K (2006) A case of amelogenesis imperfecta, cleft lip and palate and polycystic kidney disease. Orthod Craniofac Res 9: 52–56. p p p y y y 18. Salzer U, Prohaska R (2001) Stomatin, flotillin-1, and flotillin-2 are major integral proteins of erythrocyte lipid rafts. Blood 97: 1141–1143. 38. Sillibourne JE, Hurbain I, Grand-Perret T, Goud B, Tran P, et al. (2013) Primary ciliogenesis requires the distal appendage component Cep123. Biol Open 2: 535–545. integral proteins of erythrocyte lipid rafts. Blood 97: 1141–1143. 19. Neumann-Giesen C, Falkenbach B, Beicht P, Claasen S, Luers G, et al. Acknowledgments We thank the Spatiotemporal Modeling Center (STMC) at the University of New Mexico and UAB Global Center for Craniofacial Oral and Dental Disorders. Some experiments used the facilities or services provided by the Keck-UNM Genomics Resource, a facility supported by the State of New Mexico and the UNM Cancer Center. Images were generated and quantified in the University of New Mexico Cancer Center Fluorescence Microscopy Facility, supported by P30 CA118100. Diane Lidke, Genevieve Phillips, Carolyn Pehlke, Tamara Howard and Jason Byars are thanked for helpful discussions. Movie S1 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 efficiencies that were consistently ,10%. Lipofectamine 2000 (Life Technologies) resulted in transfection efficiencies that were consistently ,10%. (TIF) OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain 40. Hanaoka K, Qian F, Boletta A, Bhunia AK, Piontek K, et al. (2000) Co- assembly of polycystin-1 and -2 produces unique cation-permeable currents. Nature 408: 990–994. 52. Christensen ST, Clement CA, Satir P, Pedersen LB (2012) Primary cilia and coordination of receptor tyrosine kinase (RTK) signalling. J Pathol 226: 172– 184. 53. Manzanares D, Monzon ME, Savani RC, Salathe M (2007) Apical oxidative hyaluronan degradation stimulates airway ciliary beating via RHAMM and RON. Am J Respir Cell Mol Biol 37: 160–168. 41. Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, et al. (2003) Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat Genet 33: 129–137. 54. McCleese JK, Bear MD, Kulp SK, Mazcko C, Khanna C, et al. (2013) Met interacts with EGFR and Ron in canine osteosarcoma. Vet Comp Oncol 11: 124–139. 42. Griffiths GM, Tsun A, Stinchcombe JC (2010) The immunological synapse: a focal point for endocytosis and exocytosis. J Cell Biol 189: 399–406. 43. Baldari CT, Rosenbaum J (2010) Intraflagellar transport: it’s not just for cilia anymore. Curr Opin Cell Biol 22: 75–80. 55. Benvenuti S, Lazzari L, Arnesano A, Li Chiavi G, Gentile A, et al. (2011) Ron kinase transphosphorylation sustains MET oncogene addiction. Cancer Res 71: 1945–1955. 44. Rajendran L, Beckmann J, Magenau A, Boneberg EM, Gaus K, et al. (2009) Flotillins are involved in the polarization of primitive and mature hematopoietic cells. PLoS One 4: e8290. 56. Peace BE, Hill KJ, Degen SJ, Waltz SE (2003) Cross-talk between the receptor tyrosine kinases Ron and epidermal growth factor receptor. Exp Cell Res 289: 317–325. 45. Lui-Roberts WW, Stinchcombe JC, Ritter AT, Akhmanova A, Karakesisoglou I, et al. (2012) Cytotoxic T lymphocyte effector function is independent of nucleus- centrosome dissociation. Eur J Immunol 42: 2132–2141. 57. Liu HS, Hsu PY, Lai MD, Chang HY, Ho CL, et al. (2010) An unusual function of RON receptor tyrosine kinase as a transcriptional regulator in cooperation with EGFR in human cancer cells. Carcinogenesis 31: 1456–1464. centrosome dissociation. Eur J Immunol 42: 2132–2141. 46. Banning A, Tomasovic A, Tikkanen R (2011) Functional aspects of membrane association of reggie/flotillin proteins. Curr Protein Pept Sci 12: 725–735. g 58. Horie S, Higashihara E, Nutahara K, Mikami Y, Okubo A, et al. (1994) Mediation of renal cyst formation by hepatocyte growth factor. Lancet 344: 789–791. 47. References (2004) Membrane and raft association of reggie-1/flotillin-2: role of myristoylation, palmitoylation and oligomerization and induction of filopodia by overexpres- sion. Biochem J 378: 509–518. 39. Ye X, Zeng H, Ning G, Reiter JF, Liu A (2014) C2cd3 is critical for centriolar distal appendage assembly and ciliary vesicle docking in mammals. Proc Natl Acad Sci U S A 111: 2164–2169. September 2014 | Volume 9 | Issue 9 | e106330 September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org 13 OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain OFD1, Polycystins, EGFR and Flotillins Form Ciliary Microdomain Neumann-Giesen C, Fernow I, Amaddii M, Tikkanen R (2007) Role of EGF- induced tyrosine phosphorylation of reggie-1/flotillin-2 in cell spreading and signaling to the actin cytoskeleton. J Cell Sci 120: 395–406. 59. Qin S, Taglienti M, Cai L, Zhou J, Kreidberg JA (2012) c-Met and NF-kappaB- dependent overexpression of Wnt7a and -7b and Pax2 promotes cystogenesis in polycystic kidney disease. J Am Soc Nephrol 23: 1309–1318. 48. Meister M, Tomasovic A, Banning A, Tikkanen R (2013) Mitogen-Activated Protein (MAP) Kinase Scaffolding Proteins: A Recount. Int J Mol Sci 14: 4854– 4884. p y y y p 60. Zheleznova NN, Wilson PD, Staruschenko A (2011) Epidermal growth factor- mediated proliferation and sodium transport in normal and PKD epithelial cells. Biochim Biophys Acta 1812: 1301–1313. 49. Kurrle N, Ockenga W, Meister M, Vollner F, Kuhne S, et al. (2013) Phosphatidylinositol 3-Kinase dependent upregulation of the epidermal growth factor receptor upon Flotillin-1 depletion in breast cancer cells. BMC Cancer 13: 575. 61. MacDougall M, Thiemann F, Ta H, Hsu P, Chen LS, et al. (1995) Temperature sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell cultures: establishment of clonal odontoblast cell line. Connect Tissue Res 33: 97–103. 50. Zhang ZR, Chu WF, Song B, Gooz M, Zhang JN, et al. (2013) TRPP2 and TRPV4 form an EGF-activated calcium permeable channel at the apical membrane of renal collecting duct cells. PLoS One 8: e73424. 62. Ishikawa H, Thompson J, Yates JR 3rd, Marshall WF (2012) Proteomic analysis of mammalian primary cilia. Curr Biol 22: 414–419. 51. Marley A, Choy RW, von Zastrow M (2013) GPR88 reveals a discrete function of primary cilia as selective insulators of GPCR cross-talk. PLoS One 8: e70857. 63. Nelson DL (1995) Preparation of cilia and subciliary fractions from Paramecium. Methods Cell Biol 47: 17–24. September 2014 | Volume 9 | Issue 9 | e106330 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 14
https://openalex.org/W4388328041
https://scindeks-clanci.ceon.rs/data/pdf/0350-8501/2023/0350-85012398215Q.pdf
Serbian
null
Water usage under the concession model in the law of Republika Srpska
Zbornik radova Pravnog fakulteta u Nišu
2,023
cc-by-sa
10,222
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 заштиту водених ресурса. Литература / References Бабић, И. Медић, Д. Хашић, Е. Повлакић, М. Велић, Л. (2011). Коментар Закона о стварним правима Републике Српске. Сарајево: Привредна штампа. Бабић, И. Јотановић, Р. (2020). Грађанско право, Књига 2 – Стварно право. Бања Лука: Правни факултет Универзитета у Бањој Луци. Бабић, И. Медић, Д. Хашић, Е. Повлакић, М. Велић, Л. (2011). Коментар Закона о стварним правима Републике Српске. Сарајево: Привредна штампа. Закона о стварним правима Републике Српске. Сарајево: Привредна штампа. Бабић, И. Јотановић, Р. (2020). Грађанско право, Књига 2 – Стварно право. Бања Лука: Правни факултет Универзитета у Бањој Луци. Бабић, И. Јотановић, Р. (2020). Грађанско право, Књига 2 – Стварно право. Бања Лука: Правни факултет Универзитета у Бањој Луци. Боројевић, К. (2015). Поступак додјела концесија према Закону о 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 successful model of public-privte partnership. Revizor. 23(91−92). 23−36. Vassileva, A. Ignjatijević S. (2020). BOT (build-operate transfer) projects as a successful model of public-privte partnership. Revizor. 23(91−92). 23−36. Vodinelić, V. V. (2012). Građansko pravo: uvod u građansko pravo i opšti deo gra- 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 Саду. 2(XXXVIII). 389−409. Јотановић, Р. (2015). Посебно правно регулисање за одређене врсте ствари у праву Републике Српске. Правна ријеч. 43(XII). 187−207. Gavella, N. (2012). Građansko pravo i posebna pravna uređenja za određene vrste dobara. Zbornik Pravnog fakulteta Univerziteta u Zagrebu. 62(5−6). 1445−1490. Радић, Д. (2021). Правно уређење природних ресурса у Републици Српској. Бања Лука: Правни факултет Универзитета у Бањој Луци. Устав Републике Српске, Сл. гласник РС, бр. 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, 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, 26/21 и 44/22. Устав БиХ. Преузето 23. 2. 2023. https://www.paragraf.ba/propisi/bih/ustav- 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 hidroelektrane na teritoriji Bosne i Hercegovine. Преузето 16. 2. 2023. https:// czzs.org/wp-content/uploads/2018/11/Analiza-MHE1.pdf. 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. Документ о политици додјеле концесија. Преузето 13. 2. 2023. https://kon- cesije-rs.org/wp-content/uploads/2019/06/Politika_dodjela-Cir.pdf. Документ о политици додјеле концесија. Преузето 13. 2. 2023. https://kon- cesije-rs.org/wp-content/uploads/2019/06/Politika_dodjela-Cir.pdf. 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. 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. Zabrana izgradnje malih hidroelektrana na Zapadnom Balkanu − Uporedna ana- 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
W4386716949.txt
https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/64/e3sconf_icgee2023_01010.pdf
en
A Review Study on the Effect of Nanomaterials and Local Materials on Soil Geotechnical Properties
E3S web of conferences
2,023
cc-by
4,810
E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 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/). E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 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) . 2 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 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]. 3 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 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). 4 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 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]. 5 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 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 6 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 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]. 7 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 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]. 8 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 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. 9 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 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. 10 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 • • https://doi.org/10.1051/e3sconf/202342701010 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. REFERENCES [1] Sakr, M., Azzam, W., and El Sawwaf, M. Behavior of Soft Clay Soil Stabilized with Lime Columns. Journal of Multidisciplinary Engineering Science Studies (JMESS). 2021; 7(4): 3809–3813. [2] Ali Behnood. Soil and clay stabilization with calcium- and non-calcium-based additives: A state-of-the-art review of challenges. Approaches and techniques. 2018; 17(1): 14–32. [3] Ito, A., and Wagai, R. Data Descriptor: Global distribution of clay-size minerals on land surface for biogeochemical and climatological studies. Scientific Data. 2017; 4(1):1–11. [4] Ramos, M., R., Uhlmann, A., , Dedecek, R. A., and and Curcio, G. R. (2015). Clay mineralogy and genesis of fragipan in soils from Southeast Brazil. Catena. 2015; 135(1): 22-28. [5] KARKUSH, Mahdi O.; ALMURSHEDI, Alaa D.; KARIM, Hussein H. Investigation of the Impacts of Nanomaterials on the Micromechanical Properties of Gypseous Soils. Arabian Journal for Science and Engineering. 2023; 48(1): 665-675.. [6] Pashabavandpouri, M. A., and Jahangiri, S. Effect of nano silica on swelling, compaction and strength properties of clayey soil stabilized with lime. J. Appl. Environ. Biol. Sci. 2015; 5(7S): 538–548. www.textroad.com [7] Ghasabkolaei N, Choobbasti AJ, Roshan N, Ghasemi SE. Geotechnical properties of the soils modified with nanomaterials: A comprehensive review. Archives of civil and mechanical engineering. 2017 Sep;17:639-50. [8] Handy RL, Jordan JL, Manfre LE, Davidson DT. Chemical Treatments for Surface Hardening of SoilCement and Soil-Lime-Flyash. Highway Research Board Bulletin. 1960(241). [9] Koyluoglu, U. Soil mechanics for unsaturated soils. Soil Dynamics and Earthquake Engineering.1993; 12(7): 449–450. [10] Al-Murshedi, A. D., Karkush, M. O., and Karim, H. H. Collapsibility and shear strength of gypseous soil improved by nano silica fume (Nsf). Key Engineering Materials, 857 KEM(August), 2020; 292–301. [11] Karkush, M. O., Al-Murshedi, A. D., and Karim, H. H. Investigation of the impacts of nano-clay on the collapse potential and geotechnical properties of Gypseous soils. Jordan Journal of Civil Engineering. 2020; 14(4): 537–547. [12] JOJU, M. R.; CHANDRAKARAN, S. Comparative study on stabilization of marine clay using nano-silica and lime. In: Problematic Soils and Geoenvironmental Concerns: Proceedings of IGC 2018. Springer Singapore, 2021. [13] Sabat, A. K., and Muni, P. K. (2015). Effects of limestone dust on geotechnical properties of an expansive soil. International Journal of Applied Engineering Research. 2015; 10(17): 37724–37730. [14] IBRAHIM, Hawkar Hashim, et al. Improving the geotechnical properties of high expansive clay using limestone powder. Innovative Infrastructure Solutions. 2020; 5(3): 112. [15] Pastor, J. L., Tomás, R., Cano, M., Riquelme, A., and Gutiérrez, E. Evaluation of the improvement effect of limestone powder waste in the stabilization of Swelling Clayey Soil. Sustainability (Switzerland). 2019; 11(3). [16] Eswaramoorthi, P., Kumar, V. S., Prabhu, P. S., Prabu, T., and Lavanya, S. (2017). Influence of nanosized silica and lime particles on the behaviour of soil. International Journal of Civil Engineering and Technology. 2017; 8(9): 353–360. [17] Kalhor, A., Ghazavi, M., and Roustaei, M. Impacts of Nano-silica on Physical Properties and Shear Strength of Clayey Soil. Arabian Journal for Science and Engineering. 2022; 47(4): 5271–5279. [18] BUTRÓN, Christian; AXELSSON, Magnus; GUSTAFSON, Gunnar. Silica sol for rock grouting: Laboratory testing of strength, fracture behaviour and hydraulic conductivity. Tunnelling and Underground Space Technology. 2009; 24(6): 603-607. [19] Ren, X., and Hu, K. Effect of nanosilica on the physical and mechanical properties of silty clay. Nanoscience and Nanotechnology Letters. 2014; 6(11): 1010–1013. [20] Changizi, F., and Haddad, A. Strength properties of soft clay treated with mixture of nano-SiO2 and recycled polyester fiber. Journal of Rock Mechanics and Geotechnical Engineering. 2015; 7(4): 367–378. [21] Ghavami, S., Farahani, B., Jahanbakhsh, H., and Moghadas Nejad, F. (2018). Effects of Silica Fume and Nano-silica on the Engineering Properties of Kaolinite Clay. AUT Journal of Civil Engineering, 2(2), 135– 142. [22] Fattahi Masrour F, Naghdipour Mirsadeghi M, MolaAbasi H, Jamshidi Chenari R. Effect of nanosilica on the macro-and microbehavior of dispersive clays. Journal of Materials in Civil Engineering. 2021 Dec 1;33(12):04021349. [23] Hussan, Z. A., and Al-Janabi, A. T. Studying some mechanical properties of soft soils treated by CaCo3Nano Material. International Journal of Civil Engineering and Technology. 2018; 9(11): 870–877. [24] Kong R, Zhang F, Wang G, Peng J. Stabilization of loess using nano-SiO2. Materials. 2018 Jun 14;11(6):1014. 11 E3S Web of Conferences 427, 01010 (2023) ICGEE 2023 https://doi.org/10.1051/e3sconf/202342701010 [25] Kalhor A, Ghazavi M, Roustaei M, Mirhosseini SM. Influence of nano-SiO2 on geotechnical properties of fine soils subjected to freeze-thaw cycles. Cold Regions Science and Technology. 2019 May 1;161:12936. [26] Gelsefifi, S. A. S., and Mamaghanian, J. Stabilization of a Weak Low Plasticity Clay Soil Using Nanomaterial. 5the International Young Geotechnical Engineers Conference (IYGEC). 2013. [27] Ahmadi H, Shafiee O. Experimental comparative study on the performance of nano-SiO2 and microsilica in stabilization of clay. The European Physical Journal Plus. 2019 Sep 23;134(9):459. [28] Mohammadi M, Rajabi AM, Khodaparast M. Experimental and numerical evaluation of the effect of nano calcium carbonate on geotechnical properties of clayey sand soil. KSCE Journal of Civil Engineering. 2022 Jan;26(1):35-46. [29] Yong, L. L., Perera, S. V. A. D. N. J., Syamsir, A., Emmanuel, E., Paul, S. C., and Anggraini, V. Stabilization of a residual soil using calcium and magnesium hydroxide nanoparticles: A quick precipitation method. Applied Sciences (Switzerland).2019;9(20),1–15. https://doi.org/10.3390/app9204325 [30] Taipodia J., Dutta J., D. A. K. Effect of nanoparticles on properties of soil. Proc. of Indian Geotechnical Conference, Kochi. 2011. 12
W2167717183.txt
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0079529&type=printable
en
Selected Neurophysiological, Psychological, and Behavioral Influences on Subjective Sleep Quality in Nurses: A Structure Equation Model
PloS one
2,013
cc-by
5,811
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 PLOS ONE | www.plosone.org 1 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 PLOS ONE | www.plosone.org 2 November 2013 | Volume 8 | Issue 11 | e79529 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 3 November 2013 | Volume 8 | Issue 11 | e79529 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 PLOS ONE | www.plosone.org 4 November 2013 | Volume 8 | Issue 11 | e79529 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. PLOS ONE | www.plosone.org 5 November 2013 | Volume 8 | Issue 11 | e79529 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. References 6. Ishihara K, Miyake S, Miyasita A, Miyata Y (1988) Comparisons of sleep-wake habits of morning and evening types in Japanese worker sample. J Hum Ergol 17: 111–118. 7. Monk TH, Petrie SR, Hayes AJ, Kupfer DJ (1994) Regularity of daily life in relation to personality, age, gender, sleep quality and circadian rhythms. J Sleep Res 3: 196–205. 8. Furihata R, Uchiyama M, Takahashi S, Konno C, Suzuki M, et al. (2011) Selfhelp behaviors for sleep and depression: a Japanese nationwide general population survey. J Affect Disord 130: 75–82. 9. Ardekani ZZ, Kakooei H, Ayattollahi SM, Choobineh A, Seraji GN (2008) Prevalence of mental disorders among shift work hospital nurses in Shiraz, Iran. Pak J Biol Sci 11: 1605–1609. 10. Gonda X, Telek T, Juhasz G, Lazary J, Vargha A, et al. (2008) Patterns of mood changes throughout the reproductive cycle in healthy women without 1. Jacobs GD, Pace-Schott EF, Stickgold R, Otto MW (2004) Cognitive behavior therapy and pharmacotherapy for insomnia: a randomized controlled trial and direct comparison. Arch Intern Med 164: 1888–1896. 2. Smith MT, Perlis ML, Park A, Smith MS, Pennington J, et al. (2002) Comparative meta-analysis of pharmacotherapy and behavior therapy for persistent insomnia. Am J Psychiatry 159: 5–11. 3. Stepanski EJ, Wyatt JK (2003) Use of sleep hygiene in the treatment of insomnia. Sleep Med Rev 7: 215–225. 4. van der Heijden KB, Smits MG, Gunning WB (2006) Sleep hygiene and actigraphically evaluated sleep characteristics in children with ADHD and chronic sleep onset insomnia. J Sleep Res 15: 55–62. 5. Jefferson CD, Drake CL, Scofield HM, Myers E, McClure T, et al. (2005) Sleep hygiene practices in a population-based sample of insomniacs. Sleep 28: 611– 615. PLOS ONE | www.plosone.org 6 November 2013 | Volume 8 | Issue 11 | e79529 Sleep and Structure Equation Model 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Buysse DJ, Reynolds CF 3rd, Monk TH, Berman SR, Kupfer DJ (1989) The Pittsburgh Sleep Quality Index: a new instrument for psychiatric practice and research. Psychiatry Res 28: 193–213. 32. Backhaus J, Junghanns K, Broocks A, Riemann D, Hohagen F (2002) Test-retest reliability and validity of the Pittsburgh Sleep Quality Index in primary insomnia. J Psychosom Res 53: 737–740. 33. Beck SL, Schwartz AL, Towsley G, Dudley W, Barsevick A (2004) Psychometric evaluation of the Pittsburgh Sleep Quality Index in cancer patients. J Pain Symptom Manage 27: 140–148. 34. Ruggiero JS (2003) Correlates of fatigue in critical care nurses. Res Nurs Health 26: 434–444. 35. Tsai PS (2005) Psychometric assessment of the Chinese version of the Pittsburgh Sleep Quality Index in primary insomnia and control participants. Appl Psychophys and Biof 30: 404–404. 36. Coffman DL, MacCallum RC (2005) Using parcels to convert path analysis models into latent variable models. Multivar Behav Res 40: 235–259. 37. MacCallum RC, Widaman KF, Zhang SB, Hong SH (1999) Sample size in factor analysis. Psychol Meth 4: 84–99. 38. Kline RB (1998) Principles and practice of structural equation modelling. New York: NY: The Guilford Press. 39. Baron RM, Kenny DA (1986) The moderator-mediator variable distinction in social psychological research: conceptual, strategic, and statistical considerations. J Pers Soc Psychol 51: 1173–1182. 40. Sobel ME (1982) Asymptotic Confidence Intervals for Indirect Effects in Structural Equation Models. Sociol Methodol 13: 290–312. 41. MacKinnon DP, Fritz MS, Williams J, Lockwood CM (2007) Distribution of the product confidence limits for the indirect effect: program PRODCLIN. Behav Res Methods 39: 384–389. 42. Nakade M, Takeuchi H, Kurotani M, Harada T (2009) Effects of meal habits and alcohol/cigarette consumption on morningness-eveningness preference and sleep habits by Japanese female students aged 18–29. J Physiol Anthropol 28: 83–90. 43. Chen PH, Kuo HY, Chueh KH (2010) Sleep hygiene education: efficacy on sleep quality in working women. J Nurs Res 18: 283–289. 44. Suen LK, Tam WW, Hon KL (2010) Association of sleep hygiene-related factors and sleep quality among university students in Hong Kong. Hong Kong Med J 16: 180–185. 45. Wong ML, Lau EY, Wan JH, Cheung SF, Hui CH, et al. (2013) The interplay between sleep and mood in predicting academic functioning, physical health and psychological health: a longitudinal study. J Psychosom Res 74: 271–277. 46. Shao MF, Chou YC, Yeh MY, Tzeng WC (2010) Sleep quality and quality of life in female shift-working nurses. J Adv Nurs 66: 1565–1572. 47. Strine TW, Chapman DP, Ahluwalia IB (2005) Menstrual-related problems and psychological distress among women in the United States. J Womens Health (Larchmt) 14: 316–323. 48. Selvi Y, Gulec M, Agargun MY, Besiroglu L (2007) Mood changes after sleep deprivation in morningness-eveningness chronotypes in healthy individuals. J Sleep Res 16: 241–244. 49. Russo PM, Bruni O, Lucidi F, Ferri R, Violani C (2007) Sleep habits and circadian preference in Italian children and adolescents. J Sleep Res 16: 163– 169. premenstrual dysphoric disorders. Prog Neuropsychopharmacol Biol Psychiatry 32: 1782–1788. Lund HG, Reider BD, Whiting AB, Prichard JR (2010) Sleep patterns and predictors of disturbed sleep in a large population of college students. J Adolesc Health 46: 124–132. Baker FC, Driver HS (2007) Circadian rhythms, sleep, and the menstrual cycle. Sleep Med 8: 613–622. Saper CB, Scammell TE, Lu J (2005) Hypothalamic regulation of sleep and circadian rhythms. Nature 437: 1257–1263. Natale V, Cicogna P (2002) Morningness-eveningness dimension: is it really a continuum? Pers Individ Dif 32: 809–816. Kerkhof GA (1998) The 24-hour variation of mood differs between morningand evening-type individuals. Percept Mot Skills 86: 264–266. Chung MH, Chang FM, Yang CC, Kuo TB, Hsu N (2009) Sleep quality and morningness-eveningness of shift nurses. J Clin Nurs 18: 279–284. Selvi Y, Gulec M, Agargun MY, Besiroglu L (2007) Mood changes after sleep deprivation in morningness-eveningness chronotypes in healthy individuals. J Sleep Res 16: 241–244. Strine TW, Chapman DP, Ahluwalia IB (2005) Menstrual-related problems and psychological distress among women in the United States. J Womens Health 14: 316–323. Kline RB (2005) Principles and practice of structural equation modeling. New York: Guilford Press. Horne JA, Ostberg O (1976) A self-assessment questionnaire to determine morningness-eveningness in human circadian rhythms. Int J Chronobiol 4: 97– 110. Giannotti F, Cortesi F, Sebastiani T, Ottaviano S (2002) Circadian preference, sleep and daytime behaviour in adolescence. J Sleep Res 11: 191–199. Shieh KK (1995) Chinese version of the morningness-eveningness questionnaire conference on Ergonomics and Occupational Safety and Health. Taipei city, Taiwan, R.O.C. Moos RH (1968) The development of a menstrual distress questionnaire. Psychosom Med 30: 853–867. Lieber MR, Winans CS, Griem ML, Moossa R, Elner VM, et al. (1985) Sarcomas arising after radiotherapy for peptic ulcer disease. Dig Dis Sci 30: 593–599. Moos RH (1985) Perimenstrual symptoms: A manual and overview of research with the menstrual distress questionnaire. Palo Alto. (CA): Stanford University. Lacks P, Rotert M (1986) Knowledge and practice of sleep hygiene techniques in insomniacs and good sleepers. Behav Res Ther 24: 365–368. Brown FC, Buboltz WC, Soper B (2002) Relationship of sleep hygiene awareness, sleep hygiene practices, and sleep quality in university students. Behav Med 28: 33–38. McNair DM, Lorr M, Droppleman LF (1992) Manual of the Profile of Mood States. San Diego, CA.: Educational and Industrial Testing Service. Wang RC, Wang SJ, Chang YC, Lin CC (2007) Mood state and quality of sleep in cancer pain patients: a comparison to chronic daily headache. J Pain Symptom Manage 33: 32–39. Lin CC, Lai YL, Ward SE (2003) Effect of cancer pain on performance status, mood status, and level of hope among Taiwanese cancer patients. J Pain Symptom Manage 25: 29–37. PLOS ONE | www.plosone.org 7 November 2013 | Volume 8 | Issue 11 | e79529
https://openalex.org/W2113306639
https://opus.bibliothek.uni-augsburg.de/opus4/files/85424/pgen.1002607.pdf
English
null
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 H lth S i C t H t T 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 i l M di l S h l U i it f E t M d l R d y gy y 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 l S h l U i i f E 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 f Bi i i B U i i S h l 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, J h H ki U i it B lti M l d USA 33 Department of Epidemiology, Bloomberg School o 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 y 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 p g y 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, L d U i i U i i H i l M l M l S d 102 Department of Clinical Sciences, Diabetes and Endocrinology, 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 y g g 125 MRC Epidemiology Resource Centre, University of Southampton, y y g g 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 N i l I i f H l h B l i M l d USA 72 Lab of Cardiovascular Sciences, National Institute , 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, J h H ki U i i B l i M l d USA g Johns Hopkins University, Baltimore, Maryland, USA 116 Departments of Medicine, Epidemiology and Health Services, University of Washington, Seattle, Washington, USA. J p y y 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 p 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 y , g y 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 p 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) Associations of adiponectin, resistin, and tumor necrosis factor-alpha with insulin resistance. J Clin Endocrinol Metab 93: 3165–3172. 17. Jee SH, Sull JW, Lee JE, Shin C, Park J, et al. (2010) Adiponectin concentrations: a genome-wide association study. Am J Hum Genet 87: 545–552. a genome-wide association study. Am J Hum Genet 87: 545–552. 18. Wu Y, Li Y, Lange EM, Croteau-Chonka DC, Kuzawa CW, et al. (2010) Genome-wide association study for adiponectin levels in Filipino women identifies CDH13 and a novel uncommon haplotype at KNG1-ADIPOQ. Hum Mol Genet 19: 4955–4964. 2. Tilg H, Moschen AR (2006) Adipocytokines: mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol 6: 772–783. 2. Tilg H, Moschen AR (2006) Adipocytokines: mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol 6: 772–783. 3. Pischon T, Girman CJ, Hotamisligil GS, Rifai N, Hu FB, et al. (2004) Plasma Adiponectin Levels and Risk of Myocardial Infarction in Men. JAMA: The Journal of the American Medical Association 291: 1730–1737. 19. Voight BF, Scott LJ, Steinthorsdottir V, Morris AP, Dina C, et al. (2010) Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis. Nat Genet 42: 579–589. 4. Li S, Shin HJ, Ding EL, van Dam RM (2009) Adiponectin levels and risk of type 2 di b t t ti i d t l i JAMA 302 179 188 4. Li S, Shin HJ, Ding EL, van Dam RM (2009) Adiponectin levels and risk of type 2 diabetes: a systematic review and meta-analysis. JAMA 302: 179–188. 4. Li S, Shin HJ, Ding EL, van Dam RM (2009) Adiponectin levels and risk of type 2 diabetes: a systematic review and meta-analysis. JAMA 302: 179–188. 20. Dupuis J, Langenberg C, Prokopenko I, Saxena R, Soranzo N, et al. (2010) New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk. Nat Genet 42: 105–116. 5. Stumvoll M, Goldstein BJ, van Haeften TW (2005) Type 2 diabetes: principles of pathogenesis and therapy. Lancet 365: 1333–1346. 21. Lindgren CM, Heid IM, Randall JC, Lamina C, Steinthorsdottir V, et al. (2009) Genome-wide association scan meta-analysis identifies three Loci influencing adiposity and fat distribution. PLoS Genet 5: e1000508. doi:10.1371/ journal.pgen.1000508. 6. Nawrocki AR, Rajala MW, Tomas E, Pajvani UB, Saha AK, et al. References (2006) Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to peroxisome proliferator-activated receptor gamma agonists. J Biol Chem 281: 2654–2660. 22. Teslovich TM, Musunuru K, Smith AV, Edmondson AC, Stylianou IM, et al. (2010) Biological, clinical and population relevance of 95 loci for blood lipids. Nature 466: 707–713. 7. Wang Y, Zhou M, Lam KS, Xu A (2009) Protective roles of adiponectin in obesity-related fatty liver diseases: mechanisms and therapeutic implications. Arq Bras Endocrinol Metabol 53: 201–212. 23. Kilpela¨inen TO, Zillikens MC, Stancˇa´kova´ A, Finucane FM, Ried JS, et al. (2011) Genetic variation near IRS1 associates with reduced adiposity and an impaired metabolic profile. Nature Genetics In press. 8. Comuzzie AG, Funahashi T, Sonnenberg G, Martin LJ, Jacob HJ, et al. (2001) The genetic basis of plasma variation in adiponectin, a global endophenotype for obesity and the metabolic syndrome. Journal of Clinical Endocrinology and Metabolism 86: 4321–4325. 24. Morris AP (2011) Transethnic meta-analysis of genomewide association studies. Genetic epidemiology 35: 809–822. 9. Vasseur F, Helbecque N, Dina C, Lobbens S, Delannoy V, et al. (2002) Single- nucleotide polymorphism haplotypes in the both proximal promoter and exon 3 of the APM1 gene modulate adipocyte-secreted adiponectin hormone levels and contribute to the genetic risk for type 2 diabetes in French Caucasians. Hum Mol Genet 11: 2607–2614. 25. Nica AC, Parts L, Glass D, Nisbet J, Barrett A, et al. (2011) The Architecture of Gene Regulatory Variation across Multiple Human Tissues: The MuTHER Study. PLoS Genet 7: e1002003. doi:10.1371/journal.pgen.1002003. 26. Nyholt DR (2004) A simple correction for multiple testing for single-nucleotide polymorphisms in linkage disequilibrium with each other. Am J Hum Genet 74: 765–769. 10. Cesari M, Narkiewicz K, De Toni R, Aldighieri E, Williams CJ, et al. (2007) Heritability of plasma adiponectin levels and body mass index in twins. J Clin Endocrinol Metab 92: 3082–3088. 27. Heid IM, Jackson AU, Randall JC, Winkler TW, Qi L, et al. (2010) Meta- analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution. Nat Genet 42: 949–960. 11. Liu PH, Jiang YD, Chen WJ, Chang CC, Lee TC, et al. (2008) Genetic and environmental influences on adiponectin, leptin, and BMI among adolescents in Taiwan: a multivariate twin/sibling analysis. Twin Res Hum Genet 11: 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, Finland. 114 Clinical Pharmacology and Barts and The London Genome Centre, 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 Vej 9B, DK-5000, Odense, Denmark. PLoS Genetics | www.plosgenetics.org March 2012 | Volume 8 | Issue 3 | e1002607 March 2012 | Volume 8 | Issue 3 | e1002607 21 Adiponectin: Multiethnic Genome-Wide Meta-Analysis 34. Hu Y, Sun CY, Huang J, Hong L, Zhang L, et al. (2007) Antimyeloma effects of resveratrol through inhibition of angiogenesis. Chin Med J (Engl) 120: 1672–1677. 45. Wise A, Foord SM, Fraser NJ, Barnes AA, Elshourbagy N, et al. (2003) Molecular identification of high and low affinity receptors for nicotinic acid. J Biol Chem 278: 9869–9874. J 46. Westphal S, Borucki K, Taneva E, Makarova R, Luley C (2006) Adipokines and treatment with niacin. Metabolism 55: 1283–1285. 35. Szkudelski T, Szkudelska K (2011) Anti-diabetic effects of resveratrol. Ann N Y Acad Sci 1215: 34–39. 47. Plaisance EP, Lukasova M, Offermanns S, Zhang Y, Cao G, et al. (2009) Niacin stimulates adiponectin secretion through the GPR109A receptor. Am J Physiol Endocrinol Metab 296: E549–558. 36. Ahn J, Lee H, Kim S, Ha T (2007) Resveratrol inhibits TNF-alpha-induced changes of adipokines in 3T3-L1 adipocytes. Biochem Biophys Res Commun 364: 972–977. 48. Edmondson AC, Braund PS, Stylianou IM, Khera AV, Nelson CP, et al. (2011) Dense Genotyping of Candidate Gene Loci Identifies Variants Associated with High-Density Lipoprotein Cholesterol. Circ Cardiovasc Genet. 37. Kang L, Heng W, Yuan A, Baolin L, Fang H (2010) Resveratrol modulates adipokine expression and improves insulin sensitivity in adipocytes: Relative to inhibition of inflammatory responses. Biochimie 92: 789–796. High-Density Lipoprotein Cholesterol. Circ Cardiovasc Genet. 49. Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, et al. (2001) Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 86: 1930–1935. 38. Kiss-Toth E, Bagstaff SM, Sung HY, Jozsa V, Dempsey C, et al. (2004) Human tribbles, a protein family controlling mitogen-activated protein kinase cascades. J Biol Chem 279: 42703–42708. J 39. Sung HY, Guan H, Czibula A, King AR, Eder K, et al. (2007) Human tribbles-1 controls proliferation and chemotaxis of smooth muscle cells via MAPK signaling pathways. J Biol Chem 282: 18379–18387. 50. Lindsay RS, Funahashi T, Hanson RL, Matsuzawa Y, Tanaka S, et al. (2002) Adiponectin and development of type 2 diabetes in the Pima Indian population. The Lancet 360: 57–58. 40. Waterworth DM, Ricketts SL, Song K, Chen L, Zhao JH, et al. (2010) Genetic variants influencing circulating lipid levels and risk of coronary artery disease. Arterioscler Thromb Vasc Biol 30: 2264–2276. 51. Keating BJ, Tischfield S, Murray SS, Bhangale T, Price TS, et al. References (2009) Genetic variant near IRS1 is associated with type 2 diabetes, insulin resistance and hyperinsulinemia. Nat Genet 41: 1110–1115. 12. Menzaghi C, Trischitta V, Doria A (2007) Genetic influences of adiponectin on insulin resistance, type 2 diabetes, and cardiovascular disease. Diabetes 56: 1198–1209. 29. Takeuchi F, Serizawa M, Yamamoto K, Fujisawa T, Nakashima E, et al. (2009) Confirmation of multiple risk Loci and genetic impacts by a genome-wide association study of type 2 diabetes in the Japanese population. Diabetes 58: 1690–1699. 13. Hivert MF, Manning AK, McAteer JB, Florez JC, Dupuis J, et al. (2008) Common variants in the adiponectin gene (ADIPOQ) associated with plasma adiponectin levels, type 2 diabetes, and diabetes-related quantitative traits: the Framingham Offspring Study. Diabetes 57: 3353–3359. 30. Adachi H, Tsujimoto M (2002) FEEL-1, a novel scavenger receptor with in vitro 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 concentrations and cardiovascular risk factors in the Korean population. J Med Genet 48: 10–15. j p 52. Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, et al. (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38: 904–909. 42. Chasman DI, Pare G, Mora S, Hopewell JC, Peloso G, et al. (2009) Forty-three loci associated with plasma lipoprotein size, concentration, and cholesterol content in genome-wide analysis. PLoS Genet 5: e1000730. doi:10.1371/journal.pgen. 1000730. 53. Magi R, Morris AP (2010) GWAMA: software for genome-wide association meta-analysis. BMC Bioinformatics 11: 288. 54. Higgins JP, Thompson SG, Deeks JJ, Altman DG (2003) Measuring 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 disease. Nat Genet 40: 161–169. 55. Theodoraki EV, Nikopensius T, Suhorutsenko J, Peppes V, Fili P, et al. (2010) 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
https://openalex.org/W4385352668
https://file.sciopen.com/sciopen_public/1684832452991336450.pdf
Latin
null
Metal Nanoparticles and Nano-Filters for the Disposal of Hospital Waste: A Review
Nano biomedicine and engineering
2,023
cc-by
8,986
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 production remains a challenge. [4] L.X.Y. Liu,  X.T. Zuo,  J.J. He,  et  al. Fabrication  and characterization of 2-aminophenol-4-sulfonic acid- integrated polyamide loose nanofiltration membrane. Journal of Membrane Science, 2021, 640: 119867. https:// Conclusion and Future Work f , , p doi.org/10.1016/j.memsci.2021.119867 [5] g j  E.A. Azooz,  R.K. Ridha,  H.A. Abdulridha. The fundamentals and recent applications of micellar system extraction for nanoparticles and bioactive molecules: A review. Nano Biomedicine and Engineering, 2021, 13(3): 264–278. https://doi.org/10.5101/nbev13i3.p264- 278 This work aims to study the effective application of some  nano-substances,  such  as  metal  nanoparticles, carbon nanotubes, and nano-filters for hospital waste treatment. In  addition  to  traditional  chemical  and physical  techniques,  nanoparticle  application  acts  to prevent  any  persistent  harmful  residues  of  dragged toxic  substances  used  in  chemical  drugs  and radioactive substances used in imaging studies, which have insensible effects on humans that increase with increased concentration. Due to the unique properties of  nanomaterials,  particularly  their  high  absorption capacity and multiple active components, particularly pharmaceutical particles, nano-substances were used. In spite of its effectiveness, this method still has some disadvantages  because  it  is  difficult  to  separate substances from nanoparticles. [6] Y. Ahn,  J. Choi. Bacterial  communities  and  antibiotic resistance communities in a full-scale hospital wastewater treatment plant by high-throughput pyrosequencing. Water, 2016, 8(12): 580. https://doi.org/ 10.3390/w8120580 [6] [7] A. Kadam,  S.S. Patil,  S.V. Patil,  et  al. Pharmaceutical waste management an overview. Indian Journal of Pharmacy Practice, 2016, 9(1): 2−8. https://doi.org/10. 5530/ijopp.9.1.2 [8] E. Bertone,  R.A. Stewart,  O. Sahin,  et  al. Guidelines, barriers and strategies for energy and water retrofits of public buildings. Journal of Cleaner Production, 2018, 174: 1064−1078. https://doi.org/10.1016/j.jclepro.2017. 11.065 [8] [9] E.A. Azooz,  M. Tuzen,  W.I. Mortada,  et  al. A  critical review of selected preconcentration techniques used for selenium determination in analytical samples. Critical Reviews in Analytical Chemistry, 2022. https://doi.org/ 10.1080/10408347.2022.2153579 [9] CRediT Author Statement [10] L.T.Q. Lien, N.Q. Hao, N.T.K. Chuc, et al. Antibiotics in wastewater of a rural and an urban hospital before and after wastewater treatment, and the relationship with antibiotic use—a one-year study from Vietnam. International Journal of Environmental Research and Public Health, 2016, 13(6): 588. https://doi.org/10.3390/ ijerph13060588 [10] Ali Kaswer Musa Al-Toriahi:  Data  curation, Visualization, Writing – original draft. Ebaa Adnan Azooz:  Conceptualization,  Investigation,  Project administration,  Supervision,  Writing–review  & editing. Emad Abbas Jaffar Al-Mulla:  Data curation,  Formal  analysis,  Methodology, Investigation. Q. Liu, Y. Zhou, L. Chen, et al. Application of MBR for hospital wastewater treatment in China. Desalination, 2010, 250: 605−608. https://doi.org/10.1016/j.desal. 2009.09.033 [11] F.A. Alotaibi,  R.J. Cornett,  M.N. Herod. Rapid  and efficient autoclave digestion for the extraction of iodine- 129 from urine samples. Journal of Environmental Radioactivity, 2021, 228: 106528. https://doi.org/10. 1016/j.jenvrad.2020.106528 [12] Conflict of Interest J.P. Zotesso, E.S. Cossich, V. Janeiro, et al. Treatment of hospital laundry wastewater by UV/H2O2 process. Environmental Science and Pollution Research, 2017, 24: 6278−6287. https://doi.org/10.1007/s11356-016- 6860-5 [13] The authors declare that no competing interest exists. https://www.sciopen.com/journal/2150-5578 References org/10.1016/j.biortech.2016.11.042 [19] p g j  I. Grčić,  G.L. Puma. Six-flux  absorption-scattering models for photocatalysis under wide-spectrum irradiation sources in annular and flat reactors using catalysts with different optical properties. Applied Catalysis B: Environmenal, 2017, 211: 222−234. https:// doi.org/10.1016/j.apcatb.2017.04.014 [32] W.I. Mortada,  E.A. Azooz. Microextraction  of  metal ions based on solidification of a floating drop: basics and recent updates. Trends in Environmental Analytical Chemistry, 2022, 34: e00163. https://doi.org/10.1016/j. teac.2022.e00163 [20] g j p  Y.Q. Wang,  C. Pan,  W. Chu,  et  al. Environmental remediation applications of carbon nanotubes and graphene oxide: Adsorption and catalysis. Nanomaterials, 2019, 9(3): 439. https://doi.org/10.3390/ nano9030439 [33] K. Timraz, Y.H. Xiong, H. Al Qarni, et al. Removal of bacterial cells, antibiotic resistance genes and integrase genes by on-site hospital wastewater treatment plants: Surveillance of treated hospital effluent quality. Environmental Science: Water Research & Technology, 2017, 3(2): 293−303. https://doi.org/10.1039/ C6EW00322B [21] K,  Vijayaraghavan,  T. Ashokkumar. Plant-mediated biosynthesis of metallic nanoparticles: A review of literature, factors affecting synthesis, characterization techniques and applications. Journal of Environmental Chemical Engineering, 2017, 5(5): 4866−4883. https:// doi.org/10.1016/j.jece.2017.09.026 [34] L. Rizzo, C. Manaia, C. Merlin, et al. Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review. Science of The Total Environment, 2013, 447: 345−360. https://doi.org/10.1016/j.scitotenv.2013.01. 032 [22] g j j  R.K. Ridha,  E. Azooz,  S.S. Taresh. Rapid  palladium preconcentration and spectrophotometric determination in water and soil samples. Analytical and Bioanalytical Chemistry Research, 2022, 9(3): 251−258. [35] S. Kadhim Jawad, M. Omran Kadhim, E. Adnan Azooz. Incorporation of onium system with cloud point extraction and determination of iron(III) and mercury(II) in different samples. Oriental Journal of Chemistry, 2017, 33(4): 1879−1889. https://doi.org/10.13005/ojc/ 330433 [36] E.A. Azooz, J.R. Moslim, S.M. Hameed, et al. Aspirin in food samples for separation and micro determination of copper(II) using cloud point extraction/solvation method. Nano Biomedicine and Engineering, 2021, 13(1): 62−71. https://doi.org/10.5101/nbe.v13i1.p62-71 [23] E.A. Azooz,  M. Tuzen,  W.I. Mortada. Green microextraction approach focuses on air-assisted dispersive liquid-liquid with solidified floating organic drop for preconcentration and determination of toxic metals in water and wastewater samples. Chemical Papers, 2023, 77(6): 3427−3438. https://doi.org/10.1007 /s11696-023-02714-6 [37] ( ) p g p  K.M. Aguilar-Pérez,  J.I. Avilés-Castrillo,  G. Ruiz- Pulido. Nano-sorbent materials for pharmaceutical- based wastewater effluents-An overview. Case Studies in Chemical and Environmental Engineering, 2020, 2: 100028. https://doi.org/10.1016/j.cscee.2020.100028 [24] [25] p g j  T. Rasheed, M. Bilal, A.A. Hassan, et al. References S.F. Nadeem,  U.F. Gohar,  S.F. Tahir,  et  al. Antimicrobial resistance: more than 70 years of war between humans and bacteria. Critical Reviews in Microbiology, 2020, 46(5): 578−599. https://doi.org/10. 1080/1040841X.2020.1813 [14] H. Bagheri,  A. afkhami,  A. Noroozi. Removal  of pharmaceutical compounds from hospital wastewater using nanomaterials: A review. Analytical and Bioanalytical Chemistry Research, 2016, 3(1): 1−18. https://doi.org/10.22036/ABCR.2016.12655 [1] H. Bagheri,  A. afkhami,  A. Noroozi. Removal  of pharmaceutical compounds from hospital wastewater using nanomaterials: A review. Analytical and Bioanalytical Chemistry Research, 2016, 3(1): 1−18. https://doi.org/10.22036/ABCR.2016.12655 [1] D.G.J. Larsson, C. de Pedro, N. Paxeus. Effluent from drug manufactures contains extremely high levels of pharmaceuticals. Journal of Hazardous Materials, 2007, 148(3): 751−755. https://doi.org/10.1016/j.jhazmat.2007. 07.008 [15] p g  J.Y. Chin,  A.L. Ahmad,  S.C. Low. Evolution  of photocatalytic membrane for antibiotics degradation: Perspectives and insights for sustainable environmental remediation. Journal of Water Process Engineering, 2023, 51: 103342. https://doi.org/10.1016/j.jwpe.2022. 103342 [2] p g  J.Y. Chin,  A.L. Ahmad,  S.C. Low. Evolution  of photocatalytic membrane for antibiotics degradation: Perspectives and insights for sustainable environmental remediation. Journal of Water Process Engineering, 2023, 51: 103342. https://doi.org/10.1016/j.jwpe.2022. 103342 [2] I.D. Amoah, P. Reddy, R. Seidu, et al. Concentration of soil-transmitted helminth eggs in sludge from South Africa and Senegal: A probabilistic estimation of infection risks associated with agricultural application. Journal of Environmental Management, 2018, 206: 1020−1027. https://doi.org/10.1016/j.jenvman.2017.12. 003 [16] K.S. Obayomi,  S.Y. Lau,  I.E. Mayowa,  et  al. Recent advances in graphene-derived materials for biomedical waste treatment. Journal of Water Process Engineering, [3] https://www.sciopen.com/journal/2150-5578 Nano Biomed. Eng., 2023, 15(2) 187 R.E. Morsi,  A.M. Alsabagh,  S.A. Nasr,  et  al. Multifunctional nanocomposites of chitosan, silver nanoparticles, copper nanoparticles and carbon nanotubes for water treatment: antimicrobial characteristics. International Journal of Biological Micromolecules, 2017, 97: 264−269. https://doi.org/10. 1016/j.ijbiomac.2017.01.032 [30] L. Kovalova,  H. Siegrist,  H. Singer,  et  al. Hospital wastewater treatment by membrane bioreactor: Performance and efficiency for organic micropollutant elimination. Environmental Science & Technology, 2012, 46(3): 1536−1545. https://doi.org/10.1021/ es203495d [17] [18] R.M.A. Pedreira, K. Pahnke, P. Böning, et al. Tracking hospital effluent-derived gadolinium in Atlantic coastal waters off Brazil. Water Research, 2018, 43: 1345−1354. https://doi.org/10.1016/j.watres.2018.08. 005 [18] R. Ahmadi, E.A. Azooz, Y. Yamini, et al. Liquid-liquid microextraction techniques based on in-situ formation/decomposition of deep eutectic solvents. TrAC Trends in Analytical Chemistry, 2023, 161: 117019. https://doi.org/10.1016/j.trac.2023.117019 [31] B. Tiwari, B. Sellamuthu, Y. Ouarda, et al. Review on fate and mechanism of removal of pharmaceutical pollutants from wastewater using biological approach. Bioresource Technology, 2017, 224: 1−12. https://doi. References Environmental threatening concern and efficient removal of pharmaceutically active compounds using metal-organic frameworks as adsorbents. Environmental Research, 2020, 185: 109436. https://doi.org/10.1016/j.envres. 2020.109436 [25] R.K. Thines,  N.M. Mubarak,  S. Nizamuddin,  et  al. Application potential of carbon nanomaterials in water and wastewater treatment: A review. Journal of the Taiwan Institute of Chemical Engineers, 2017, 72: 116−133. https://doi.org/10.1016/j.jtice.2017.01.018 [38] [39] I. Mohmood,  C.B. Lopes,  I. Lopes,  et  al. Nanoscale materials and their use in water contaminants removal—a review. Environmental Science and Pollution Research, 2013, 20: 1239−1260. https://doi. org/10.1007/s11356-012-1415-x [26] R.K. Mohapatra,  M.M. El-ajaily,  F.S. Alassbaly,  et  al. DFT, anticancer, antioxidant and molecular docking investigations of some ternary Ni(II) complexes with 2- [(E)-[4-(dimethylamino)phenyl]methyleneamino]phenol. Chemical Papers, 2021, 75(3): 1005−1019. https://doi. org/10.1007/s11696-020-01342-8 [39] g  C. Santhosh,  V. Velmurugan,  G. Jacob,  et  al. Role  of nanomaterials in water treatment applications: A review. Chemical Engineering Journal, 2016, 306: 1116−1137. https://doi.org/10.1016/j.cej.2016.08.053 [27] g  S. Banerjee, S.C. Pillai, P. Falaras, et al. New insights into the mechanism of visible light photocatalysis. The Journal of Physical Chemistry Letters, 2014, 5(15): 2543−2554. https://doi.org/10.1021/jz501030x [40] A. Cincinelli,  T. Martellini,  E. Coppini,  et  al. Nanotechnologies for removal of pharmaceuticals and personal care products from water and wastewater. A review. Journal of Nanoscience and Nanotechnology, 2015, 15(5): 3333−3347. https://doi.org/10.1166/jnn. 2015.10036 [28] R. Rienzie,  S. Ramanayaka,  N.M. Adassooriya. Nanotechnology applications for the removal of environmental contaminants from pharmaceuticals and personal care products. In: Pharmaceuticals and Personal Care Products: Waste Management and Treatment Technology. Amsterdam: Elsevier, 2019: 279–296. https:// doi.org/10.1016/B978-0-12-816189-0.00012-3 [41] [29] T. Rasheed,  M. Adeel,  F. Nabeel,  et  al. TiO2/SiO2 decorated carbon nanostructured materials as a multifunctional platform for emerging pollutants removal. Science of The Total Environment, 2019, 688: 299−311. https://doi.org/10.1016/j.scitotenv.2019.06.200 [29] H. Karimnezhad,  A.H. Navarchian,  T. Tavakoli Gheinani, et al. Amoxicillin removal by Fe-based nanoparticles immobilized on polyacrylonitrile [42] https://www.sciopen.com/journal/2150-5578 https://www.sciopen.com/journal/2150-5578 Nano Biomed. Eng., 2023, 15(2) 188 nanotubes with selected properties for dynamic filtration of pharmaceuticals and personal care products. Water Research, 2016, 92: 104−112. https://doi.org/10.1016/j. watres.2016.01.038 membrane: Individual nanofiltration or Fenton reaction, vs. engineered combined process. Chemical Engineering Research and Design, 2020, 153: 187−200. https://doi. org/10.1016/j.cherd.2019.10.031 [43] [55] Y. Wei,  Y.S. Zhang,  X.L. Gao,  et  al. Multilayered graphene oxide membranes for water treatment: A review. Carbon, 2018, 139: 964−981. https://doi.org/10. 1016/j.carbon.2018.07.040 [55] g j  A. Balati,  A. Matta,  H.J. Shipley. Heterojunction  of vertically aligned MoS2 layers to Hydrogenated Black TiO2 and Rutile Based Inorganic Hollow Microspheres for the highly enhanced visible light arsenic photooxidation. References Composites Part B: Engineering, 2020, 185: 107785. https://doi.org/10.1016/j.compositesb.2020. 107785 [43] j  Y. Wang, J. Fang, J.C. Crittenden, et al. Novel RGO/α- FeOOH supported catalyst for Fenton oxidation of phenol at a wide pH range using solar-light-driven irradiation. Journal of Hazardous Materials, 2017, 329: 321−329. https://doi.org/10.1016/j.jhazmat.2017.01.041 [56] E.A. Azooz, H.S.A. Al-Wani, M.S. Gburi, et al. Recent modified air-assisted liquid–liquid microextraction applications for medicines and organic compounds in various samples: A review. Open Chemistry, 2022, 20(1): 525−540. https://doi.org/10.1515/chem-2022- 0174 [44] A. Al-Atta, F. Sher, A. Hazafa, et al. Supercritical water oxidation of phenol and process enhancement with in situ formed Fe2O3 nano catalyst. Environmental Science and Pollution Research, 2022, 29(41): 61896−61904. https://doi.org/10.1007/s11356-021-16390-0 [57] C. Zhao,  M. Pelaez,  D.D. Dionysiou,  et  al. UV  and visible light activated TiO2 photocatalysis of 6- hydroxymethyl uracil, a model compound for the potent cyanotoxin cylindrospermopsin. Catalysis Today, 2014, 224: 70−76. https://doi.org/10.1016/j.cattod.2013.09.042 [45] J.A. MiR-Tutusaus,  M. Sarrà,  G. Caminal. Continuous treatment of non-sterile hospital wastewater by Trametes versicolor: How to increase fungal viability by means of operational strategies and pretreatments. Journal of Hazardous Materials, 2016, 318: 561−570. https://doi. org/10.1016/j.jhazmat.2016.07.036 [58] p g j  A.S. Adeleye, J.R. Conway, K. Garner, et al. Engineered nanomaterials for water treatment and remediation: Costs, benefits, and applicability. Chemical Engineering Journal, 2016, 286: 640−662. https://doi.org/10.1016/j. cej.2015.10.105 [46] [59] g j j  Y. Wang,  M.X. Liang,  J.S. Fang,  et  al. Visible-light photo-Fenton oxidation of phenol with rGO-α-FeOOH supported on Al-doped mesoporous silica (MCM-41) at neutral pH: Performance and optimization of the catalyst. Chemosphere, 2017, 182: 468−476. https://doi. org/10.1016/j.chemosphere.2017.05.037 [59] j  E.A. Azooz,  G.S. Jawad,  E.H.B. Al-Muhanna,  et  al. Displacement cloud point extraction procedure for preconcentration of iron(III) in water and fruit samples prior to spectrophotometric determination. Bulletin of the Chemical Society of Ethiopia, 2022, 37(1): 1−10. https://doi.org/10.4314/bcse.v37i1.1 [47] g j p  F.A. Wannas, E.A. Azooz, R.K. Ridha, et al. Separation and micro determination of zinc(II) and cadmium(II) in food samples using cloud point extraction method. Iraqi Journal of Science, 2023, 64(3): 1046−1061. https://doi. org/10.24996/ijs.2023.64.3.2 [60] [48] N.S.K. Gowthaman,  J.W. Chen,  C.F. Dee,  et  al. Nanostructural dimension and oxygen vacancy synergistically induced photoactivity across high surface a r e a   m o n o d i s p e r s e d   A u N P s / Z n O   n a n o r o d s heterojunction. Journal of Alloys and Compounds, 2022, 920: 165836. https://doi.org/10.1016/j.jallcom.2022. 165836 [48] [61] S. Ali, S.A.U. https://www.sciopen.com/journal/2150-5578 References Rehman, H.Y. Luan, et al. Challenges and opportunities in functional carbon nanotubes for membrane-based water treatment and desalination. Science of the Total Environment, 2019, 646: 1126−1139. https://doi.org/10.1016/j.scitotenv.2018.07. 348 [61] [49] A.R. Hussein,  M.S. Gburi,  N.M. Muslim,  et  al. A greenness evaluation and environmental aspects of solidified floating organic drop microextraction for metals: A review. Trends in Environmental Analytical Chemistry, 2023, 37: e00194. https://doi.org/10.1016/j. teac.2022.e00194 [49] J.Q. Wang, P. Zhang, B. Liang, et al. Graphene oxide as an effective barrier on a porous nanofibrous membrane for water treatment. ACS Applied Materials & Interfaces, 2016, 8(9): 6211−6218. https://doi.org/10. 1021/acsami.5b12723 [62] [50] [63] S. Rahman, R. Nawaz, J.A. Khan, et al. Synthesis and characterization of carbon and carbon-nitrogen doped black TiO2 nanomaterials and their application in sonophotocatalytic remediation of treated agro-industrial wastewater. Materials, 2021, 14(20): 6175. https://doi. org/10.3390/ma14206175 [50] C.-Y. Wang,  W.-J. Zeng,  T.-T. Jiang,  et  al. Incorporating attapulgite nanorods into graphene oxide nanofiltration membranes for efficient dyes wastewater treatment. Separation and Purification Technology, 2019, 214: 21−30. https://doi.org/10.1016/j.seppur.2018. 04.079 [63] X. Wang,  H.X. Wang,  Y.M. Wang,  et  al. Hydrotalcite/graphene oxide hybrid nanosheets functionalized nanofiltration membrane for desalination. Desalination, 2019, 451: 209−218. https://doi.org/10. [64] g  F.X. Kong,  Y.K. Wang,  J.M. Cao,  et  al. The performance of three typical single and two coupled electrochemical processes for drilling wastewater treatment: Comparison and implication. Environmental Science: Water Research & Technology, 2021, 7(12): 2390−2396. https://doi.org/10.1039/D1EW00523E [51] 1016/j.desal.2017.05.012 F.H.J. Al-Shemmari,  E.A.J. Al-Mulla,  A.A. Rabah. A comparative study of different surfactants for natural rubber clay nanocomposite preparation. Rendiconti Lincei, 2014, 25: 409−413. https://doi.org/10.1007/ s12210-014-0307-z [65] P. Kumari,  M. Alam,  W.A. Siddiqi. Usage  of nanoparticles as adsorbents for waste water treatment: An emerging trend. Sustainable Materials and Technologies, 2019, 22: e00128. https://doi.org/10.1016/ j.susmat.2019.e00128 [52] Z.O. Dou,  T. Wang,  W.S. Chen,  et  al. Self-driven membrane filtration by core–shell polymer composites. Journal of Materials Chemistry A, 2020, 8(31): 15942−15950. https://doi.org/10.1039/D0TA03617J [66] j  S.K. Jawad,  M.U. Kadhium,  E.A. Azooz. Application cloud point extraction method joined with liquid ion exchange for selective determination of Fe3+and Hg2+. Journal of Engineering and Applied Sciences, 2019, 14(11): 3514−3521. https://doi.org/10.36478/jeasci.2019. 3514.3521 [53] p g  F.A. Wannas,  E.A. Azooz,  S.K. Jawadc. Liquid  ion exchange with spectrophotometric method for separation and determination W (VI). Journal of Advanced Research in Dynamical and Control Systems, 2019, [67] Y. Wang,  J. Ma,  J. Zhu,  et  al. Multi-walled  carbon [54] https://www.sciopen.com/journal/2150-5578 Nano Biomed. Eng., 2023, 15(2) 189 11(07-Special): 260−269. (IPCA): Experimental studies and kinetics analysis. Photochemical & Photobiological Sciences, 2011, 10(6): 1014−1022. https://doi.org/10.1039/c0pp00368a 11(07-Special): 260−269. Q.L. Li,  R.R. Jia,  J.H. Shao,  et  al. Photocatalytic degradation of amoxicillin via TiO2 nanoparticle coupling with a novel submerged porous ceramic membrane reactor. Journal of Cleaner Production, 2019, 209: 755−761. https://doi.org/10.1016/j.jclepro. [68] [81] A. Seid-Mohammadi, G. Asgarai, Z. Ghorbanian, et al. The removal of cephalexin antibiotic in aqueous solutions by ultrasonic waves/hydrogen peroxide/nickel oxide nanoparticles (US/H2O2/NiO) hybrid process. Separation Science and Technology, 2020, 55(8): 1558−1568. https://doi.org/10.1080/01496395.2019. 1603241 [81] [69] R.R. Kalantary,  M. Farzadkia,  M. Kermani,  et  al. Heterogeneous electro-Fenton process by nano-Fe3O4 for catalytic degradation of amoxicillin: Process optimization using response surface methodology. Journal of Environmental Chemical Engineering, 2018, 6(4): 4644−4652. https://doi.org/10.1016/j.jece.2018.06. 043 [69] [82] P. Rai,  K.P. Singh. Valorization  of  Poly  (ethylene) terephthalate (PET) wastes into magnetic carbon for adsorption of antibiotic from water: Characterization and application. Journal of Environmental Management, 2018, 207: 249−261. https://doi.org/10.1016/j.jenvman. 2017.11.047 [82] N. Olama, M. Dehghani, M. Malakootian. The removal of amoxicillin from aquatic solutions using the TiO2/UV- C nanophotocatalytic method doped with trivalent iron. Applied Water Science, 2018, 8(4): 97. https://doi.org/10. 1007/s13201-018-0733-7 [70] [83] M. Leili, M. Fazlzadeh, A. Bhatnagar. Green synthesis of nano-zero-valent iron from Nettle and Thyme leaf extracts and their application for the removal of cephalexin antibiotic from aqueous solutions. Environmental Technology, 2018, 39(9): 1158−1172. https://doi.org/10.1080/09593330.2017.1323956 [83] S. Ramasundaram,  M.G. Seid,  W. Lee,  et  al. 1016/j.desal.2017.05.012 Preparation, characterization, and application of TiO2- patterned polyimide film as a photocatalyst for oxidation of organic contaminants. Journal of Hazardous Materials, 2017, 340: 300−308. https://doi.org/10.1016/j. jhazmat.2017.06.069 [71] p g  E. Azooz, J. Moslim, S. Jawad. Cloud point extraction methodology for separation, extraction and preconcentration of Mn (VII) coupled with spectroscopy for determination in different samples. Biochemical and Cellular Archives, 2020, 20(1): 2641−2648. [84] X.Y. Wang,  A. Wang,  J. Ma. Visible-light-driven photocatalytic removal of antibiotics by newly designed C3N4@MnFe2O4-graphene nanocomposites. Journal of Hazardous Materials, 2017, 336: 81−92. https://doi.org/ 10.1016/j.jhazmat.2017.04.012 [72] ( )  E.A. Azooz, F.A. Wannas, S.K. Jawad. Developed cloud point extraction coupled with onium system for separation and determination cobalt in biological samples. Research Journal of Pharmacy and Technology, 2021, 14(2): 594−598. https://doi.org/10. 5958/0974-360X.2021.00106.2 [85] B. Amraei,  R.R. Kalantary,  A. Jonidi  jafari,  et  al. Efficiency of CuFe 20 4 bimetallic in removing amoxicillin from aqueous solutions. Journal of Mazandaran University of Medical Sciences, 2017, 27(147): 259−275. [73] [86] N.T. Nguyen, T.H. Dao, T.T. Truong, et al. Adsorption characteristic of ciprofloxacin antibiotic onto synthesized alpha alumina nanoparticles with surface modification by polyanion. Journal of Molecular Liquids, 2020, 309: 113150. https://doi.org/10.1016/j. molliq.2020.113150 [86] S. Machado, J.G. Pacheco, H.P.A. Nouws, et al. Green zero-valent iron nanoparticles for the degradation of amoxicillin. International Journal of Environmental Science and Technology, 2017, 14(5): 1109−1118. https:// doi.org/10.1007/s13762-016-1197-7 [74] q  M. Malakootian,  A. Nasiri,  M. Amiri  Gharaghani. Photocatalytic degradation of ciprofloxacin antibiotic by TiO2 nanoparticles immobilized on a glass plate. Chemical Engineering Communications, 2020, 207(1): 56−72. https://doi.org/10.1080/00986445.2019.1573168 [87] S. Pourmoslemi,  A. Mohammadi,  F. Kobarfard,  et  al. Photocatalytic removal of doxycycline from aqueous solution using ZnO nano-particles: A comparison between UV-C and visible light. Water Science and Technology, 2016, 74(7): 1658−1670. https://doi.org/10. 2166/wst.2016.339 [75] [88] p g  X.Y. Mi,  Y. Li,  X.M. Ning,  et  al. Electro-Fenton degradation of ciprofloxacin with highly ordered mesoporous MnCo2O4-CF cathode: Enhanced redox capacity and accelerated electron transfer. Chemical Engineering Journal, 2019, 358: 299−309. https://doi. org/10.1016/j.cej.2018.10.047 [88] E.A. Azooz, G.J. Shabaa, E.A.J. Al-Mulla. Methodology for preconcentration and determination of silver in aqueous samples using cloud point extraction. Brazilian Journal of Analytical Chemistry, 2021, 9(35): 39−48. https://doi.org/10.30744/brjac.2179-3425.AR-61-2021 [76] [89] A.R. Yazdanbakhsh,  H. Daraei,  M. Rafiee,  et  al. Performance of iron nano particles and bimetallic Ni/Fe nanoparticles in removal of amoxicillin trihydrate from synthetic wastewater. Water Science and Technology, 2016, 73(12): 2998−3007. https://doi.org/10.2166/wst. 2016.157 [77] M. Malakootian, H. Mahdizadeh, A. Dehdarirad, et al. 1016/j.desal.2017.05.012 Mechanism  of  the  OH  radical generation in photocatalysis with TiO2 of different crystalline types. The Journal of Physical Chemistry C, 2014, 118(20): 10824−10832. https://doi.org/10.1021/ jp501214m [106] p g j q  E.A. Azooz,  F.A. Wannas,  R.K. Ridha,  et  al. A  green approach for micro determination of silver (I) in water and soil samples using vitamin C. Analytical and Bioanalytical Chemistry Research, 2022, 9(2): 133−140. https://doi.org/10.22036/ABCR.2021.277834.1609 [95] J. Li, J.-L. Gong, G.-M. Zeng, et al. Thin-film composite polyester nanofiltration membrane with high flux and efficient dye/salts separation fabricated from precise molecular sieving structure of β-cyclodextrin. Separation and Purification Technology, 2021, 276(9): 119352. https://doi.org/10.1016/j.seppur.2021.119352 [107] p g  T.H. Dao,  T.T. Tran,  V.R. Nguyen,  et  al. Removal  of antibiotic from aqueous solution using synthesized TiO2 nanoparticles: Characteristics and mechanisms. Environmental Earth Sciences, 2018, 77(10): 359. https:// doi.org/10.1007/s12665-018-7550-z [96] p g j pp  K.Z. Li,  B. Lee,  Y. Kim. High  performance  reverse osmosis membrane with carbon nanotube support layer. Journal of Membrane Science, 2019, 592: 117358. https:// doi.org/10.1016/j.memsci.2019.117358 [108] Z. Lin,  X.L. Weng,  G. Owens,  et  al. Simultaneous removal of Pb(II) and rifampicin from wastewater by iron nanoparticles synthesized by a tea extract. Journal of Cleaner Production, 2020, 242: 118476. https://doi. org/10.1016/j.jclepro.2019.118476 [97] R.S. Li,  W. Sun,  L.F. Xia,  et  al. Adsorption  of  toxic tetracycline, thiamphenicol and sulfamethoxazole by a granular activated carbon (GAC) under different conditions. Molecules, 2022, 27(22): 7980. https://doi. org/10.3390/molecules27227980 [109] K.V.G. Ravikumar,  H. Kubendiran,  K. Ramesh,  et  al. Batch and column study on tetracycline removal using green synthesized NiFe nanoparticles immobilized alginate beads. Environmental Technology & Innovation, 2019, 17(1): 100520. https://doi.org/10.1016 /j.eti.2019.100520 [98] M. Fathi, E.A. Gomaa, S.E. Salem, et al. Parameters for the conductometric association for lump and nano CoSO4·7H2O in the presence and absence of fuchsin acid in water at different temperature. Bulletin of the Chemical Society of Ethiopia, 2023, 37(3): 789−804. https://doi.org/10.4314/bcse.v37i3.20 [110] j  M. SefidSiahbandi,  O. Moradi,  B. Akbari-adergani,  et al. Fabrication and implementation of bimetallic Fe/Zn nanoparticles (mole ratio 1: 1) loading on hydroxyethylcellulose–Graphene oxide for removal of tetracycline antibiotic from aqueous solution. Chemosphere, 2023, 312: 137184. https://doi.org/10. 1016/j.chemosphere.2022.137184 [99] p g  M.M. Radhi,  E.A.J. Al-Mulla,  W.T. Tan. Electrochemical characterization of the redox couple of Fe(III)/Fe(II) mediated by grafted polymer electrode. Research on Chemical Intermediates, 2014, 40(1): 179–192. https://doi.org/10.1007/s11164-012-0954-6 [111] W. Shi,  L. Wang,  J. Wang,  et  al. 1016/j.desal.2017.05.012 Photocatalytic ozonation degradation of ciprofloxacin using ZnO nanoparticles immobilized on the surface of stones. Journal of Dispersion Science and Technology, 2019, 40(6): 846−854. https://doi.org/10.1080/01932691. 2018.1485580 [89] [90] X.L. Weng,  Q. Sun,  S. Lin,  et  al. Enhancement  of catalytic degradation of amoxicillin in aqueous solution using clay supported bimetallic Fe/Ni nanoparticles. Chemosphere, 2014, 103: 80−85. https://doi.org/10.1016/ j.chemosphere.2013.11.033 [78] X.Y.,  Zheng,  S.P. Xu,  Y. Wang,  et  al. Enhanced degradation of ciprofloxacin by graphitized mesoporous carbon (GMC)-TiO2 nanocomposite: Strong synergy of adsorption-photocatalysis and antibiotics degradation mechanism. Journal of Colloid and Interface Science, 2018, 527: 202−213. https://doi.org/10.1016/j.jcis.2018. 05.054 [90] H.R. Pouretedal,  N. Sadegh. Effective  removal  of Amoxicillin, Cephalexin, Tetracycline and Penicillin G from aqueous solutions using activated carbon nanoparticles prepared from vine wood. Journal of Water Process Engineering, 2014, 1: 64−73. https://doi. org/10.1016/j.jwpe.2014.03.006 [79] J.M. Chaba,  P.N. Nomngongo. Preparation  of  V2O5- ZnO coated carbon nanofibers: Application for removal of selected antibiotics in environmental matrices. Journal of Water Process Engineering, 2018, 23: 50−60. https://doi.org/10.1016/j.jwpe.2018.03.003 [91] S. Basha,  C. Barr,  D. Keane,  et  al. On  the adsorption/photodegradation of amoxicillin in aqueous solutions by an integrated photocatalytic adsorbent [80] p g j j p  D. Li, X.L. Guo, H.R. Song, et al. Preparation of RuO2- T i O 2 / N a n o - g r a p h i t e   c o m p o s i t e   a n o d e   f o r [92] https://www.sciopen.com/journal/2150-5578 https://www.sciopen.com/journal/2150-5578 Nano Biomed. Eng., 2023, 15(2) 190 chemosphere.2021.129852 chemosphere.2021.129852 electrochemical degradation of ceftriaxone sodium. Journal of Hazardous Materials, 2018, 351: 250−259. https://doi.org/10.1016/j.jhazmat.2018.03.007 p  N. Pugazhenthiran,  P. Sathishkumar,  O. Albormani,  et al. Silver nanoparticles modified ZnO nanocatalysts for effective degradation of ceftiofur sodium under UV–vis light illumination. Chemosphere, 2023, 313: 137515. https://doi.org/10.1016/j.chemosphere.2022.137515 [104] [93] g j j  L.M.F. Oliveira, M.A. Nascimento, Y.M. Guimarães, et al. Removal of beta-lactams antibiotics through zero- valent copper nanoparticles. Journal of the Brazilian Chemical Society, 2018, 29(8): 1630−1637. https://doi. org/10.21577/0103-5053.20180034 [93] p g j p  J. Barasarathi, P. S. Abdullah, E.C. Uche. Application of magnetic carbon nanocomposite from agro-waste for the removal of pollutants from water and wastewater. Chemosphere, 2022, 305: 135384. https://doi.org/10. 1016/j.chemosphere.2022.135384 [105] g  M.Y. Badi, A. Azari, H. Pasalari, et al. Modification of activated carbon with magnetic Fe3O4 nanoparticle composite for removal of ceftriaxone from aquatic solutions. Journal of Molecular Liquids, 2018, 261: 146−154. https://doi.org/10.1016/j.molliq.2018.04.019 [94] J. Zhang,  Y. Nosaka. https://www.sciopen.com/journal/2150-5578 1016/j.desal.2017.05.012 Magnetically retrievable CdS/reduced graphene oxide/ZnFe2O4 ternary nanocomposite for self-generated H2O2 towards Photo-Fenton removal of tetracycline under visible light irradiation. Separation and Purification Technology, 2022, 292: 120987. https://doi.org/10.1016/j.seppur. 2022.120987 [100] p g  E.A.J. Al-Mulla,  K.W.S. Al-Janabi. Extraction  of cobalt(II) from aqueous solution by N, N′-carbonyl difatty amides. Chinese Chemical Letters, 2011, 22(4): 469–472. https://doi.org/10.1016/j.cclet.2010.10.037 [112] E.A.J. Al-Mulla. Preparation  of  polylactic acid/epoxidized palm oil/fatty nitrogen compounds modified clay nanocomposites by melt blending. Polymer Science Series A, 2011, 53(2): 149–157. https:// doi.org/10.1134/S0965545X11020015 [113] C.M. El-Maraghy,  O.M. El-Borady,  O.A. El-Naem. Effective removal of levofloxacin from pharmaceutical wastewater using synthesized zinc oxid, graphen oxid nanoparticles compared with their combination. Scientific Reports, 2020, 10: 5914. https://doi.org/10. 1038/s41598-020-61742-4 [101] g  E.A.J. Al-Mulla,  W.M.d. Zin  Wan  Yunus,  N.A. Bt Ibrahim, et al. Epoxidized palm oil plasticized polylactic acid/fatty nitrogen compound modified clay nanocomposites: Preparation and characterization. Polymers and Polymer Composites, 2010, 18(8): 451–460. https://doi.org/10.1177/096739111001800806 [114] P.X. Liu, H.M. Zhang, Y.J. Feng, et al. Removal of trace antibiotics from wastewater: A systematic study of nanofiltration combined with ozone-based advanced oxidation processes. Chemical Engineering Journal, 2014, 240: 211−220. https://doi.org/10.1016/j.cej.2013. 11.057 [102] © The author(s) 2023. This is an open-access article distributed under  the  terms  of  the  Creative  Commons  Attribution  4.0 International  License  (CC  BY)  (http://creativecommons.org/ licenses/by/4.0/),  which  permits  unrestricted  use,  distribution, and reproduction in any medium, provided the original author and source are credited. R. Neha,  S. Adithya,  R.S. Jayaraman,  et  al. Nano- adsorbents an effective candidate for removal of toxic pharmaceutical compounds from aqueous environment: A critical review on emerging trends. Chemosphere, 2021, 272: 129852. https://doi.org/10.1016/j. [103] https://www.sciopen.com/journal/2150-5578
https://openalex.org/W4385671734
https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/39/e3sconf_transsiberia2023_04014.pdf
English
null
Study of the parameters for a parallel hybrid electric vehicle
E3S web of conferences
2,023
cc-by
4,544
* 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, 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) 3. K.C. Agatay Bayindir, et.al., Energy Conversion and Management 52(2), 1305–1313 (2011) 8 E3S Web of Conferences 402, 04014 (2023) TransSiberia 2023 https://doi.org/10.1051/e3sconf/202340204014 4. S. Kermani, S. Delprat, T.M. Guerra, R. Trigui, B. Jeanneret, Control Engineering Practice 20(4), 408–420 (2012) 5. J.S. Guo, J.M. Wang, L. Yang, B. Zhuo, China Journal of Highway and Transport 21(5), 115–120 (2008) 6. L. Wang, Y. Zhang, C. Yin, H. Zhang, C. Wang, Simulation Modelling Practice and Theory 25, 148–162 (2012) 7. T. Banjac, F. Trenc, T. Katrasnik, Energy Conversion and Management 50(12), 2865– 2878 (2009) 8. H. Zhong, F. Wang, G.Q. Aoetal, Proceedings of the Institution of Mechanical Engineers D 222(1), 79–92 (2008) 9. M. Cipek, D. Pavkovi´c, J. Petri´c, Applied Energy 101, 121–133 (2013) 10. E. Pennestri, L. Mariti, P.P. Valentini, V.H. Mucino, Mechanism and Machine Theory 49, 157–176 (2012) 11. T. Horiba, K. Hironaka, T. Matsumura, T. Kai, M. Koseki, Y. Muranaka, Journal of Power Sources 119- 121, 893–896 (2003) 12. X. Hu, N. Murgovski, L. Johannesson, B. Egardt, Applied Energy 111, 1001–1009 (2013) 9 9
https://openalex.org/W2126288864
https://publications.goettingen-research-online.de/bitstream/2/8908/2/1742-2094-9-80.pdf
English
null
Immunoadsorption therapy in patients with multiple sclerosis with steroid-refractory optical neuritis
Journal of neuroinflammation
2,012
cc-by
7,443
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, Lassmann H: Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann Neurol 2000, 47:707–717. 2. 2. Reindl M, Linington C, Brehm U, Egg R, Dilitz E, Deisenhammer F, Poewe W, Berger T: Antibodies against the myelin oligodendrocyte glycoprotein and the myelin basic protein in multiple sclerosis and other neurological diseases: a comparative study. Brain 1999, 122:2047–2056. 2. Reindl M, Linington C, Brehm U, Egg R, Dilitz E, Deisenhammer F, Poewe W, Berger T: Antibodies against the myelin oligodendrocyte glycoprotein and the myelin basic protein in multiple sclerosis and other neurological diseases: a comparative study. Brain 1999, 122:2047–2056. Author details 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. Ruprecht K, Klinker E, Dintelmann T, Rieckmann P, Gold R: Plasma exchange for severe optic neuritis: treatment of 10 patients. Neurology 2004, 63:1081–1083. Koziolek et al. Journal of Neuroinflammation 2012, 9:80 http://www.jneuroinflammation.com/content/9/1/80 Page 10 of 10 Page 10 of 10 8. Keegan M, Konig F, McClelland R, Brück W, Morales Y, Bitsch A, Panitch H, Lassmann H, Weinshenker B, Rodriguez M, Parisi J, Lucchinetti CF: Relation between humoral pathological changes in multiple sclerosis and response to therapeutic plasma exchange. Lancet 2005, 366:579–582. 28. Whitaker JN: Myelin basic protein in cerebrospinal fluid and other body fluids. Mult Scler 1998, 4:16–21. doi:10.1186/1742-2094-9-80 Cite this article as: Koziolek et al.: Immunoadsorption therapy in patients with multiple sclerosis with steroid-refractory optical neuritis. Journal of Neuroinflammation 2012 9:80. doi:10.1186/1742-2094-9-80 Cite this article as: Koziolek et al.: Immunoadsorption therapy in patients with multiple sclerosis with steroid-refractory optical neuritis. Journal of Neuroinflammation 2012 9:80. 9. Klingel R, Heibges R, Fassbender C: Plasma exchange and immunoadsorption for autoimmune neurologic diseases - current guidelines and future perspectives. Atheroscler Suppl 2009, 10:129–132. 10. Schilling S, Linker RA, König FB, Koziolek M, Bähr M, Müller GA, Paulus W, Gärtner J, Brück W, Chan A, Gold R: Plasma exchange therapy for steroid-unresponsive multiple sclerosis relapses: clinical experience with 16 patients. Nervenarzt 2006, 77:430–438. 11. Hetherington R: The Snellen chart as a test of visual acuity. Psychol Forsch 1954, 24:349–357. 11. Hetherington R: The Snellen chart as a test of visual acuity. Psychol Forsch 1954, 24:349–357. 12. Stegmayr B, Ptak J, Wikstroem B, Berlin G, Axelsson CG, Griskevicius A, Centoni P, Liumbruno G, Molfettini P, Audzijoniene J, Mokvist K, Nilsson B, Norda R, Knutson F, Ramlow W, Blaha M, Witt V, Evergren M, Tomaz J: World apheresis registry 2003-2007 data. Trans Apher Sci 2008, 39:247–254. 13. Kurtzke JF: Rating neurologic impairment in multiple sclerosis: an expanded disability scale (EDSS). Neurology 1983, 33:1444–1452. 14. Sivieri S, Ferrarini AM, Gallo P: Multiple sclerosis: IL-2 and sIL-2R levels in cerebrospinal fluid and serum. Review of literature and critical analysis of ELISA pitfalls. Mult Scler 1998, 4:7–11. 15. Bramlage C, Schröder C, Bramlage P, Ahrens K, Zapf A, Müller GA, Koziolek MJ: Predictors of complications of therapeutical plasma exchange. J Clin Apher 2009, 24:225–231. 16. Dihazi H, Koziolek MJ, Söllner T, Kahler E, Klingel R, Neuhoff R, Strutz F, Mueller GA: Protein adsorption during LDL-apheresis: proteomic analysis. Nephrol Dial Transplant 2008, 23:2925–2935. 17. Koziolek MJ, Müller GA, Zapf A, Patschan D, Koschnick S, Bramlage C, Vasko R, Strutz F: Role of CX3C-chemokine CX3C-L/fractalkine expression in a model of slowly progressive renal failure. Nephrol Dial Transplant 2010, 25:684–698. 18. Conclusions l l 3. Lehmann HC, Hartung HP, Hetzel GR, Stüve O, Kieseier BC: Plasma exchange in neuroimmunological disorders: Part 1: Rationale and treatment of inflammatory central nervous system disorders. Arch Neurol 2006, 63:930–935. Our clinical results show a high clinical efficacy of tryptophan-IA comparable to PE in the treatment of MS relapses refractory to corticosteroids. Furthermore, our experimental data suggest several possible effects on MS pathogenesis: not only removal of immunoglobulins and complement from plasma, but also reduced levels of circulating autoantigens and regulatory proteins. We suggest that more prospective studies are needed to confirm and extend our results, to give new insights into this treatment approach, to optimize therapeutic IA protocols and, lastly, to investigate further therapeutic principles, for example, T-cell reactivity to MBP before and after IA. 4. Weinshenker BG, O'Brien PC, Petterson TM, Noseworthy JH, Lucchinetti CF, Dodick DW, Pineda AA, Stevens LN, Rodriguez M: A randomized trial of plasma exchange in acute central nervous system inflammatory demyelinating disease. Ann Neurol 1999, 46:878–886. 5. Gold R, Hartung HP: Towards individualised multiple-sclerosis therapy. Lancet Neurol 2005, 4:693–694. 5. Gold R, Hartung HP: Towards individualised multiple-sclerosis therapy. Lancet Neurol 2005, 4:693–694. 6. Ruprecht K, Klinker E, Dintelmann T, Rieckmann P, Gold R: Plasma exchange for severe optic neuritis: treatment of 10 patients. Neurology 2004, 63:1081–1083. 6. Ruprecht K, Klinker E, Dintelmann T, Rieckmann P, Gold R: Plasma exchange for severe optic neuritis: treatment of 10 patients. Neurology 2004, 63:1081–1083. 7. Wiendl H, Toyka KV, Rieckmann P, Gold R, Hartung HP, Hohlfeld R, Multiple Sclerosis Therapy Consensus Group (MSTCG): Basic and escalating immunomodulatory treatments in multiple sclerosis: current therapeutic recommendations. J Neurol 2008, 255:1449–1463. 7. Wiendl H, Toyka KV, Rieckmann P, Gold R, Hartung HP, Hohlfeld R, Multiple Sclerosis Therapy Consensus Group (MSTCG): Basic and escalating immunomodulatory treatments in multiple sclerosis: current therapeutic recommendations. J Neurol 2008, 255:1449–1463. 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 Trebst C, Bronzlik P, Kielstein JT, Schmidt BMW, Stangel M: Immunoadsorption therapy for steroid-unresponsive relapses in patients with multiple sclerosis. Blood Purif 2012, 33:1–6. 19. Mauch E, Zwanzger J, Hettich R, Fassbender C, Klingel R, Heigl F: [Immunoadsorption for steroid-unresponsive multiple sclerosis-relapses: clinical data of 14 patients]. Nervenarzt 2011, 82(12):1590–1595. In German. 20. Galldiks N, Dohmen C, Neveling M, Fink GR, Haupt WF: Selective immune adsorption treatment of severe Guillain-Barré-syndrome in the intensive care unit. Neurocrit Care 2009, 11:317–321. 21. Samtleben W, Blumenstein M, Liebl L, Gurland HJ: Membrane plasma separation for treatment of immunologically mediated diseases. Trans Am Soc Artif Intern Organs 1980, 26:12–16. 22. Oda S, Hirasawa H, Shiga H, Nakanishi K, Matsuda K, Nakamura M, Ikeda H, Sakai M: Cytokine adsorptive property of various adsorbents in immunoadsorption columns and a newly developed adsorbent: an in vitro study. Blood Purif 2004, 22:530–536. 23. Yoshida M, Tamura M, Yamada Y, Yamawaki N, Yamashita Y, Immusorba TR, Immusorba PH: Basics of design and features of functions. Ther Apher 1998, 2:185–192. 23. Yoshida M, Tamura M, Yamada Y, Yamawaki N, Yamashita Y, Immusorba TR, Immusorba PH: Basics of design and features of functions. Ther Apher 1998, 2:185–192. 24. Rithidech KN, Honikel L, Milazzo M, Madigan D, Troxell R, Krupp LB: Protein expression profiles in pediatric multiple sclerosis: potential biomarkers. Mult Scler 2009, 15:455–464. 24. Rithidech KN, Honikel L, Milazzo M, Madigan D, Troxell R, Krupp LB: Protein expression profiles in pediatric multiple sclerosis: potential biomarkers. Mult Scler 2009, 15:455–464. Koziolek et al. Journal of Neuroinflammation 2012, 9:80 http://www.jneuroinflammation.com/content/9/1/80 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 25. Harp CT, Ireland S, Davis LS, Remington G, Cassidy B, Cravens PD, Stuve O, Lovett-Racke AE, Eagar TN, Greenberg BM, Racke MK, Cowell LG, Karandikar NJ, Frohman EM, Monson NL: Memory B cells from a subset of treatment-naïve relapsing-remitting multiple sclerosis patients elicit CD4(+) T-cell proliferation and IFN-γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Eur J Immunol 2010, 40:2942–2956. Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission 26. Hellings N, Bare’e M, Verhoeven C, Beatrijs D'hooghe M, Medaer R, Bernard CCA, Raus J, Stinissen P: T-cell reactivity to multiple myelin antigens in multiple sclerosis patients and healthy controls. J Neurosci Res 2001, 63:290–302. 27. Axtell RC, Webb MS, Barnum SR, Raman C: Cutting edge: critical role for CD5 in experimental autoimmune encephalomyelitis: inhibition of engagement reverses disease in mice. J Immunol 2004, 173:2928–2932.
https://openalex.org/W4388494199
https://www.cambridge.org/core/services/aop-cambridge-core/content/view/C1D1CC30B3A18BC246CEACAF5C53E0DA/S0007114523002520a.pdf/div-class-title-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-div.pdf
English
null
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
British journal of nutrition
2,023
cc-by
8,187
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 3 / 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 adults. Geriatr Gerontol Int 17, 937–944. R. P., E. O. V., R. G., C. D., N. D. and A. F. J. conceived and designed the ORCHID study. R. P. and A. F. J. performed the statistical analysis, interpreted results and wrote the manuscript. E. O. V., R. G. and C. F. supported the analyses and writing of the paper. All authors reviewed the manuscript and contributed to the discussion. 14. Ruel MT (2003) Operationalizing dietary diversity: a review of measurement issues and research priorities. J Nutr 133, Suppl. 2, 3911S–3926S. 15. Verger EO, Le Port A, Borderon A, et al. (2021) Dietary diversity indicators and their associations with dietary adequacy and health outcomes: a systematic scoping review. Adv Nutr 12, 1659–1672. There are no conflicts of interest. 16. Kant AK (1996) Indexes of overall diet quality: a review. J Am Diet Assoc 96, 785–791. Conclusion The ORCHID score is the first healthy dietary diversity score developed to assess diet based on the rating of consumption occurrences. There is strong evidence that it will be a useful and pragmatic tool, that is, one that does not require overly sophisticated dietary data to assess dietary diversity and diet quality indicators. Not only is it the first healthy dietary diversity score for older adults, but it could also be easily adapted to other population groups through rating systems and food group modifications. 8. de Sire A, Ferrillo M, Lippi L, et al. (2022) Sarcopenic dysphagia, malnutrition, and oral frailty in elderly: a comprehensive review. Nutrients 14, 982. 9. Seligman HK, Levi R, Adebiyi VO, et al. (2023) Assessing and monitoring nutrition security to promote healthy dietary intake and outcomes in the United States. Annu Rev Nutr 43, 409–429. 10. Otsuka R, Tange C, Nishita Y, et al. (2020) Dietary diversity and all-cause and cause-specific mortality in Japanese community- dwelling older adults. Nutrients 12, 1052. g 11. Tao L, Xie Z & Huang T (2020) Dietary diversity and all-cause mortality among Chinese adults aged 65 or older: a community- based cohort study. Asia Pac J Clin Nutr 29, 152–160. Healthy ageing The ORCHID score is adapted to healthy ageing and the prevention of malnutrition: it focuses on protein intake by having eleven ORCHID components from ORCHID food groups about rich-protein food, with seven ORCHID components that are calculated only with the positive rating system, and promotes a A.-F. Jacquemot et al. 1062 4. Shlisky J, Bloom DE, Beaudreault AR, et al. (2017) Nutritional considerations for healthy aging and reduction in age-related chronic disease. Adv Nutr 8, 17–26. advanced age, participants may be more prone to memory issues. This could potentially affect data accuracy(36). No correlation was found between the ORCHID score and total monthly household income and living with someone. However, it is offset by the others correlations found (smocking status, level of physical activity, level of study and BMI). Finally, the data did not include a biological sample. 5. Chaltiel D, Adjibade M, Deschamps V, et al. (2019) Programme national nutrition santé – guidelines score 2 (PNNS-GS2): development and validation of a diet quality score reflecting the 2017 French dietary guidelines. Br J Nutr 122, 331–342. 2017 French dietary guidelines. Br J Nutr 122, 331–342. 6. Guenther PM, Reedy J, Krebs-Smith SM, et al. (2008) Evaluation of the healthy eating index-2005. J Am Diet Assoc 108, 1854–1864. Finally, there is no ‘gold’ standard against which the ORCHID score can be calibrated. However, the use of a detailed strategy based on other measures with converging results strengthened confidence in the ORCHID score’s reliability. 7. Martin G (2008) Sociologie de la Vieillesse et du Vieillissement. (Sociology of old age and ageing). Vincent Caradec, Paris, Armand Colin, Coll. « 128 », 2e Édition, 128 Pages ISBN: 2200353391. Idées Économiques et Sociales 154, 79. https:// www.cairn.info/revue-idees-economiques-et-sociales-2008-4- page-79.htm (accessed June 2022). Supplementary material 17. Vadiveloo M, Dixon LB, Mijanovich T, et al. (2014) Development and evaluation of the US healthy food diversity index. Br J Nutr 112, 1562–1574. For supplementary materials referred to in this article, please visit https://doi.org/10.1017/S0007114523002520 18. Drescher LS, Thiele S & Mensink GBM (2007) A new index to measure healthy food diversity better reflects a healthy diet than traditional measures. J Nutr 137, 647–651. 19. HCSP (2021) Avis Relatif à la Révision des Repères Alimentaires Pour les Personnes Âgées. (Notice concerning the revision of the dietary guidelines for older people) https://www.hcsp.fr/ explore.cgi/avisrapportsdomaine?clefr=1087 (accessed June 2022). 22. Bland JM & Altman DG (2002) Validating scales and indexes. BMJ 324, 606–607. y 2. Béland D & Viriot Durandal JP (2013) Aging in France: population trends, policy issues, and research institutions. Gerontologist 53, 191–197. References 1. Insee (2020) 3.2 Population par Âge. (Population by age.) In: Tableaux de l’Économie Française. 88 Avenue Verdier, 92541 MONTROUGE CEDE. Institut National de la Statistique et des Études Économiques. p. 24. (Insee Références). https://www. insee.fr/fr/statistiques/4277619?sommaire=4318291 (accessed Febrary 2021). 20. Katsas K, Mamalaki E, Kontogianni MD, et al. (2020) Malnutrition in older adults: correlations with social, diet- related, and neuropsychological factors. Nutrition 71, 110640. y 2. Béland D & Viriot Durandal JP (2013) Aging in France: population trends, policy issues, and research institutions. Gerontologist 53, 191–197. 21. Verger EO, Mariotti F, Holmes BA, et al. (2012) Evaluation of a diet quality index based on the probability of adequate nutrient intake (PANDiet) using national French and US dietary surveys. PLOS ONE 7, e42155. 3. Stenholm S, Westerlund H, Head J, et al. (2015) Comorbidity and functional trajectories from midlife to old age: the health and retirement study. J Gerontol A Biol Sci Med Sci 70, 332–338. 22. Bland JM & Altman DG (2002) Validating scales and indexes. BMJ 324, 606–607. Occurrence-based dietary diversity score 1063 31. Toft U, Kristoffersen LH, Lau C, et al. (2007) The dietary quality score: validation and association with cardiovascular risk factors: the Inter99 study. Eur J Clin Nutr 61, 270–278. 23. Allès B, Samieri C, Lorrain S, et al. (2016) Nutrient patterns and their food sources in older persons from France and Quebec: dietary and lifestyle characteristics. Nutrients 8, 225. 32. Waijers PMCM, Feskens EJM & Ocké MC (2007) A critical review of predefined diet quality scores. Br J Nutr 97, 219–231. 24. Estaquio C, Kesse-Guyot E, Deschamps V, et al. (2009) Adherence to the French programme national nutrition Santé guideline score is associated with better nutrient intake and nutritional status. J Am Diet Assoc 109, 1031–1041. 33. Larrieu S, Letenneur L, Berr C, et al. (2004) Sociodemographic differences in dietary habits in a population-based sample of elderly subjects: the 3C study. J Nutr Health Aging 8, 497–502. 25. Féart C, Jutand MA, Larrieu S, et al. (2007) Energy, macro- nutrient and fatty acid intake of French elderly community dwellers and association with socio-demographic character- istics: data from the Bordeaux sample of the three-city study. Br J Nutr 98, 1046–1057. 34. Bocquier A, Jacquemot AF, Dubois C, et al. (2022) Study protocol for a pragmatic cluster randomized controlled trial to improve dietary diversity and physical fitness among older people who live at home (the “ALAPAGE study”). References BMC Geriatr 22, 643. J 26. Dallongeville J, Marécaux N, Fruchart JC, et al. (1998) Cigarette smoking is associated with unhealthy patterns of nutrient intake: a meta-analysis. J Nutr 128, 14501457. 35. Équipe de Surveillance et d’Épidémiologie Nutritionnelle (2017) Volet nutrition. Chapitre Consommations alimentaires (Nutrition section. Food consumption chapter). In Santé publique France, Étude de Santé sur l’Environnement, la Biosurveillance, l’activité Physique et la Nutrition (Esteban) 2014–2016, pp. 193. Saint-Maurice: Santé Publique France. 27. Ledikwe JH, Blanck HM, Khan LK, et al. (2006) Low-energy- density diets are associated with high diet quality in adults in the United States. J Am Diet Assoc 106, 1172–1180. 28. Vieux F, Dubois C, Duchêne C, et al. (2018) Nutritional quality of school meals in France: impact of guidelines and the role of protein dishes. Nutrients 10, 205. p 29. Lumley T (2004) Survey: Analysis of Complex Survey Samples. https://r-survey.r-forge.r-project.org/survey/ (accessed Febrary 2021). 36. Johansson G, Wikman A, Ahrén AM, et al. (2001) Underreporting of energy intake in repeated 24-h recalls related to gender, age, weight status, day of interview, educational level, reported food intake, smoking habits and area of living. Public Health Nutr 4, 919–927. y 30. R Core Team (2019) R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing.
https://openalex.org/W2963433445
https://europepmc.org/articles/pmc6678647?pdf=render
English
null
Effects of Stress on Phase Transformations in Grinding by FE Modeling and Experimental Approaches
Materials
2,019
cc-by
11,177
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 temperature fields and hardened layer thickness Int J Mach Tools Manuf 2010 50 901–910 [CrossRef] p y J f , , [ ] 6. Cong, M.; Zhou, Z.; Jian, Z.; Huang, X.; Gu, D. An experimental investigation of affected layers formed in grinding of AISI 52100 steel. Int. J. Adv. Manuf. Technol. 2011, 54, 515–523. [CrossRef] 6. Cong, M.; Zhou, Z.; Jian, Z.; Huang, X.; Gu, D. An experimental investigation of affected layers formed in grinding of AISI 52100 steel. Int. J. Adv. Manuf. Technol. 2011, 54, 515–523. [CrossRef] 7. Liu, J.; Wei, Y.; Huang, S.; Xu, Z. Experimental study on grinding-hardening of 1060 Steel. Energy Procedia 2012, 16, 103–108. [CrossRef] 7. Liu, J.; Wei, Y.; Huang, S.; Xu, Z. Experimental study on grinding-hardening of 1060 Steel. Energy Procedia 2012, 16, 103–108. [CrossRef] 8. Yaakoubi, M.; Kchaou, M.; Dammak, F. Simulation of the thermomechanical and metallurgical behavior of steels by using ABAQUS software. Comput. Mater. Sci. 2013, 68, 297–306. [CrossRef] 8. Yaakoubi, M.; Kchaou, M.; Dammak, F. 11. Brosse, A.; Hamdi, H.; Bergheau, J.M. A numerical study of phase transformation during grinding. Int. J. Mach. Mach. Mater. 2008, 4, 148. [CrossRef] References Material behaviour in conditions similar to metal cutting: Flow stress in primary shear zone. J. Mater. Process. Technol. 2002, 122, 322–330. [CrossRef] 21. Ding, H.; Shin, Y.C. A metallo-thermomechanically coupled analysis of orthogonal cutting of AISI 1045 steel. J. Manuf. Sci. Eng. 2012, 134, 051014. [CrossRef] 22. Avrami, M. Granulation, phase change, and microstructure kinetics of phase change. III. J. Chem. Phys. 1941, 9, 177–184. [CrossRef] 23. Cheng, H.; Huang, X.; Wang, H. Calculation of the residual stress of a 45 steel cylinder with a non-linear surface heat-transfer coefficient including phase transformation during quenching. J. Mater. Process. Technol. 1999, 89–90, 339–343. [CrossRef] 24. Fernandes, F.; Denis, S.; Simon, A. Mathematical model coupling phase transformation and temperature evolution during quenching of steels. Mater. Sci. Technol. 1985, 1, 838–844. [CrossRef] g q g 5. Scheil, E. Anlaufzeit der austenitumwandlung. Archiv für das Eisenhüttenwesen 1935, 8, 565–567. [Cross g f 26. Koistinen, D.P.; Marburger, R.E. A general equation prescribing the extent of the austenite-martensite transformation in pure iron-carbon alloys and plain carbon steels. Acta Metall. 1959, 7, 59–60. [CrossRef] 27. Griffiths, B. Mechanisms of white layer generation with reference to machining and deformation processes. J. Tribol. 1987, 109, 525–530. [CrossRef] 28. Ramesh, A.; Melkote, S.N. Modeling of white layer formation under thermally dominant conditions in orthogonal machining of hardened AISI 52100 steel. Int. J. Mach. Tools Manuf. 2008, 48, 402–414. [CrossRef] 28. Ramesh, A.; Melkote, S.N. Modeling of white layer formation under thermally dominant conditions in orthogonal machining of hardened AISI 52100 steel. Int. J. Mach. Tools Manuf. 2008, 48, 402–414. [CrossRef] orthogonal machining of hardened AISI 52100 steel. Int. J. Mach. Tools Manuf. 2008, 48, 402–414. [CrossRef] 29. Han, H.N.; Lee, C.G.; Oh, C.S.; Lee, T.H.; Kim, S.J. A model for deformation behavior and mechanically induced martensitic transformation of metastable austenitic steel. Acta Mater. 2004, 52, 5203–5214. [CrossRef] 30. Denis, S.; Sjöström, S.; Simon, A. Coupled temperature, stress, phase transformation calculation model numerical illustration of the internal stress evolution during cooling of a eutectoid steel cylinder. Metall. 29. Han, H.N.; Lee, C.G.; Oh, C.S.; Lee, T.H.; Kim, S.J. A model for deformation behavior and mechanically induced martensitic transformation of metastable austenitic steel. Acta Mater. 2004, 52, 5203–5214. [CrossRef] 29. Han, H.N.; Lee, C.G.; Oh, C.S.; Lee, T.H.; Kim, S.J. A model for deformation behavior and mechanically induced martensitic transformation of metastable austenitic steel. Acta Mater. 2004, 52, 5203–5214. [CrossRef] 30. References Simulation of the thermomechanical and metallurgical behavior of steels by using ABAQUS software. Comput. Mater. Sci. 2013, 68, 297–306. [CrossRef] 9. Wang, M.; Jiang, S.; Zhang, Y. Phase transformation, twinning, and detwinning of NiTi shape-memory alloy subject to a shock wave based on molecular-dynamics simulation. Materials 2018, 11, 2334. [CrossRef] 9. Wang, M.; Jiang, S.; Zhang, Y. Phase transformation, twinning, and detwinning of NiTi shape-memory alloy subject to a shock wave based on molecular-dynamics simulation. Materials 2018, 11, 2334. [CrossRef] 10. Zhang, L.; Mahdi, M. Applied mechanics in grinding-IV. The mechanism of grinding induced phase transformation. Int. J. Mach. Tools Manuf. 1995, 35, 1397–1409. [CrossRef] 10. Zhang, L.; Mahdi, M. Applied mechanics in grinding-IV. The mechanism of grinding induced phase transformation. Int. J. Mach. Tools Manuf. 1995, 35, 1397–1409. [CrossRef] 11. Brosse, A.; Hamdi, H.; Bergheau, J.M. A numerical study of phase transformation during grinding. Int. J. Mach. Mach. Mater. 2008, 4, 148. [CrossRef] 11. Brosse, A.; Hamdi, H.; Bergheau, J.M. A numerical study of phase transformation during grinding. Int. J. Mach. Mach. Mater. 2008, 4, 148. [CrossRef] Materials 2019, 12, 2327 18 of 18 18 of 18 12. Ding, Z.; Li, B.; Liang, S. Maraging steel phase transformation in high strain rate grinding. Int. J. Adv. Manuf. Technol. 2015, 80, 711–718. [CrossRef] 13. Deng, Y.; Xiu, S. Research on microstructure evolution of austenitization in grinding hardening by cellular automata simulation and experiment. Int. J. Adv. Manuf. Technol. 2017, 93, 2599–2612. [CrossRef] 14. Salonitis, K. A hybrid cellular automata-finite element model for the simulation of the grind-hardening process. Int. J. Adv. Manuf. Technol. 2017, 93, 4007–4013. [CrossRef] 15. Serajzadeh, S. Modelling of temperature history and phase transformations during cooling of steel. J. Mater. Process. Technol. 2004, 146, 311–317. [CrossRef] 16. Bojinovi´c, M.; Mole, N.; Štok, B. A computer simulation study of the effects of temperature change rate on austenite kinetics in laser hardening. Surf. Coat. Technol. 2015, 273, 60–76. [CrossRef] 17. Guo, C.; Malkin, S. Analytical and experimental investigation of burnout in creep-feed grinding. CIRP Ann-Manuf. Technol. 1994, 43, 283–286. [CrossRef] 18. Svoboda, J.; Gamsjäger, E.; Fischer, F. Relaxation of the elastic strain energy of misfitting inclusions due to diffusion of vacancies. Philos. Mag. Lett. 2005, 85, 473–479. [CrossRef] 9. Leblond, J.B.; Devaux, J.; Devaux, J. Mathematical modelling of transformation plasticity in steels I: Cas ideal-plastic phases. Int. J. Plast. 1989, 5, 551–572. [CrossRef] 0. Jaspers, S.; Dautzenberg, J. References Denis, S.; Sjöström, S.; Simon, A. Coupled temperature, stress, phase transformation calculation model numerical illustration of the internal stress evolution during cooling of a eutectoid steel cylinder. Metall. Trans. A 1987, 18, 1203–1212. [CrossRef] 30. Denis, S.; Sjöström, S.; Simon, A. Coupled temperature, stress, phase transformation calculation model numerical illustration of the internal stress evolution during cooling of a eutectoid steel cylinder. Metall. Trans. A 1987, 18, 1203–1212. [CrossRef] , , [ ] 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/).
https://openalex.org/W4236805817
https://ora.ox.ac.uk/objects/uuid:dacafb1c-a5e4-4c1a-92ba-282b16c0441e/files/mfe1bfe73a3c38b7083260661f0a094cf
English
null
CONSORT 2010 Statement: updated guidelines for reporting parallel group randomised trials
BMJ. British medical journal
2,010
cc-by
6,612
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 and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W4391105333
https://ojs.reformedjournals.co.za/stj/article/download/2621/3536
English
null
Mary-Anne Plaatjies-van Huffel
Stellenbosch theological journal
2,024
cc-by
6,814
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 Company. Schüssler Fiorenza, E. 2011. Transforming Vision: Explorations In Feminist The*logy. Philadelphia: Fortess Press. Schüssler Fiorenza, E. 2014. Between Movement and Academy: Feminist Biblical Studies. In E. (. Schüssler Fiorenza. Feminist Biblical Studies 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.
https://openalex.org/W3199114375
https://europepmc.org/articles/pmc8469425?pdf=render
English
null
RNA-Sequencing Based microRNA Expression Signature of Colorectal Cancer: The Impact of Oncogenic Targets Regulated by miR-490-3p
International journal of molecular sciences
2,021
cc-by
15,088
  * 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 incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018, 68, 394–424. [CrossRef] 2 Dekker E ; Tanis PJ ; Vleugels J L A ; Kasi PM ; Wallace M B Colorectal cancer Lancet 2019 394 1467 1480 [CrossRef] J g J [ ] 3. Siegel, R.L.; Miller, K.D.; Goding Sauer, A.; Fedewa, S.A.; Butterly, L.F.; Anderson, J.C.; Cercek, A.; Smith, R.A.; Jemal, A. Colorectal cancer statistics, 2020. CA Cancer J. Clin. 2020, 70, 145–164. [CrossRef] 4. Carlomagno, C.; De Stefano, A.; Rosanova, M.; De Falco, S.; Attademo, L.; Fiore, G.; De Placido, S. Multiple treatment lines and prognosis in metastatic colorectal cancer patients. Cancer Metastasis Rev. 2019, 38, 307–313. [CrossRef] [PubMed] arey, F.A.; Pratt, N.R.; Steele, R.J. The colorectal adenoma–carcinoma sequence. BJS 2002, 89, 845–860. [Cr 5. Leslie, A.; Carey, F.A.; Pratt, N.R.; Steele, R.J. The colorectal adenoma–carcinoma sequence. BJS 2002 [PubMed] [ ] 6. Grady, W.M.; Markowitz, S.D. The Molecular Pathogenesis of Colorectal Cancer and Its Potential Application to Colorectal Cancer Screening. Dig. Dis. Sci. 2015, 60, 762–772. [CrossRef] [PubMed] 6. Grady, W.M.; Markowitz, S.D. The Molecular Pathogenesis of Colorectal Cancer and Its Potenti Cancer Screening. Dig. Dis. Sci. 2015, 60, 762–772. [CrossRef] [PubMed] 7. The Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012, 487, 330–337. [CrossRef] [PubMed] 8. Bala, P.; Singh, A.K.; Kavadipula, P.; Kotapalli, V.; Sabarinathan, R.; Bashyam, M.D. Exome sequencing identifies ARID2 as a novel tumor suppressor in early-onset sporadic rectal cancer. Oncogene 2021, 40, 863–874. [CrossRef] 9. Anfossi, S.; Babayan, A.; Pantel, K.; Calin, G.A. Clinical utility of circulating non-coding RNAs—An update. Nat. Rev. Clin. Oncol. 2018, 15, 541–563. [CrossRef] 10. Esteller, M. Non-coding RNAs in human disease. Nat. Rev. Genet. 2011, 12, 861–874. [CrossRef] [PubMed] 11. Panni, S.; Lovering, R.C.; Porras, P.; Orchard, S. Non-coding RNA regulatory networks. Biochim. Biophys. Acta Bioenerg. 2020, 1863, 194417. [CrossRef] 12 A i d E J b L S Sl k F J N di RNA k i N t R C 2018 18 5 18 [C R f] u, E.; Jacob, L.S.; Slack, F.J. Non-coding RNA networks in cancer. Nat. Rev. Cancer 2018, 18, 5–18. [CrossRef] 12. Anastasiadou, E.; Jacob, L.S.; Slack, F.J. Non-coding RNA networks in cancer. Nat. Rev. Cancer 2018, 18 13. 5. Conclusions We created new RNA sequencing-based CRC miRNA signatures that revealed novel miRNAs that were aberrantly expressed. This miRNA signature laid the foundation for exploring a new molecular RNA network for CRC. Moreover, we revealed that IRAK1, regulated by miR-490-3p, may be a novel diagnostic and therapeutic target in CRC. Our approach, which utilized the analysis of miRNA signatures, could contribute to the eluci- dation of the molecular pathogenesis of cancer. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ijms22189876/s1. References Xing, X.L.; Yao, Z.Y.; Zhang, T.; Zhu, N.; Liu, Y.W.; Peng, J. MicroRNA-Related Prognosis Biomar Sequencing Data of Colorectal Cancer. Biomed. Res. Int. 2020, 2020, 7905380. [CrossRef] 24. Neerincx, M.; Sie, D.; Buffart, T.E.; Verheul, H.M.W.; Van Der Wiel, M.A.; Van Grieken, N.C.T.; Burggraaf, J.D.; Dekker, H.; Eijk, P.P.; Ylstra, B.; et al. MiR expression profiles of paired primary colorectal cancer and metastases by next-generation sequencing. Oncogenesis 2015, 4, e170. [CrossRef] g 25. Hamfjord, J.; Stangeland, A.M.; Hughes, T.; Skrede, M.L.; Tveit, K.M.; Ikdahl, T.; Kure, E.H. Differential Expression of miRNAs in Colorectal Cancer: Comparison of Paired Tumor Tissue and Adjacent Normal Mucosa Using High-Throughput Sequencing. PLoS ONE 2012, 7, e34150. [CrossRef] [PubMed] 26. Tanaka, T.; Okada, R.; Hozaka, Y.; Wada, M.; Moriya, S.; Satake, S.; Idichi, T.; Kurahara, H.; Ohtsuka, T.; Seki, N. Molecular Pathogenesis of Pancreatic Ductal Adenocarcinoma: Impact of miR-30c-5p and miR-30c-2-3p Regulation on Oncogenic Genes. Cancers 2020, 12, 2731. [CrossRef] 27. Kawagoe, K.; Wada, M.; Idichi, T.; Okada, R.; Yamada, Y.; Moriya, S.; Okubo, K.; Matsushita, D.; Arigami, T.; Kurahara, H.; et al. Regulation of aberrantly expressed SERPINH1 by antitumor miR-148a-5p inhibits cancer cell aggressiveness in gastric cancer. J. Hum. Genet. 2020, 65, 647–656. [CrossRef] [PubMed] 28. Del Vecchio, F.; Mastroiaco, V.; Di Marco, A.; Compagnoni, C.; Capece, D.; Zazzeroni, F.; Capalbo, C.; Alesse, E.; Tessitore, A. Next-generation sequencing: Recent applications to the analysis of colorectal cancer. J. Transl. Med. 2017, 15, 1–19. [CrossRef] h C k h A h A h f k l k A l h l S l 29. Röhr, C.; Kerick, M.; Fischer, A.; Kühn, A.; Kashofer, K.; Timmermann, B.; Daskalaki, A.; Meinel, T.; Drichel, D.; Börno, S.T.; et al. High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal, Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications. PLoS ONE 2013, 8, e67461. [CrossRef] 30. Lee, J.; Hong, H.K.; Peng, S.B.; Kim, T.W.; Lee, W.Y.; Yun, S.H.; Kim, H.C.; Liu, J.; Ebert, P.J.; Aggarwal, A.; et al. Identifying metastasis-initiating miRNA-target regulations of colorectal cancer from expressional changes in primary tumors. Sci. Rep. 2020, 10, 14919. [CrossRef] 31. Bandrés, E.; Cubedo, E.; Agirre, X.; Malumbres, R.; Zárate, R.; Ramirez, N.; Abajo, A.; Navarro, A.; Moreno, I.; Monzó, M.; et al. Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues. Mol. Cancer 2006, 5, 29. [CrossRef] 32. References Liu, X.; Hao, L.; Li, D.; Zhu, L.; Hu, S. Long Non-coding RNAs and Their Biological Roles in Plants. Genom. Proteom. Bioinform. 2015, 13, 137–147. [CrossRef] , , [ ] 14. Ha, M.; Kim, V.N. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 2014, 15, 509–524. [CrossRef] 15. He, L.; Hannon, G.J. MicroRNAs: Small RNAs with a big role in gene regulation. Nat. Rev. Genet. 2004, 5, 522–531. [CrossRef] 14. Ha, M.; Kim, V.N. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 2014, 15, 509–524. [CrossRef] Int. J. Mol. Sci. 2021, 22, 9876 16 of 17 16 of 17 16. Idichi, T.; Seki, N.; Kurahara, H.; Fukuhisa, H.; Toda, H.; Shimonosono, M.; Yamada, Y.; Arai, T.; Kita, Y.; Kijima, Y.; et al. Involvement of anti-tumor miR-124-3p and its targets in the pathogenesis of pancreatic ductal adenocarcinoma: Direct regulation of ITGA3 and ITGB1 by miR-124-3p. Oncotarget 2018, 9, 28849–28865. [CrossRef] y p g 17. Goto, Y.; Kojima, S.; Nishikawa, R.; Enokida, H.; Chiyomaru, T.; Kinoshita, T.; Nakagawa, M.; Naya, Y.; Ichikawa, T.; Seki, N. The microRNA-23b/27b/24-1 cluster is a disease progression marker and tumor suppressor in prostate cancer. Oncotarget 2014, 5, 7748–7759. [CrossRef] 18. Xie, N.; Meng, Q.; Zhang, Y.; Luo, Z.; Xue, F.; Liu, S.; Li, Y.; Huang, Y. MicroRNA-142-3p suppresses cell proliferation, invasion and epithelial-to-mesenchymal transition via RAC1-ERK1/2 signaling in colorectal cancer. Mol. Med. Rep. 2021, 24, 1–12. [CrossRef] [PubMed] 19. Wada, M.; Goto, Y.; Tanaka, T.; Okada, R.; Moriya, S.; Idichi, T.; Noda, M.; Sasaki, K.; Kita, Y.; Kurahara, H.; et al. RNA sequencing-based microRNA expression signature in esophageal squamous cell carcinoma: Oncogenic targets by antitumor miR-143-5p and miR-143-3p regulation. J. Hum. Genet. 2020, 65, 1019–1034. [CrossRef] [PubMed] 20. Toda, H.; Kurozumi, S.; Kijima, Y.; Idichi, T.; Shinden, Y.; Yamada, Y.; Arai, T.; Maemura, K.; Fujii, T.; Horiguchi, J.; et al. Molecular pathogenesis of triple-negative breast cancer based on microRNA expression signatures: Antitumor miR-204-5p targets AP1S3. J. Hum. Genet. 2018, 63, 1197–1210. [CrossRef] [PubMed] 21. Yonemori, K.; Seki, N.; Idichi, T.; Kurahara, H.; Osako, Y.; Koshizuka, K.; Arai, T.; Okato, A.; Kita, Y.; Arigami, T.; et al. The microRNA expression signature of pancreatic ductal adenocarcinoma by RNA sequencing: Anti-tumour functions of the microRNA-216 cluster. Oncotarget 2017, 8, 70097–70115. [CrossRef] g 22. Pantaleão, L.C.; Ozanne, S. Small RNA Sequencing: A Technique for miRNA Profiling. Methods Mol. Biol. 2018, 1735, 321–330. [CrossRef] [PubMed] 23. References MicroRNA-490 inhibits tumorigenesis and progression in breast cancer. Oncol. Targets Ther. 2016, 9, 4505–4516. [CrossRef] 45. Jia, Z.; Liu, Y.; Gao, Q.; Han, Y.; Zhang, G.; Xu, S.; Cheng, K.; Zou, W. miR-490-3p inhibits the growth and invasiveness in triple-negative breast cancer by repressing the expression of TNKS2. Gene 2016, 593, 41–47. [CrossRef] 46. Li, Z.; Jiang, D.; Yang, S. MiR-490-3p Inhibits the Malignant Progression of Lung Adenocarcinoma. Cancer Manag. Res. 2020, 12, 10975–10984. [CrossRef] Z.; Huang, N.; Wu, X.; Li, S.; Li, Y.; Wu, S. MicroRNA-490-3p inhibits colorectal cancer metastasis by targeting er 2015, 15, 1–10. [CrossRef] 47. Xu, X.; Chen, R.; Li, Z.; Huang, N.; Wu, X.; Li, S.; Li, Y.; Wu, S. MicroRNA-490-3p inhibits colorectal canc TGFβR1. BMC Cancer 2015, 15, 1–10. [CrossRef] 48. Wang, B.; Yin, M.; Cheng, C.; Jiang, H.; Jiang, K.; Shen, Z.; Ye, Y.; Wang, S. Decreased expression of miR-490-3p in colorectal cancer predicts poor prognosis and promotes cell proliferation and invasion by targeting RAB14. Int. J. Oncol. 2018, 53, 1247–1256. [CrossRef] 49. Flannery, S.; Bowie, A.G. The interleukin-1 receptor-associated kinases: Critical regulators of innate immune signalling. Biochem. Pharmacol. 2010, 80, 1981–1991. [CrossRef] 49. Flannery, S.; Bowie, A.G. The interleukin 1 receptor associated kinases: Critical regulators of innate immune signalling. Biochem. Pharmacol. 2010, 80, 1981–1991. [CrossRef] 50 Rh G W St ki D T IRAK i lli i B J C 2015 112 232 237 [C R f] Starczynowski, D.T. IRAK signalling in cancer. Br. J. Cancer 2015, 112, 232–237. [CrossRef] 50. Rhyasen, G.W.; Starczynowski, D.T. IRAK signalling in cancer. Br. J. Cancer 2015, 112, 232–237. [Cro 51. Wee, Z.N.; Yatim, S.M.; Kohlbauer, V.K.; Feng, M.; Goh, J.Y.; Bao, Y.; Lee, P.L.; Zhang, S.; Wang, P.P.; Lim, E.; et al. IRAK1 is a therapeutic target that drives breast cancer metastasis and resistance to paclitaxel. Nat. Commun. 2015, 6, 8746. [CrossRef] [PubMed] 52. Wang, Y.; Wang, Y.; Duan, X.; Wang, Y.; Zhang, Z. Interleukin-1 receptor-associated kinase 1 correlates with metastasis and invasion in endometrial carcinoma. J. Cell Biochem. 2018, 119, 2545–2555. [CrossRef] 53. Adams, A.K.; Bolanos, L.C.; Dexheimer, P.J.; Karns, R.A.; Aronow, B.J.; Komurov, K.; Jegga, A.G.; Casper, K.A.; Patil, Y.J.; Wilson, K.M.; et al. IRAK1 is a novel DEK transcriptional target and is essential for head and neck cancer cell survival. Oncotarget 2015, 6, 43395–43407. [CrossRef] 54. Singer, J.W.; Fleischman, A.; Al-Fayoumi, S.; Mascarenhas, J.O.; Yu, Q.; Agarwal, A. References Exploration of small RNA-seq data for small non-coding RNAs in Human Colorectal Cancer. Int. J. Genom. 2017, 5, 16–31. [CrossRef] J 39. Yu, D.; Liu, H.; Qin, J.; Huangfu, M.; Guan, X.; Li, X.; Zhou, L.; Dou, T.; Liu, Y.; Wang, L.; et al. Curcumol inhibits the viability and J 39. Yu, D.; Liu, H.; Qin, J.; Huangfu, M.; Guan, X.; Li, X.; Zhou, L.; Dou, T.; Liu, Y.; Wang, L.; et al. Curcumol inhibits the viability and invasion of colorectal cancer cells via miR-30a-5p and Hippo signaling pathway. Oncol. Lett. 2021, 21, 1–13. [CrossRef] 39. Yu, D.; Liu, H.; Qin, J.; Huangfu, M.; Guan, X.; Li, X.; Zhou, L.; Dou, T.; Liu, Y.; Wang, L.; et al. Curcumo invasion of colorectal cancer cells via miR-30a-5p and Hippo signaling pathway. Oncol. Lett. 2021, 21, g g ion of colorectal cancer cells via miR-30a-5p and Hippo signaling pathway. Oncol. Lett. 2021, 21, 1–13. [Cros p pp g g p y 40. Mitra, R.; Adams, C.M.; Jiang, W.; Greenawalt, E.; Eischen, C.M. Pan-cancer analysis reveals cooperativity of both strands of microRNA that regulate tumorigenesis and patient survival. Nat. Commun. 2020, 11, 1–15. [CrossRef] 40. Mitra, R.; Adams, C.M.; Jiang, W.; Greenawalt, E.; Eischen, C.M. Pan-cancer analysis reveals cooper microRNA that regulate tumorigenesis and patient survival. Nat. Commun. 2020, 11, 1–15. [CrossRef] 40. Mitra, R.; Adams, C.M.; Jiang, W.; Greenawalt, E.; Eischen, C.M. Pan cancer analysis reveals cooperativity of both strands of microRNA that regulate tumorigenesis and patient survival. Nat. Commun. 2020, 11, 1–15. [CrossRef] J g y p microRNA that regulate tumorigenesis and patient survival. Nat. Commun. 2020, 11, 1–15. [CrossRef] 41. Idichi, T.; Seki, N.; Kurahara, H.; Fukuhisa, H.; Toda, H.; Shimonosono, M.; Okato, A.; Arai, T.; Kita, Y.; Mataki, Y.; et al. Molecular pathogenesis of pancreatic ductal adenocarcinoma: Impact of passenger strand of pre-miR-148a on gene regulation. Cancer Sci. 2018, 109, 2013–2026. [CrossRef] [PubMed] 2018, 109, 2013–2026. [CrossRef] [PubMed] 42. Shimomura, H.; Okada, R.; Tanaka, T.; Hozaka, Y.; Wada, M.; Moriya, S.; Idichi, T.; Kita, Y.; Kurahara, H.; Ohtsuka, T.; et al. Role of miR-30a-3p Regulation of Oncogenic Targets in Pancreatic Ductal Adenocarcinoma Pathogenesis. Int. J. Mol. Sci. 2020, 21, 6459. [CrossRef] [ ] 43. Li, Y.; Tian, D.; Chen, H.; Cai, Y.; Chen, S.; Duan, S. MicroRNA-490-3p and -490-5p in carcinogenesis: Separate or the same goal? Oncol. Lett. 2021, 22, 678. [CrossRef] [PubMed] 44. Zheng, X.; Zhao, L. References Sarver, A.L.; French, A.J.; Borralho, P.M.; Thayanithy, V.; Oberg, A.L.; Silverstein, K.A.; Morlan, B.W.; Riska, S.M.; Boardman, L.A.; Cunningham, J.M.; et al. Human colon cancer profiles show differential microRNA expression depending on mismatch repair status and are characteristic of undifferentiated proliferative states. BMC Cancer 2009, 9, 401. [CrossRef] 33. Schetter, A.J.; Leung, S.Y.; Sohn, J.J.; Zanetti, K.A.; Bowman, E.D.; Yanaihara, N.; Yuen, S.T.; Chan, T.L.; Kwong, D.L.W.; Au, G.K.H.; et al. MicroRNA Expression Profiles Associated with Prognosis and Therapeutic Outcome in Colon Adenocarcinoma. JAMA 2008, 299, 425–436. [CrossRef] J [ ] 34. Cummins, J.M.; He, Y.; Leary, R.J.; Pagliarini, R.; Diaz, L.; Sjoblom, T.; Barad, O.; Bentwich, Z.; Szafranska, A.E.; Labourier, E.; et al. The colorectal microRNAome. Proc. Natl. Acad. Sci. USA 2006, 103, 3687–3692. [CrossRef] J 34. Cummins, J.M.; He, Y.; Leary, R.J.; Pagliarini, R.; Diaz, L.; Sjoblom, T.; Barad, O.; Bentwich, Z.; Szafranska, A.E.; Labourier, E.; et al. The colorectal microRNAome. Proc. Natl. Acad. Sci. USA 2006, 103, 3687–3692. [CrossRef] 35. Li, J.; Mo, R.; Zheng, L. MicroRNA-490-3p inhibits migration and chemoresistance of colorectal cancer cells via targeting TNKS2. et al. The colorectal microRNAome. Proc. Natl. Acad. Sci. USA 2006, 103, 3687–3692. [CrossRef] 35. Li, J.; Mo, R.; Zheng, L. MicroRNA-490-3p inhibits migration and chemoresistance of colorectal cancer cells via targeting TNKS2. World J Surg Oncol 2021 19 117 [CrossRef] [PubMed] 35. Li, J.; Mo, R.; Zheng, L. MicroRNA-490-3p inhibits migration and chemoresistance of colorectal cancer cells via targeting TNKS2. World J. Surg. Oncol. 2021, 19, 117. [CrossRef] [PubMed] 35. Li, J.; Mo, R.; Zheng, L. MicroRNA-490-3p inhibits migration and World J. Surg. Oncol. 2021, 19, 117. [CrossRef] [PubMed] 35. Li, J.; Mo, R.; Zheng, L. MicroRNA-490-3p inhibits migration and chemoresistance of colorectal cancer World J. Surg. Oncol. 2021, 19, 117. [CrossRef] [PubMed] 36. Liu, X.; He, B.; Xu, T.; Pan, Y.; Hu, X.; Chen, X.; Wang, S. MiR-490-3p Functions as a Tumor Suppressor by Inhibiting Oncogene VDAC1 Expression in Colorectal Cancer. J. Cancer 2018, 9, 1218–1230. [CrossRef] 37. Wang, L.; Guo, J.; Zhou, J.; Wang, D.; Kang, X.; Zhou, L. NF-κB maintains the stemness of colon cancer cells by downregulating miR-195-5p/497-5p and upregulating MCM2. J. Exp. Clin. Cancer Res. 2020, 39, 225. [CrossRef] Int. J. Mol. Sci. 2021, 22, 9876 17 of 17 17 of 17 38. Koduru, S.V.; Tiwari, A.K.; Hazard, S.W.; Mahajan, M.; Ravnic, D.J. References Inhibition of interleukin-1 receptor-associated kinase 1 (IRAK1) as a therapeutic strategy. Oncotarget 2018, 9, 33416–33439. [CrossRef] 55. Feng, Z.; Duan, Z.; Shi, G.; Wang, Q.; Zhou, J.; Chen, Y. Pharmacological inhibition of IRAK1 attenuates colitis-induced tumorigenesis in mice by inhibiting the inflammatory response and epithelial–mesenchymal transition. J. Biochem. Mol. Toxicol. 2021, e22838. [CrossRef] 56. Toda, H.; Seki, N.; Kurozumi, S.; Shinden, Y.; Yamada, Y.; Nohata, N.; Moriya, S.; Idichi, T.; Maemura, K.; Fujii, T.; et al. RNA-sequence-based microRNA expression signature in breast cancer: Tumor-suppressive miR-101-5p regulates molecular pathogenesis. Mol. Oncol. 2020, 14, 426–446. [CrossRef]
https://openalex.org/W2791849371
https://www.scielo.br/j/reben/a/zT6J4GnDjH4jYdc93V9bT5b/?lang=en&format=pdf
English
null
Obstacles in the detection and reporting of tuberculosis cases: a discursive analysis
Revista Brasileira de Enfermagem
2,018
cc-by
7,390
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
https://openalex.org/W3011341999
https://europepmc.org/articles/pmc7070067?pdf=render
English
null
Temporospatial shifts in the human gut microbiome and metabolome after gastric bypass surgery
npj biofilms and microbiomes
2,020
cc-by
13,653
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 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. REFERENCES 30. Courcoulas, A. P. et al. Weight change and health outcomes at 3 years after bariatric surgery among individuals with severe obesity. JAMA 310, 2416–2425 (2013). 1. Benotti, P. N. et al. Preoperative weight loss before bariatric surgery. Arch. Surg. 144, 1150–1155 (2009). 31. Iannelli, A. et al. Impact of laparoscopic roux-en-Y gastric bypass on metabolic syndrome, inflammation, and insulin resistance in super versus morbidly obese women. Obes. Surg. 19, 577–582 (2009). 2. Korner, J. et al. Prospective study of gut hormone and metabolic changes after adjustable gastric banding and Roux-en-Y gastric bypass. Int. J. Obes. 33, 786–795 (2009). 32. Nassour, I., Almandoz, J. P., Adams-Huet, B., Kukreja, S. & Puzziferri, N. Metabolic syndrome remission after Roux-en-Y gastric bypass or sleeve gastrectomy. Dia- betes Metab. Syndr. Obes. Targets Ther. 10, 393–402 (2017). 3. Ryan, K. K. et al. FXR is a molecular target for the effects of vertical sleeve gastrectomy. Nature 509, 183–188 (2014). 4. Furet, J. P. et al. Differential adaptation of human gut microbiota to bariatric surgery-induced weight loss: links with metabolic and low-grade inflammation markers. Diabetes 59, 3049–3057 (2010). 33. Lozupone, C., Hamady, M. & Knight, R. UniFrac-an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 7, 371 (2006). 5. Graessler, J. et al. Metagenomic sequencing of the human gut microbiome before and after bariatric surgery in obese patients with type 2 diabetes: correlation with inflammatory and metabolic parameters. Pharmacogenomics J. 13, 514–522 (2013). 34. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010). 35. Hooper, L. V., Midtvedt, T. & Gordon, J. I. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu. Rev. Nutr. 22, 283–307 (2002). 6. Ilhan, Z. E. et al. Distinctive microbiomes and metabolites linked with weight loss after gastric bypass, but not gastric banding. ISME J. 11, 2047–2058 (2017). 36. Espey, M. G. Role of oxygen gradients in shaping redox relationships between the human intestine and its microbiota. Free Radic. Biol. Med. 55, 130–140 (2013). 7. Palleja, A. et al. Roux-en-Y gastric bypass surgery of morbidly obese patients induces swift and persistent changes of the individual gut microbiota. Genome Med 8, 67 (2016). 37. Conlon, M. A. & Bird, A. R. The impact of diet and lifestyle on gut microbiota and human health. Nutrients 7, 17–44 (2015). 8. Tremaroli, V. et al. REFERENCES Roux-en-Y gastric bypass and vertical banded gastroplasty induce long-term changes on the human gut microbiome contributing to fat mass regulation. Cell Metab. 22, 228–238 (2015). 38. Antharam, V. C. et al. Intestinal dysbiosis and depletion of butyrogenic bacteria in Clostridium difficile infection and nosocomial diarrhea. J. Clin. Microbiol. 51, 2884–2892 (2013). 9. Zhang, H. et al. Human gut microbiota in obesity and after gastric bypass. Proc. Natl. Acad. Sci. USA 106, 2365–2370 (2009). 39. Jost, T., Lacroix, C., Braegger, C. & Chassard, C. Assessment of bacterial diversity in breast milk using culture-dependent and culture-independent approaches. Br. J. Nutr. 110, 1253–1262 (2013). 10. Aron-Wisnewsky, J. et al. Major microbiota dysbiosis in severe obesity: fate after bariatric surgery. Gut 68, 70–82 (2019). y 11. Liu, R. et al. Gut microbiome and serum metabolome alterations in obesity and after weight-loss intervention. Nat. Med. 23, 859–868 (2017). 40. Krishna Rao, R. & Samak, G. Protection and restitution of gut barrier by probiotics: nutritional and clinical implications. Curr. Nutr. Food Sci. 9, 99–107 (2013). 12. Medina, D. A. et al. Distinct patterns in the gut microbiota after surgical or medical therapy in obese patients. PeerJ 2017, e3443 (2017). 41. Bourriaud, C. et al. Lactate is mainly fermented to butyrate by human intestinal microfloras but inter-individual variation is evident. J. Appl. Microbiol. 99, 201–212 (2005). 13. Kong, L. C. et al. O27 adaptation du microbiote intestinal après la perte du poids: effets du bypass gastrique (GBP) chez les sujets massivement obèses. Diabetes Metab. 36, A7–A8 (2010). 42. Amar, J. et al. Energy intake is associated with endotoxemia in apparently healthy men. Am. J. Clin. Nutr. 87, 1219–1223 (2008). 14. Sanmiguel, C. P. et al. Surgically induced changes in gut microbiome and hedonic eating as related to weight loss: preliminary findings in obese women under- going bariatric surgery. Psychosom. Med. 79, 880–887 (2017). 43. Everard, A. et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc. Natl Acad. Sci. USA 110, 9066–9071 (2013). 15. Farin, W. et al. Laparoscopic Roux-en-Y gastric bypass profoundly changes gut microbiota compared to laparoscopic sleeve gastrectomy: a metagenomic comparative analysis. bioRxiv 425074. https://doi.org/10.1101/425074 (2018). 44. Geurts, L., Neyrinck, aM., Delzenne, N. M., Knauf, C. & Cani, P. D. Gut microbiota controls adipose tissue expansion, gut barrier and glucose metabolism: novel insights into molecular targets and interventions using prebiotics. Beneficial Microbes 5, 3–17 (2014). 16. Durbán, A. et al. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 24. Chambers, E. S. et al. Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in over- weight adults. Gut 64, 1744–1754 (2015). Received: 12 August 2019; Accepted: 14 February 2020; 28. Alvarado, R. et al. The impact of preoperative weight loss in patients undergoing laparoscopic Roux-en-Y gastric bypass. Obes. Surg. 15, 1282–1286 (2005). 29. Dadan, J. et al. The activity of gastric ghrelin positive cells in obese patients treated surgically. Folia Histochem. Cytobiol. 47, 307–313 (2009). Z.E. Ilhan et al. 11 Ethics approval and consent to participate 21. Pellegrini, S. et al. Duodenal mucosa of patients with type 1 diabetes shows distinctive inflammatory profile and microbiota. J. Clin. Endocrinol. Metab. 102, 1468–1477 (2017). 21. Pellegrini, S. et al. Duodenal mucosa of patients with type 1 diabetes shows distinctive inflammatory profile and microbiota. J. Clin. Endocrinol. Metab. 102, 1468–1477 (2017). All study participants provided written informed consent and all procedures were approved by the Institutional Review Boards of Mayo Clinic and Arizona State University (IRB# 10-008725). 22. Liou, A. P. et al. Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity. Sci. Transl. Med 5, 178ra41 (2013). 23. Nilsson, N. E., Kotarsky, K., Owman, C. & Olde, B. Identification of a free fatty acid receptor, FFA2R, expressed on leukocytes and activated by short-chain fatty acids. Biochem. Biophys. Res. Commun. 303, 1047–1052 (2003). Published in partnership with Nanyang Technological University npj Biofilms and Microbiomes (2020) 12 GC-MS analysis of fecal metabolites Metabolites were extracted from 10 mg of lyophilized stool samples using methanol with sonication. Extracted metabolites were completely dried in vacuo and derivatized by methoxylamination and trimethylsilyation and analyzed by GC-MS as reported previously68. GC-MS raw data files were processed using the Metabolite Detector software, version 2.5 beta69. All raw GC-MS data will be made available via the MetaboLights metabo- lomics data repository (http://www.ebi.ac.uk/metabolights/index). Statistical analyses of microbiome and metabolome data sets We used Statistical Package for Social Sciences (SPSS) and R packages70 for all statistical analyses. The medians of the groups along with median absolute deviation values were calculated and reported. Shapiro–Wilk test was used to test normality of the data sets. For the longitudinal cohort, 16S rRNA gene relative abundance comparisons were tested with Wilcoxon signed-rank test. For cross-sectional cohort comparisons, Mann–Whitney’s U-test was used. The p values were corrected using Benjamini and Hochberg method71 and corrected p values less than 0.05 were accepted as significant. Same tests were utilized to analyze NMR, GC-MS, and LC-MS data sets. For the LC-MS data analysis, the data were analyzed after they were log2 transformed. We performed ADONIS test72 on microbiome and metabolome distance matrices to quantify the variation explained by defined variables based on 999 permutations. To reveal associations between bile acid concentrations and the relative abundance of taxonomic groups, we calculated Spearman’s rank correlation coefficient and accepted significance above critical values with Bonferroni corrected p values less than 0.05. DNA extraction, 16S rRNA gene sequencing, and analysis We extracted microbial DNA from feces and biopsy (mucosal) samples using MOBIO PowerSoil DNA extraction kit (MOBIO Laboratories, Carlsbald, CA, USA). We prepared sequencing libraries using the protocols from Earth npj Biofilms and Microbiomes (2020) 12 DATA AVAILABILITY 25. Kohli, R. et al. A surgical model in male obese rats uncovers protective effects of bile acids post-bariatric surgery. Endocrinology 154, 2341–2351 (2013). The 16S rRNA gene sequences were deposited in the Sequence Read Archive (SRA) database (BioSample IDs = SAMN08684029-SAMN08684111). The 16S rRNA gene sequences were deposited in the Sequence Read Archive (SRA) database (BioSample IDs = SAMN08684029-SAMN08684111). 26. Li, J. V. et al. Metabolic surgery profoundly influences gut microbial-host meta- bolic cross-talk. Gut 60, 1214–1223 (2011). 27. Guida, C. et al. PYY plays a key role in the resolution of diabetes followin bariatric surgery in humans. EBioMedicine 40, 67–76 (2019). Received: 12 August 2019; Accepted: 14 February 2020; ADDITIONAL INFORMATION 61. Gripeteg, L., Lindroos, A. K., Peltonen, M., Sjöström, L. & Narbro, K. Effects of bariatric surgery on disability pension in Swedish obese subjects. Int. J. Obes. 36, 356–362 (2012). Supplementary information is available for this paper at https://doi.org/10.1038/ s41522-020-0122-5. 62. Gilbert, J. A., Jansson, J. K. & Knight, R. The Earth Microbiome project: successes and aspirations. BMC Biol. 12, 69 (2014). Correspondence and requests for materials should be addressed to Z.E.I. or R.K.-B. 63. Masella, A. P., Bartram, A. K., Truszkowski, J. M., Brown, D. G. & Neufeld, J. D. PANDAseq: paired-end assembler for illumina sequences. BMC Bioinformatics 13, 31 (2012). Reprints and permission information is available at http://www.nature.com/ reprints 64. Mcmurdie, P. J. et al. Advancing our understanding of the human microbiome using QIIME. Methods Enzymol. 531, 371–444 (2015). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 65. Faith, D. P. Conservation evaluation and phylogenetic diversity. Biol. Conserv. 61, 1–10 (1992). 66. Metsalu, T. & Vilo, J. ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic Acids Res. 43, W566–W570 (2015). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. 67. Humbert, L. et al. Bile acid profiling in human biological samples: comparison of extraction procedures and application to normal and cholestatic patients. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 899, 135–145 (2012). 68. Snijders, A. M. et al. Influence of early life exposure, host genetics and diet on the mouse gut microbiome and metabolome. Nat. Microbiol. 2, 16221 (2016). 69. Hiller, K. et al. Metabolite detector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Anal. COMPETING INTERESTS The authors declare no competing interests. The authors declare no competing interests. 60. Duboc, H. et al. Increase in fecal primary bile acids and dysbiosis in patients with diarrhea-predominant irritable bowel syndrome. Neurogastroenterol. Motil. 24, 513–520 (2012). REFERENCES Assessing gut microbial diversity from feces and rectal mucosa. Microb. Ecol. 61, 123–133 (2011). 45. Dao, M. C. et al. Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology. Gut 65, 426–436 (2016). 17. Sartor, R. B. Gut microbiota: optimal sampling of the intestinal microbiota for research. Nat. Rev. Gastroenterol. Hepatol. 12, 253–254 (2015). 18. Zoetendal, E. G. et al. Mucosa-associated bacteria in the human gastrointestinal tract are uniformly distributed along the colon and differ from the community recovered from feces. Appl. Environ. Microbiol. 68, 3401–3407 (2002). 46. Dao, M. C. et al. Akkermansia muciniphila abundance is lower in severe obesity, but its increased level after bariatric surgery is not associated with metabolic health improvement. Am. J. Physiol. Endocrinol. Metab. 317, E446–E459 (2019). 19. Macfarlane, S. & Dillon, J. F. Microbial biofilms in the human gastrointestinal tract. J. Appl. Microbiol. 102, 1187–1196 (2007). 47. Lin, H. V. et al. Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent mechanisms. PLoS ONE 7, e35240 (2012). 20. Chen, W., Liu, F., Ling, Z., Tong, X. & Xiang, C. Human intestinal lumen and mucosa-associated microbiota in patients with colorectal cancer. PLoS ONE 7, e39743 (2012). npj Biofilms and Microbiomes (2020) 12 Published in partnership with Nanyang Technological University Z.E. Ilhan et al. 12 72. McArdle, B. H. & Anderson, M. J. Fitting multivariate models to community data: a comment on distance-based redundancy analysis. Ecology 82, 290–297 (2001). 48. Newgard, C. B. et al. A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance. Cell Metab. 9, 311–326 (2009). 49. Bhutta, H. Y. et al. Effect of Roux-en-Y gastric bypass surgery on bile acid 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, No. 1 (1995). J. R. Stat. Soc. 57, 289–300 (1995). © The Author(s) 2020 npj Biofilms and Microbiomes (2020) 12 Published in partnership with Nanyang Technological University
https://openalex.org/W1968091104
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0050416&type=printable
English
null
A Single Lys Residue on the First Intracellular Loop Modulates the Endoplasmic Reticulum Export and Cell-Surface Expression of α2A-Adrenergic Receptor
PloS one
2,012
cc-by
8,238
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) Enhancement of the recycling and activation of beta-adrenergic receptor by Rab4 GTPase in cardiac myocytes. J Biol Chem 281: 11097–11103. 1. Pierce KL, Premont RT, Lefkowitz RJ (2002) Seven-transmembrane receptors. Nat Rev Mol Cell Biol 3: 639–650. 2. Qin K, Dong C, Wu G, Lambert NA (2011) Inactive-state preassembly of G(q)- coupled receptors and G(q) heterotrimers. Nat Chem Biol 7: 740–747. 13. Moore CA, Milano SK, Benovic JL (2007) Regulation of receptor trafficking by GRKs and arrestins. Annu Rev Physiol 69: 451–482. 3. Wess J (1997) G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J 11: 346– 354. 14. Lan TH, Kuravi S, Lambert NA (2011) Internalization dissociates beta2- adrenergic receptors. PLoS One 6: e17361. 4. Kobilka BK (2011) Structural insights into adrenergic receptor function and pharmacology. Trends Pharmacol Sci 32: 213–218. 15. Duvernay MT, Dong C, Zhang X, Zhou F, Nichols CD, et al. (2009) Anterograde trafficking of G protein-coupled receptors: function of the C- terminal F(X)6LL motif in export from the endoplasmic reticulum. Mol Pharmacol 75: 751–761. 5. Dong C, Filipeanu CM, Duvernay MT, Wu G (2007) Regulation of G protein- coupled receptor export trafficking. Biochim Biophys Acta 1768: 853–870. 16. Conn PM, Ulloa-Aguirre A, Ito J, Janovick JA (2007) G protein-coupled receptor trafficking in health and disease: lessons learned to prepare for therapeutic mutant rescue in vivo. Pharmacol Rev 59: 225–250. 6. Petaja-Repo UE, Hogue M, Laperriere A, Bhalla S, Walker P, et al. (2001) Newly synthesized human delta opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome. J Biol Chem 276: 4416–4423. 17. Ulloa-Aguirre A, Janovick JA, Brothers SP, Conn PM (2004) Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease. Traffic 5: 821–837. degraded by the proteasome. J Biol Chem 276: 4416–4423. 7. Wang G, Wu G (2012) Small GTPase regulation of GPCR anterograde trafficking. Trends Pharmacol Sci 33: 28–34. 18. Tai AW, Chuang JZ, Bode C, Wolfrum U, Sung CH (1999) Rhodopsin’s carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1. Cell 97: 877–887. 8. Discussion It is possible that mutation of Lys to Arg stabilizes 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 Lys65 to Arg. a2-AR Export and Cell-Surface Expression Kallal L, Benovic JL (2000) Using green fluorescent proteins to study G-protein- coupled receptor localization and trafficking. Trends Pharmacol Sci 21: 175– 180. 29. Robert J, Auzan C, Ventura MA, Clauser E (2005) Mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin V1b/ V3 receptor by a pharmacological chaperone. J Biol Chem 280: 42198–42206. 44. Wu G, Zhao G, He Y (2003) Distinct pathways for the trafficking of angiotensin II and adrenergic receptors from the endoplasmic reticulum to the cell surface: Rab1-independent transport of a G protein-coupled receptor. J Biol Chem 278: 47062–47069. 30. Hirota N, Yasuda D, Hashidate T, Yamamoto T, Yamaguchi S, et al. (2010) Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor. Journal of Biological Chemistry 285: 5931–5940. 45. Dong C, Yang L, Zhang X, Gu H, Lam ML, et al. (2010) Rab8 interacts with distinct motifs in {alpha}2B- and {beta}2-adrenergic receptors and differentially modulates their transport. J Biol Chem 285: 20369–20380. y 31. Schulein R, Hermosilla R, Oksche A, Dehe M, Wiesner B, et al. (1998) A dileucine sequence and an upstream glutamate residue in the intracellular carboxyl terminus of the vasopressin V2 receptor are essential for cell surface transport in COS.M6 cells. Mol Pharmacol 54: 525–535. 46. Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224: 213–232. 47. Dong C, Li C, Wu G (2011) Regulation of alpha2B-Adrenergic Receptor- mediated Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Activation by ADP-ribosylation Factor 1. J Biol Chem 286: 43361–43369. p 32. Hammad MM, Kuang YQ, Morse A, Dupre DJ (2012) Rab1 interacts directly with the beta2-adrenergic receptor to regulate receptor anterograde trafficking. Biol Chem 393: 541–546. 48. Dong C, Wu G (2007) Regulation of anterograde transport of adrenergic and angiotensin II receptors by Rab2 and Rab6 GTPases. Cell Signal 19: 2388– 2399. 33. Zhang X, Dong C, Wu QJ, Balch WE, Wu G (2011) Di-acidic motifs in the membrane-distal C termini modulate the transport of angiotensin II receptors from the endoplasmic reticulum to the cell surface. J Biol Chem 286: 20525– 20535. 49. Duvernay MT, Wang H, Dong C, Guidry JJ, Sackett DL, et al. (2011) {alpha}2B-Adrenergic receptor interaction with tubulin controls its transport from the endoplasmic reticulum to the cell surface. J Biol Chem 286: 14080– 14089. 34. Dong C, Nichols CD, Guo J, Huang W, Lambert NA, et al. a2-AR Export and Cell-Surface Expression 37. Duvernay MT, Zhou F, Wu G (2004) A conserved motif for the transport of G protein-coupled receptors from the endoplasmic reticulum to the cell surface. J Biol Chem 279: 30741–30750. teins, on k Opioid Receptor Expression. Journal of Biological Chemistry 286: 15106–15115. 23. Ge X, Loh HH, Law PY (2009) mu-Opioid receptor cell surface expression is regulated by its direct interaction with Ribophorin I. Mol Pharmacol 75: 1307– 1316. J 38. Duvernay MT, Dong C, Zhang X, Robitaille M, Hebert TE, et al. (2009) A single conserved leucine residue on the first intracellular loop regulates ER export of G protein-coupled receptors. Traffic 10: 552–566. 24. Carrel D, Hamon M, Darmon M (2006) Role of the C-terminal di-leucine motif of 5-HT1A and 5-HT1B serotonin receptors in plasma membrane targeting. J Cell Sci 119: 4276–4284. export of G protein-coupled receptors. Traffic 10: 552–566. 39. Zhou F, Filipeanu CM, Duvernay MT, Wu G (2006) Cell-surface targeting of alpha2-adrenergic receptors – inhibition by a transport deficient mutant through dimerization. Cell Signal 18: 318–327. J 25. Sawyer GW, Ehlert FJ, Shults CA (2010) A conserved motif in the membrane proximal C-terminal tail of human muscarinic m1 acetylcholine receptors affects plasma membrane expression. J Pharmacol Exp Ther 332: 76–86. 40. Dong C, Wu G (2006) Regulation of anterograde transport of alpha2-adrenergic receptors by the N termini at multiple intracellular compartments. J Biol Chem 281: 38543–38554. plasma membrane expression. J Pharmacol Exp Ther 332: 76–8 26. Guo Y, Jose PA (2011) C-Terminal Di-leucine Motif of Dopamine D(1) Receptor Plays an Important Role in Its Plasma Membrane Trafficking. PLoS One 6: e29204. 41. Dong C, Zhang X, Zhou F, Dou H, Duvernay MT, et al. (2010) ADP- ribosylation factors modulate the cell surface transport of G protein-coupled receptors. J Pharmacol Exp Ther 333: 174–183. 27. Donnellan PD, Kimbembe CC, Reid HM, Kinsella BT (2011) Identification of a novel endoplasmic reticulum export motif within the eighth alpha-helical domain (alpha-H8) of the human prostacyclin receptor. Biochim Biophys Acta 1808: 1202–1218. 42. Lu R, Li Y, Zhang Y, Chen Y, Shields AD, et al. (2009) Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of a2- Adrenergic Responses Is because of Ligand-selective Internalization. Journal of Biological Chemistry 284: 13233–13243. 28. Bermak JC, Li M, Bullock C, Zhou QY (2001) Regulation of transport of the dopamine D1 receptor by a new membrane-associated ER protein. Nat Cell Biol 3: 492–498. 43. References Petaja-Repo UE, Hogue M, Laperriere A, Walker P, Bouvier M (2000) Export from the endoplasmic reticulum represents the limiting step in the maturation and cell surface expression of the human delta opioid receptor. J Biol Chem 275: 13727–13736. 19. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, et al. (1998) RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393: 333–339. 9. Hanyaloglu AC, von Zastrow M (2008) Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annu Rev Pharmacol Toxicol 48: 537–568. p 20. Colley NJ, Baker EK, Stamnes MA, Zuker CS (1991) The cyclophilin homolog ninaA is required in the secretory pathway. Cell 67: 255–263. 10. Wu G, Krupnick JG, Benovic JL, Lanier SM (1997) Interaction of arrestins with intracellular domains of muscarinic and alpha2-adrenergic receptors. J Biol Chem 272: 17836–17842. 21. Xia Z, Gray JA, Compton-Toth BA, Roth BL (2003) A Direct Interaction of PSD-95 with 5-HT2A Serotonin Receptors Regulates Receptor Trafficking and Signal Transduction. Journal of Biological Chemistry 278: 21901–21908. Signal Transduction. Journal of Biological Chemistry 278: 2190 11. Wu G, Bogatkevich GS, Mukhin YV, Benovic JL, Hildebrandt JD, et al. (2000) Identification of Gbetagamma binding sites in the third intracellular loop of the M(3)-muscarinic receptor and their role in receptor regulation. J Biol Chem 275: 9026–9034. 22. Chen C, Wang Y, Huang P, Liu-Chen L-Y (2011) Effects of C-terminal Modifications of GEC1 Protein and c-Aminobutyric Acid Type A (GABAA) Receptor-associated Protein (GABARAP), Two Microtubule-associated Pro- December 2012 | Volume 7 | Issue 12 | e50416 PLOS ONE | www.plosone.org 8 December 2012 | Volume 7 | Issue 12 | e50416 a2-AR Export and Cell-Surface Expression (2012) A triple Arg motif mediates alpha2B-adrenergic receptor interaction with Sec24C/D and export. Traffic 13: 857–868. 50. Sanchez-Laorden BL, Herraiz C, Valencia JC, Hearing VJ, Jimenez-Cervantes C, et al. (2009) Aberrant trafficking of human melanocortin 1 receptor variants associated with red hair and skin cancer: Steady-state retention of mutant forms in the proximal golgi. J Cell Physiol 220: 640–654. p 35. Janovick JA, Park BS, Conn PM (2011) Therapeutic rescue of misfolded mutants: validation of primary high throughput screens for identification of pharmacoperone drugs. PLoS One 6: e22784. p p g 36. Daunt DA, Hurt C, Hein L, Kallio J, Feng F, et al. (1997) Subtype-specific 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
https://openalex.org/W4380631097
https://zenodo.org/record/8033972/files/24-13-2023.pdf
English
null
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
2,023
cc-by
7,410
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 Page No. 3301-3308 Volume 06 Issue 06 June 2023 Available at: www.ijcsrr.org 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 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 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 Page No. 3301-3308 Volume 06 Issue 06 June 2023 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
https://openalex.org/W2551509862
https://europepmc.org/articles/pmc5101197?pdf=render
English
null
Ovarian Sex Hormones Modulate Compulsive, Affective and Cognitive Functions in A Non-Induced Mouse Model of Obsessive-Compulsive Disorder
Frontiers in behavioral neuroscience
2,016
cc-by
12,256
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 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 REFERENCES Ahmari, S. E., Spellman, T., Douglass, N. L., Kheirbek, M. A., Simpson, H. B., Deisseroth, K., et al. (2013). Repeated cortico-striatal stimulation generates persistent OCD-like behavior. Science 340, 1234–1239. doi: 10.1126/science. 1234733 Alonso, P., Gratacòs, M., Segalàs, C., Escaramís, G., Real, E., Bayés, M., et al. (2011). Variants in estrogen receptor alpha gene are associated with phenotypical expression of obsessive-compulsive disorder. Psychoneuroendocrinology 36, 473–483. doi: 10.1016/j.psyneuen.2010.07.022 Frontiers in Behavioral Neuroscience | www.frontiersin.org November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 11 Mitra et al. Ovarian Steroids in Surgical Menopause de Mathis, M. A., do Rosario, M. C., Diniz, J. B., Torres, A. R., Shavitt, R. G., Ferrão, Y. A., et al. (2008). Obsessive—compulsive disorder: influence of age at onset on comorbidity patterns. Eur. Psychiatry 23, 187–194. doi: 10.1016/j. eurpsy.2008.01.002 Antunes, M., and Biala, G. (2012). The novel object recognition memory: neurobiology, test procedure and its modifications. Cogn. Process. 13, 93–110. doi: 10.1007/s10339-011-0430-z Arnold, P. D., Rosenberg, D. R., Mundo, E., Tharmalingam, S., Kennedy, J. L., and Richter, M. A. (2004). Association of a glutamate (NMDA) subunit receptor gene (GRIN2B) with obsessive-compulsive disorder: a preliminary study. Psychopharmacology (Berl) 174, 530–538. doi: 10.1007/s00213-004- 1847-1 Demeter, G., Racsmány, M., Csigó, K., Harsányi, A., Németh, A., and Döme, L. (2013). Intact short-term memory and impaired executive functions in obsessive compulsive disorder. Ideggyogy. Sz. 66, 35–41. Dirson, S., Bouvard, M., Cottraux, J., and Martin, R. (1995). Visual memory impairment in patients with obsessive-compulsive disorder: a controlled study. Psychother. Psychosom. 63, 22–31. doi: 10.1159/0002 88933 Bachmann, G. (2001). Physiologic aspects of natural and surgical menopause. J. Reprod. Med. 46, 307–315. Barth, C., Villringer, A., and Sacher, J. (2015). Sex hormones affect neurotransmitters and shape the adult female brain during hormonal transition periods. Front. Neurosci. 9:37. doi: 10.3389/fnins.2015. 00037 Dreher, J. C., Schmidt, P. J., Kohn, P., Furman, D., Rubinow, D., and Berman, K. F. (2007). Menstrual cycle phase modulates reward-related neural function in women. Proc. Natl. Acad. Sci. U S A 104, 2465–2470. doi: 10.1073/pnas. 0605569104 Bastos, C. P., Pereira, L. M., Ferreira-Vieira, T. H., Drumond, L. E., Massensini, A. R., Moraes, M. F., et al. (2015). Object recognition memory deficit and depressive-like behavior caused by chronic ovariectomy can be transitorialy recovered by the acute activation of hippocampal estrogen receptors. Psychoneuroendocrinology 57, 14–25. doi: 10.1016/j.psyneuen.2015. 03.020 Dumas, J. A., Kutz, A. M., McDonald, B. C., Naylor, M. R., Pfaff, A. REFERENCES C., Saykin, A. J., et al. (2013). Increased working memory-related brain activity in middle-aged women with cognitive complaints. Neurobiol. Aging 34, 1145–1147. doi: 10. 1016/j.neurobiolaging.2012.08.013 Eaton, W. W., Martins, S. S., Nestadt, G., Bienvenu, O. J., Clarke, D., and Alexandre, P. (2008). The burden of mental disorders. Epidemiol. Rev. 30, 1–14. doi: 10.1093/epirev/mxn011 Benmansour, S., Piotrowski, J. P., Altamirano, A. V., and Frazer, A. (2009). Impact of ovarian hormones on the modulation of the serotonin transporter by fluvoxamine. Neuropsychopharmacology 34, 555–564. doi: 10.1038/npp. 2008.23 Egashira, N., Abe, M., Shirakawa, A., Niki, T., Mishima, K., Iwasaki, K., et al. (2013). Effects of mood stabilizers on marble-burying behavior in mice: involvement of GABAergic system. Psychopharmacology (Berl) 226, 295–305. doi: 10.1007/s00213-012-2904-9 Bogetto, F., Venturello, S., Albert, U., Maina, G., and Ravizza, L. (1999). Gender- related clinical differences in obsessive-compulsive disorder. Eur. Psychiatry 14, 434–441. doi: 10.1016/s0924-9338(99)00224-2 Erekson, E. A., Martin, D. K., and Ratner, E. S. (2013). Oophorectomy: the debate between ovarian conservation and elective oophorectomy. Menopause 20, 110–114. doi: 10.1097/gme.0b013e31825a27ab Brandes, M., Soares, C. N., and Cohen, L. S. (2004). Postpartum onset obsessive-compulsive disorder: diagnosis and management. Arch. Womens Ment. Health 7, 99–110. doi: 10.1007/s00737-003-0035-3 Faubion, S. S., Kuhle, C. L., Shuster, L. T., and Rocca, W. A. (2015). Long-term health consequences of premature or early menopause and considerations for management. Climacteric 18, 483–491. doi: 10.3109/13697137.2015. 1020484 Bult, A., and Lynch, C. B. (1996). Multiple selection responses in house mice bidirectionally selected for thermoregulatory nest-building behavior: crosses of replicate lines. Behav. Genet. 26, 439–446. doi: 10.1007/bf023 59488 Fernández-Guasti, A., Agrati, D., Reyes, R., and Ferreira, A. (2006). Ovarian steroids counteract serotonergic drugs actions in an animal model of obsessive-compulsive disorder. Psychoneuroendocrinology 31, 924–934. doi: 10. 1016/j.psyneuen.2006.05.003 Bult, A., and Lynch, C. B. (1997). Nesting and fitness: lifetime reproductive success in house mice bidirectionally selected for thermoregulatory nest-building behavior. Behav. Genet. 27, 231–240. doi: 10.1023/A:10256101 30282 Flaisher-Grinberg, S., Albelda, N., Gitter, L., Weltman, K., Arad, M., and Joel, D. (2009). Ovarian hormones modulate ‘compulsive’ lever-pressing in female rats. Horm. Behav. 55, 356–365. doi: 10.1016/j.yhbeh.2008.10.002 Bult, A., and Lynch, C. B. (2000). Breaking through artificial selection limits of an adaptive behavior in mice and the consequences for correlated responses. Behav. Genet. 30, 193–206. doi: 10.1023/A:1001962124005 Fonseca, C. S., Gusmão, I. D., Raslan, A. C., Monteiro, B. M., Massensini, A. R., Moraes, M. F., et al. (2013). REFERENCES Object recognition memory and temporal lobe activation after delayed estrogen replacement therapy. Neurobiol. Learn. Mem. 101, 19–25. doi: 10.1016/j.nlm.2012.12.016 Cabrera, R. J., and Bregonzio, C. (1996). Turnover rate and stimulus-evoked release of dopamine by progesterone and N-methyl-D-aspartic acid in rat striatum during pregnancy. Eur. J. Pharmacol. 317, 55–59. doi: 10.1016/s0014- 2999(96)00704-2 Fontenelle, L. F., Mendlowicz, M. V., and Versiani, M. (2005). Clinical subtypes of obsessive-compulsive disorder based on the presence of checking and washing compulsions. Rev. Bras. Psiquiatr. 27, 201–207. doi: 10.1590/s1516- 44462005000300008 Capettini, S. B., Moraes, M. F., Prado, V. F., Prado, M. A., and Pereira, G. S. (2011). Vesicular acetylcholine transporter knock-down mice show sexual dimorphism on memory. Brain Res. Bull. 85, 54–57. doi: 10.1016/j.brainresbull.2011. 02.005 Forray, A., Focseneanu, M., Pittman, B., McDougle, C. J., and Epperson, C. N. (2010). Onset and exacerbation of obsessive-compulsive disorder in pregnancy and the postpartum period. J. Clin. Psychiatry 71, 1061–1068. doi: 10.4088/JCP. 09m05381blu Chen, X., Guo, T., and Li, B. (2013). Influence of prophylactic oophorectomy on mood and sexual function in women of menopausal transition or postmenopausal period. Arch. Gynecol. Obstet. 288, 1101–1106. doi: 10. 1007/s00404-013-2865-1 Frye, C. A., Koonce, C. J., Edinger, K. L., Osborne, D. M., and Walf, A. A. (2008). Androgens with activity at estrogen receptor beta have anxiolytic and cognitive-enhancing effects in male rats and mice. Horm. Behav. 54, 726–734. doi: 10.1016/j.yhbeh.2008.07.013 Clayton, A. H., and Ninan, P. T. (2010). Depression or menopause? Presentation and management of major depressive disorder in perimenopausal and postmenopausal women. Prim. Care Companion J. Clin. Psychiatry 12, e1–e13. doi: 10.4088/PCC.08r00747blu Grados, M., and Riddle, M. A. (2008). Do all obsessive-compulsive disorder subtypes respond to medication? Int. Rev. Psychiatry 20, 189–193. doi: 10. 1080/09540260801889153 Cox, L., and Liu, J. H. (2014). Primary ovarian insufficiency: an update. Int. J. Womens Health 6, 235–243. doi: 10.2147/IJWH.S37636 Greene-Schloesser, D. M., Van der Zee, E. A., Sheppard, D. K., Castillo, M. R., Gregg, K. A., Burrow, T., et al. (2011). Predictive validity of a non-induced mouse model of compulsive-like behavior. Behav. Brain Res. 221, 55–62. doi: 10.1016/j.bbr.2011.02.010 Crawley, J. N. (1985). Exploratory behavior models of anxiety in mice. Neurosci. Biobehav. Rev. 9, 37–44. doi: 10.1016/0149-7634(85)90030-2 Cyr, M., Bossé, R., and Di Paolo, T. (1998). Gonadal hormones modulate 5-hydroxytryptamine2A receptors: emphasis on the rat frontal cortex. Neuroscience 83, 829–836. doi: 10.1016/s0306-4522(97)00445-4 Gresack, J. E., and Frick, K. M. (2006). REFERENCES 66, 602–618. doi: 10.1016/j.yhbeh.2014.08.011 Rodriguez, M., and Shoupe, D. (2015). Surgical menopause. Endocrinol. Metab. Clin. North Am. 44, 531–542. doi: 10.1016/j.ecl.2015.05.003 Lynch, C. B. (1980). Response to divergent selection for nesting behavior in Mus musculus. Genetics 96, 757–765. Rodríguez-Landa, J. F., Puga-Olguín, A., Germán-Ponciano, L. J., García- Ríos, R.-I., and Soria-Fregozo, C. (2015). ‘‘Anxiety in natural and surgical menopause—physiologic and therapeutic bases’’, in A Fresh Look at Anxiety Disorders, ed. F. Durbano, (Rijeka: InTech), 173–198. doi: 10.5772/ 60621 Maio, T. P., Filgueiras, G. B., Cunha, D. C., and Estanislau, C. (2014). Animal models of obsessive-compulsive disorder: strain differences. World J. Neurosci. 4, 240–246. doi: 10.4236/wjns.2014.43027 Roth, R. M., Baribeau, J., Milovan, D., O’Connor, K., and Todorov, C. (2004). Procedural and declarative memory in obsessive-compulsive disorder. J. Int. Neuropsychol. Soc. 10, 647–654. doi: 10.1017/s1355617704105018 Martin, A., Wiggs, C. L., Altemus, M., Rubenstein, C., and Murphy, D. L. (1995). Working memory as assessed by subject-ordered tasks in patients with obsessive-compulsive disorder. J. Clin. Exp. Neuropsychol. 17, 786–792. doi: 10. 1080/01688639508405167 Ruscio, A. M., Stein, D. J., Chiu, W. T., and Kessler, R. C. (2010). The epidemiology of obsessive-compulsive disorder in the national comorbidity survey replication. Mol. Psychiatry 15, 53–63. doi: 10.1038/mp.2008.94 McClearn, G. E., Wilson, J. R., and Meredith, J. E. (1970). The Use of Isogenic and Heterogenic Mouse Stocks in Behavioral Research. New York, NY: Applenton- Century-Crofts. Schilman, E. A., Klavir, O., Winter, C., Sohr, R., and Joel, D. (2010). The role of the striatum in compulsive behavior in intact and orbitofrontal- cortex-lesioned rats: possible involvement of the serotonergic system. Neuropsychopharmacology 35, 1026–1039. doi: 10.1038/npp.2009.208 McDonald, A. J. (1991). Organization of amygdaloid projections to the prefrontal cortex and associated striatum in the rat. Neuroscience 44, 1–14. doi: 10. 1016/0306-4522(91)90247-l Schmidt, P. J., Nieman, L. K., Danaceau, M. A., Adams, L. F., and Rubinow, D. R. (1998). Differential behavioral effects of gonadal steroids in women with and in those without premenstrual syndrome. N. Engl. J. Med. 338, 209–216. doi: 10. 1056/NEJM199801223380401 Meerlo, P., Horvath, K. M., Nagy, G. M., Bohus, B., and Koolhaas, J. M. (1999). The influence of postnatal handling on adult neuroendocrine and behavioural stress reactivity. J. Neuroendocrinol. 11, 925–933. doi: 10.1046/j.1365-2826. 1999.00409.x Shuster, L. T., Rhodes, D. J., Gostout, B. S., Grossardt, B. R., and Rocca, W. A. (2010). Premature menopause or early menopause: long-term health consequences. Maturitas 65, 161–166. doi: 10.1016/j.maturitas.2009.08.003 Mundo, E., Bareggi, S. REFERENCES Post-training estrogen enhances spatial and object memory consolidation in female mice. Pharmacol. Biochem. Behav. 84, 112–119. doi: 10.1016/j.pbb.2006.04.013 Cyr, M., Ghribi, O., and Di Paolo, T. (2000). Regional and selective effects of oestradiol and progesterone on NMDA and AMPA receptors in the rat brain. J. Neuroendocrinol. 12, 445–452. doi: 10.1046/j.1365-2826.2000. 00471.x Guglielmi, V., Vulink, N. C., Denys, D., Wang, Y., Samuels, J. F., and Nestadt, G. (2014). Obsessive-compulsive disorder and female reproductive cycle events: November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 12 Mitra et al. Ovarian Steroids in Surgical Menopause Nestadt, G., Samuels, J., Riddle, M. A., Liang, K.-Y., Bienvenu, O. J., Hoehn- Saric, R., et al. (2001). The relationship between obsessive-compulsive disorder and anxiety and affective disorders: results from the Johns Hopkins OCD Family Study. Psychol. Med. 31, 481–487. doi: 10.1017/s003329170100 3579 results from the OCD and reproduction collaborative study. Depress. Anxiety 31, 979–987. doi: 10.1002/da.22234 results from the OCD and reproduction collaborative study. Depress. Anxiety 31, 979–987. doi: 10.1002/da.22234 Hay, A. G., Bancroft, J., and Johnstone, E. C. (1994). Affective symptoms in women attending a menopause clinic. Br. J. Psychiatry 164, 513–516. doi: 10.1192/bjp. 164.4.513 Neziroglu, F., Anemone, R., and Yaryura-Tobias, J. A. (1992). Onset of obsessive-compulsive disorder in pregnancy. Am. J. Psychiatry 149, 947–950. doi: 10.1176/ajp.149.7.947 Hill, R. A., McInnes, K. J., Gong, E. C., Jones, M. E., Simpson, E. R., and Boon, W. C. (2007). Estrogen deficient male mice develop compulsive behavior. Biol. Psychiatry 61, 359–366. doi: 10.1016/j.biopsych.2006.01.012 Noshirvani, H. F., Kasvikis, Y., Marks, I. M., Tsakiris, F., and Monteiro, W. O. (1991). Gender-divergent aetiological factors in obsessive-compulsive disorder. Br. J. Psychiatry 158, 260–263. doi: 10.1192/bjp.158.2.260 Karakaya, S., Kipp, M., and Beyer, C. (2007). Oestrogen regulates the expression and function of dopamine transporters in astrocytes of the nigrostriatal system. J. Neuroendocrinol. 19, 682–690. doi: 10.1111/j.1365-2826.2007.01575.x Peris, T. S., Bergman, R. L., Asarnow, J. R., Langley, A., McCracken, J. T., and Piacentini, J. (2010). Clinical and cognitive correlates of depressive symptoms among youth with obsessive compulsive disorder. J. Clin. Child Adolesc. Psychol. 39, 616–626. doi: 10.1080/15374416.2010.501285 Kashyap, H., Kumar, J. K., Kandavel, T., and Reddy, Y. C. (2013). Neuropsychological functioning in obsessive-compulsive disorder: are executive functions the key deficit? Compr. Psychiatry 54, 533–540. doi: 10. 1016/j.comppsych.2012.12.003 Kato, A., Hojo, Y., Higo, S., Komatsuzaki, Y., Murakami, G., Yoshino, H., et al. (2013). REFERENCES Female hippocampal estrogens have a significant correlation with cyclic fluctuation of hippocampal spines. Front. Neural Circuits 7:149. doi: 10. 3389/fncir.2013.00149 Porton, B., Greenberg, B. D., Askland, K., Serra, L. M., Gesmonde, J., Rudnick, G., et al. (2013). Isoforms of the neuronal glutamate transporter gene, SLC1A1/EAAC1, negatively modulate glutamate uptake: relevance to obsessive-compulsive disorder. Transl. Psychiatry 3:e259. doi: 10.1038/tp. 2013.35 Koss, W. A., Einat, H., Schloesser, R. J., Manji, H. K., and Rubinow, D. R. (2012). Estrogen effects on the forced swim test differ in two outbred rat strains. Physiol. Behav. 106, 81–86. doi: 10.1016/j.physbeh.2012.01.004 Quinlan, M. G., Almey, A., Caissie, M., LaChappelle, I., Radiotis, G., and Brake, W. G. (2013). Estradiol and striatal dopamine receptor antagonism influence memory system bias in the female rat. Neurobiol. Learn. Mem. 106, 221–229. doi: 10.1016/j.nlm.2013.08.018 Labad, J., Menchon, J. M., Alonso, P., Segalas, C., Jimenez, S., Jaurrieta, N., et al. (2008). Gender differences in obsessive-compulsive symptom dimensions. Depress. Anxiety 25, 832–838. doi: 10.1002/da.20332 Radomsky, A. S., and Rachman, S. (1999). Memory bias in obsessive-compulsive disorder (OCD). Behav. Res. Ther. 37, 605–618. doi: 10.1016/S0005- 7967(98)00151-X Labad, J., Menchón, J. M., Alonso, P., Segalàs, C., Jiménez, S., and Vallejo, J. (2005). Female reproductive cycle and obsessive-compulsive disorder. J. Clin. Psychiatry 66, 428–435; quiz 546. doi: 10.4088/jcp.v66n0404 Ramos, A. (2008). Animal models of anxiety: do I need multiple tests? Trends Pharmacol. Sci. 29, 493–498. doi: 10.1016/j.tips.2008.07.005 Leckman, J. F., Bloch, M. H., and King, R. A. (2009). Symptom dimensions and subtypes of obsessive-compulsive disorder: a developmental perspective. Dialogues Clin. Neurosci. 11, 21–33. Remijnse, P. L., van den Heuvel, O. A., Nielen, M. M., Vriend, C., Hendriks, G. J., Hoogendijk, W. J., et al. (2013). Cognitive inflexibility in obsessive-compulsive disorder and major depression is associated with distinct neural correlates. PLoS One 8:e59600. doi: 10.1371/journal.pone.0059600 Lensi, P., Cassano, G. B., Correddu, G., Ravagli, S., Kunovac, J. L., and Akiskal, H. S. (1996). Obsessive-compulsive disorder. Familial-developmental history, symptomatology, comorbidity and course with special reference to gender-related differences. Br. J. Psychiatry 169, 101–107. doi: 10.1192/bjp.169. 1.101 j p Rhodes, M. E., and Frye, C. A. (2006). ERbeta-selective SERMs produce mnemonic-enhancing effects in the inhibitory avoidance and water maze tasks. Rhodes, M. E., and Frye, C. A. (2006). ERbeta-selective SERMs produce mnemonic-enhancing effects in the inhibitory avoidance and water maze tasks. Neurobiol. Learn. Mem. 85, 183–191. doi: 10.1016/j.nlm.2005.10.003 Luine, V. N. (2014). Estradiol and cognitive function: past, present and future. Horm. Behav. REFERENCES R., Pirola, R., and Bellodi, L. (1999). Effect of acute intravenous clomipramine and antiobsessional response to proserotonergic drugs: is gender a predictive variable? Biol. Psychiatry 45, 290–294. doi: 10. 1016/s0006-3223(98)00027-4 Soares, C. N., and Zitek, B. (2008). Reproductive hormone sensitivity and risk for depression across the female life cycle: a continuum of vulnerability? J. Psychiatry Neurosci. 33, 331–343. Nakao, T., Nakagawa, A., Nakatani, E., Nabeyama, M., Sanematsu, H., Yoshiura, T., et al. (2009). Working memory dysfunction in obsessive-compulsive disorder: a neuropsychological and functional MRI study. J. Psychiatr. Res. 43, 784–791. doi: 10.1016/j.jpsychires.2008.10.013 Takeuchi, H., Yatsugi, S., and Yamaguchi, T. (2002). Effect of YM992, a novel antidepressant with selective serotonin re-uptake inhibitory and 5-HT 2A receptor antagonistic activity, on a marble-burying behavior test as an obsessive-compulsive disorder model. Jpn. J. Pharmacol. 90, 197–200. doi: 10. 1254/jjp.90.197 Nestadt, G., Grados, M., and Samuels, J. F. (2010). Genetics of obsessive-compulsive disorder. Psychiatr. Clin. North Am. 33, 141–158. doi: 10.1016/j.psc.2009.11.001 November 2016 | Volume 10 | Article 215 Frontiers in Behavioral Neuroscience | www.frontiersin.org 13 Mitra et al. Ovarian Steroids in Surgical Menopause Thomas, A., Burant, A., Bui, N., Graham, D., Yuva-Paylor, L. A., and Paylor, R. (2009). Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology (Berl) 204, 361–373. doi: 10.1007/s00213-009-1466-y Walf, A. A., Koonce, C. J., and Frye, C. A. (2008b). Estradiol or diarylpropionitrile decrease anxiety-like behavior of wildtype, but not estrogen receptor beta knockout, mice. Behav. Neurosci. 122, 974–981. doi: 10.1037/a0012749 Walf, A. A., Rhodes, M. E., and Frye, C. A. (2006). Ovarian steroids enhance object recognition in naturally cycling and ovariectomized, hormone-primed rats. Neurobiol. Learn. Mem. 86, 35–46. doi: 10.1016/j.nlm.2006.01.004 Torresan, R. C., Ramos-Cerqueira, A. T., de Mathis, M. A., Diniz, J. B., Ferrão, Y. A., Miguel, E. C., et al. (2009). Sex differences in the phenotypic expression of obsessive-compulsive disorder: an exploratory study from Brazil. Compr. Psychiatry 50, 63–69. doi: 10.1016/j.comppsych.2008. 05.005 Warneke, L. (1993). Anxiety disorders. Focus on obsessive-compulsive disorder. Can. Fam. Physician 39, 1612–1621. Weber, M. T., Mapstone, M., Staskiewicz, J., and Maki, P. M. (2012). Reconciling subjective memory complaints with objective memory performance in the menopausal transition. Menopause 19, 735–741. doi: 10.1097/gme. 0b013e318241fd22 Uguz, F., Gezginc, K., Zeytinci, I. E., Karatayli, S., Askin, R., Guler, O., et al. (2007). Obsessive-compulsive disorder in pregnant women during the third trimester of pregnancy. Compr. Psychiatry 48, 441–445. doi: 10.1016/j.comppsych.2007. 05.001 Wheaton, M. G., Abramowitz, J. REFERENCES S., Berman, N. C., Riemann, B. C., and Hale, L. R. (2010). The relationship between obsessive beliefs and symptom dimensions in obsessive-compulsive disorder. Behav. Res. Ther. 48, 949–954. doi: 10.1016/j. brat.2010.05.027 Uguz, F., Sahingoz, M., Gezginc, K., and Karatayli, R. (2010). Obsessive- compulsive disorder in postmenopausal women: prevalence, clinical features and comorbidity. Aust. N Z J. Psychiatry 44, 183–187. doi: 10. 3109/00048670903393639 Willard, S. L., Daunais, J. B., Cline, J. M., and Shively, C. A. (2011). Hippocampal volume in postmenopausal cynomolgus macaques with behavioral depression. Menopause 18, 582–586. doi: 10.1097/gme.0b013e3181fcb47e Umathe, S. N., Vaghasiya, J. M., Jain, N. S., and Dixit, P. V. (2009). Neurosteroids modulate compulsive and persistent behavior in rodents: implications for obsessive-compulsive disorder. Prog. Neuropsychopharmacol. Biol. Psychiatry 33, 1161–1166. doi: 10.1016/j.pnpbp.2009.06.013 Williams, K. E., and Koran, L. M. (1997). Obsessive-compulsive disorder in pregnancy, the puerperium and the premenstruum. J. Clin. Psychiatry 58, 330–334; quiz 335–336. doi: 10.4088/jcp.v58n0709 van der Wee, N. J., Ramsey, N. F., van Megen, H. J., Denys, D., Westenberg, H. G., and Kahn, R. S. (2007). Spatial working memory in obsessive-compulsive disorder improves with clinical response: a functional MRI study. Eur. Neuropsychopharmacol. 17, 16–23. doi: 10.1016/j.euroneuro.2006. 04.012 Yang, H., Sampson, M. M., Senturk, D., and Andrews, A. M. (2015). Sex- and SERT-mediated differences in stimulated serotonin revealed by fast microdialysis. ACS Chem. Neurosci. 6, 1487–1501. doi: 10.1021/acschemneuro. 5b00132 Vulink, N. C., Denys, D., Bus, L., and Westenberg, H. G. (2006). Female hormones affect symptom severity in obsessive-compulsive disorder. Int. Clin. Psychopharmacol. 21, 171–175. doi: 10.1097/01.yic.0000199454. 62423.99 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. 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
https://openalex.org/W4298268930
https://zenodo.org/records/6164196/files/344-346.pdf
English
null
Kinetics and Mechanism of Oxidation of Diphenyl Sulphoxide by Chloramine-B in Aqueous Acid Medium
Zenodo (CERN European Organization for Nuclear Research)
1,990
cc-by
2,860
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
W2934402352.txt
https://jmedicalcasereports.biomedcentral.com/track/pdf/10.1186/s13256-019-1984-0
en
Endoscopic removal of a gastric pharmacobezoar induced by clomipramine, lorazepam, and domperidone overdose: a case report
Journal of medical case reports
2,019
cc-by
2,921
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 published maps and institutional affiliations. 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 References 1. Simpson SE. Pharmacobezoars described and demystified. Clin Toxicol. 2011;49(2):72–89. 2. Stack PE, Thomas E. Pharmacobezoar: an evolving new entity. Dig Dis. 1995;13(6):356–64. 3. Magdalan J, Zawadzki M, Sloka T, Sozanski T. Suicidal overdose with relapsing clomipramine concentrations due to a large gastric pharmacobezoar. Forensic Sci Int. 2013;229(1–3):e19–22. 4. Kerr GW, McGuffie AC, Wilkie S. Tricyclic antidepressant overdose: a review. Emerg Med J. 2001;18(4):236–41. 5. Body R, Bartram T, Azam F, Mackway-Jones K. Guidelines in Emergency Medicine Network (GEMNet): guideline for the management of tricyclic antidepressant overdose. Emerg Med J. 2011;28(4):347–68. 6. Iwamuro M, Okada H, Matsueda K, Inaba T, Kusumoto C, Imagawa A, et al. Review of the diagnosis and management of gastrointestinal bezoars. World J Gastrointest Endosc. 2015;7(4):336–45. Hojer J, Personne M. Endoscopic removal of slow release clomipramine bezoars in two cases of acute poisoning. Clin Toxicol. 2008;46(4):317–9. Lapostolle F, Finot MA, Adnet F, Borron SW, Baud FJ, Bismuth C. Radiopacity of clomipramine conglomerations and unsuccessful endoscopy: report of 4 cases. J Toxicol Clin Toxicol. 2000;38(5):477–82. Iwamuro M, Yunoki N, Tomoda J, Nakamura K, Okada H, Yamamoto K. Gastric bezoar treatment by endoscopic fragmentation in combination with Pepsi-Cola administration. Am J Case Rep. 2015;16:445–8. Olson KR, Kearney TE, Dyer JE, Benowitz NL, Blanc PD. Seizures associated with poisoning and drug overdose. Am J Emerg Med. 1994;12(3):392–5. Ellison DW, Pentel PR. Clinical features and consequences of seizures due to cyclic antidepressant overdose. Am J Emerg Med. 1989;7(1):5–10. Ramasubbu B, James D, Scurr A, Sandilands EA. Serum alkalinisation is the cornerstone of treatment for amitriptyline poisoning. BMJ Case Rep. 2016;2016:bcr2016214685. Santana NO, Gois AF. Rhabdomyolysis as a manifestation of clomipramine poisoning. Sao Paulo Med J. 2013;131(6):432–5.
https://openalex.org/W2884368478
http://www.scielo.br/pdf/rounesp/v47n3/1807-2577-rounesp-47-3-131.pdf
Portuguese
null
Efeito da contaminação por saliva na resistência adesiva de braquetes cerâmicos utilizando uma resina ortodôntica hidrofílica
Revista de odontologia da UNESP/Revista de Odontologia da UNESP
2,018
cc-by
4,637
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
https://openalex.org/W2221846482
https://conflictandhealth.biomedcentral.com/counter/pdf/10.1186/s13031-015-0064-3
English
null
Providing surgery in a war-torn context: the Médecins Sans Frontières experience in Syria
Conflict and health
2,015
cc-by
8,270
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 reporting observational studies. Bull World Health Organ. 2007;85:867–72. 22. Edginton M, Enarson D, Zachariah R, Reid T, Satyanarayana S, Bissell K. Why ethics is indispensable for good-quality operational research. Public Health Action. 2012;2:21–2. Funding 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 quality care be ensured? Int Orthop. 2015;39:1901–8. Competing interests Competing interests The authors declare that they have no competing interests. 17. Hasanin A, Mukhtar A, Mokhtar A, Radwan A. Syrian revolution: a field hospital under attack. Am J Disaster Med. 2013;8(4):259–65. 17. Hasanin A, Mukhtar A, Mokhtar A, Radwan A. Syrian revolution: a field hospital under attack. Am J Disaster Med. 2013;8(4):259–65. Acknowledgements l l We are particularly grateful to staff in the field for all their hard work. 23. Sidel VW, Levy BS. The health impact of war. Int J Inj Contr Saf Promot. 2008;15:189–95. 23. Sidel VW, Levy BS. The health impact of war. Int J Inj Contr Saf Promot. 2008;15:189–95. 24. Donabedian A. Evaluating the quality of medical care. Milbank Mem Fund Q. 1996;44:166–203. Conclusion 10. Attar S. Field hospitals in Syria. Lancet. 2014;383:303. In conclusion, surgical needs in a conflict-afflicted set- 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 shifting. Confl Health. 2011;5:1–5. 13. Tajsic NB, Husum H. Reconstructive surgery including freeflap transfers can be performed in low-resource settings: experiences from a wartime scenario. J Trauma Inj Infect CritCare. 2008;65:1463–7. 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. 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.
https://openalex.org/W4200607299
https://www.notulaebotanicae.ro/index.php/nbha/article/download/12550/9291
English
null
The different responses of rice genotypes to heat stress associated with morphological, chlorophyll and yield characteristics
Notulae botanicae Horti Agrobotanici Cluj-Napoca
2,021
cc-by
1,279
◦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
https://openalex.org/W2935955186
https://iconline.ipleiria.pt/bitstream/10400.8/5005/1/Avelelas%20et%20al%202019_marinedrugs.pdf
English
null
Antifungal and Antioxidant Properties of Chitosan Polymers Obtained from Nontraditional Polybius henslowii Sources
Marine drugs
2,019
cc-by
10,883
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 d DNA damage in obesity-related tumorigenesis. Adv. Exp. Med. Biol. 2014, 824, 5–17. [PubMed] 10. Wölfle, U.; Seelinger, G.; Bauer, G.; Meinke, M.C.; Lademann, J.; Schempp, C.M. Reactive molecule species and antioxidative mechanisms in normal skin and skin aging. Ski. Pharmacol. Physiol. 2014, 27, 316–332. [CrossRef] [PubMed] 11. Ito, N.; Hirose, M.; Fukushima, H.; Tsuda, T.; Shirai, T.; Tatenatsu, M. Studies on antioxidants: Their carcinogenic and modifying effects on chemical carcinogens. Food Chem. Toxicol. 1986, 24, 1071–1092. [CrossRef] 12. Ferraro, V.; Cruz, I.B.; Jorge, R.F.; Malcata, F.X.; Pintado, M.E.; Castro, P.M.L. Valorisation of natural extracts from marine source focused on marine by-products: A review. Food Res. Int. 2010, 43, 2221–2233. [CrossRef] 13. Cartes, J.E.; Abelló, P.; Lloris, D.; Carbonell, A.; Torres, P.; Maynou, F.; Sola, L.G.D. Feeding guilds of western M dit d l fih d t A l i b d i S i M 2002 590 12. Ferraro, V.; Cruz, I.B.; Jorge, R.F.; Malcata, F.X.; Pintado, M.E.; Castro, P.M.L. Valorisation of natural extracts from marine source focused on marine by-products: A review. Food Res. Int. 2010, 43, 2221–2233. [CrossRef] , ; , ; J g , ; , ; , ; , from marine source focused on marine by-products: A review. Food Res. Int. 2010, 43, 2221–2233. [CrossRef] 13. Cartes, J.E.; Abelló, P.; Lloris, D.; Carbonell, A.; Torres, P.; Maynou, F.; Sola, L.G.D. Feeding guilds of western Mediterranean demersal fish and crustaceans: An analysis based on a spring survey. Sci. Mar. 2002, 590, 209–220. [CrossRef] 13. Cartes, J.E.; Abelló, P.; Lloris, D.; Carbonell, A.; Torres, P.; Maynou, F.; Sola, L.G.D. Feeding guilds of western Mediterranean demersal fish and crustaceans: An analysis based on a spring survey. Sci. Mar. 2002, 590, 209–220. [CrossRef] 14. Serrano, A.; Sánchez, F.; Punzón, A.; Velasco, F.; Olaso, I. Deep sea megafaunal assemblages offthe northern Iberian slope related to environmental factors. Sci. Mar. 2011, 75, 425–437. [CrossRef] 15. Hayward, P.J.; Ryland, J.S. Handbook of the Marine Fauna of North–West Europe; Oxford University Press: Oxford, UK, 1995; p. 454. 16. Signa, G.; Cartes, J.E.; Solé, M.; Serrano, A.; Sánchez, F. Trophic ecology of the swimming crab Polybius henslowii Leach, 1820 in Galician and Cantabrian Seas: Influences of natural variability and the Prestige oil spill. Cont. Shelf Res. 4. Conclusions Acknowledgments: The authors would like to thank their colleagues Rui Albuquerque and Pedro Sá (Mare—IP Leiria) for the assistance provided during the preparation of the raw material, as well as Purse seine fishing vessel “Mestre Comboio—Peniche” and Pedro Murraças for providing the swimming crab Conflicts of Interest: The authors declare no conflict of interest. 12 of 15 Mar. Drugs 2019, 17, 239 References 1. Moreira, A.C.; Caetano, P.; Correia, S.; Brasier, C.M.; Ferraz, J.F.P. Phytophthora species associated with cork oak decline in a Mediterranean forest in in southern Portugal. In Proceedings of the 6th International Congress of Plant Pathology, Montréal, QC, Canada, 28 July–6 August 1993. 1. Moreira, A.C.; Caetano, P.; Correia, S.; Brasier, C.M.; Ferraz, J.F.P. Phytophthora species associated with cork oak decline in a Mediterranean forest in in southern Portugal. In Proceedings of the 6th International Congress of Plant Pathology, Montréal, QC, Canada, 28 July–6 August 1993. 2. Droby, S.; Lichter, A. Post-Harvest Botrytis Infection: Etiology, Development and Management. In Botrytis: Biology, Pathology and Control; Elad, Y., Williamson, B., Tudzynski, P., Delen, N., Eds.; Springer: Dordrecht, The Netherlands, 2007; pp. 349–367, ISBN 978-1-4020-2626-3. 2. Droby, S.; Lichter, A. Post-Harvest Botrytis Infection: Etiology, Development and Management. In Botrytis: Biology, Pathology and Control; Elad, Y., Williamson, B., Tudzynski, P., Delen, N., Eds.; Springer: Dordrecht, The Netherlands, 2007; pp. 349–367, ISBN 978-1-4020-2626-3. pp 3. Nuss, D.L. Biological control of chestnut blight: An example of virus-mediated attenuation of pathogenesis. Microbiol. Rev. 1992, 56, 561–576. 4. Niemelä, T.; Korhonen, K. Taxonomy of the genus Heterobasidion. In Heterobasidion Annosum: Biology, Ecology, 1988, Impact and Control; Woodward, S., Stenlid, J., Karjalainen, R., Hu¨ttermann, A., Eds.; CAB International: Wallingford, NY, USA, 2000; pp. 27–33. 5. Smilanick, J.L.; Harvey, J.M.; Hartsell, P.L.; Henson, D.J.; Harris, C.M.; Fouse, D.C.; Assemi, M. Influence of sulfur dioxide fumigant dose on residues and control of postharvest decay of grapes. Plant Dis. J. 1990, 74, 418–421. [CrossRef] 6. Muzzarelli, R.A.; Rochetti, R.; Stanic, V.; Weckx, M. Methods for the determination of the degree of acetylation of chitin and chitosan. In Chitin Handbook; Muzzarelli, R.A.A., Peter, M.G., Eds.; ATEC: College Park, MD, USA, 1997; pp. 109–119. 7. Dutta, P.K.; Tripathi, S.; Mehrotra, G.K.; Dutta, J. Perspectives for chitosan based antimicrobial films in food applications. Food Chem. 2009, 114, 1173–1182. [CrossRef] . Kumar, R.M.N.V. A review of chitin and chitosan applications. Reactive and Functional. React. Funct. Po 2000, 46, 1–27. [CrossRef] 2000, 46, 1 27. [CrossRef] 9. Cerdá, C.; Sánchez, C.; Climent, B.; Vázquez, A.; Iradi, A.; El Amrani, F.; Bediaga, A.; Sáez, G.T. Oxidative stress and DNA damage in obesity-related tumorigenesis Adv Exp Med Biol 2014 824 5–17 [PubMed] . Cerdá, C.; Sánchez, C.; Climent, B.; Vázquez, A.; Iradi, A.; El Amrani, F.; Bediaga, A.; Sáez, G.T. Oxidative st and DNA damage in obesity-related tumorigenesis. Adv. References C b h d P l 2009 75 15 21 [C R f] , ; , ; , ; , ; , ; , y chitin, chitosan and its oligomers prepared from shrimp shell waste. Food Hydrol. 2012, 29, 48–56. [CrossRef] 29. Yen, M.-T.; Yang, J.-H.; Mau, J.-L. Physicochemical characterization of chitin and chitosan from crab shells. Carbohydr. Polym. 2009, 75, 15–21. [CrossRef] 29. Yen, M.-T.; Yang, J.-H.; Mau, J.-L. Physicochemical characterization of chitin and chitosan from crab shells. Carbohydr. Polym. 2009, 75, 15–21. [CrossRef] 30. Al-Sagheer, F.A.; Al-Sughayer, M.A.; Muslim, S.; Elsabee, M.Z. Extraction and characterization of chitin and chitosan from marine sources in Arabian Gulf. Carbohydate. Polymerst 2009, 77, 410–419. 31. Barroca-Aubry, N.; Pernet-Poil-Chevrier, A.; Domard, A.; Trombotto, S. Towards a modular synthesis of well-defined chitooligosaccharides: Synthesis of the four chitodisaccharides. Carbohydr. Res. 2010, 345, 1685–1697. [CrossRef] 32. Du, Y.; Zhao, Y.; Dai, S.; Yang, B. Preparation of water-soluble chitosan from shrimp shell and its antibacterial activity. Innov. Food Sci. Emerg. Technol. 2009, 10, 103–107. [CrossRef] 33. Tian, F.; Liu, Y.; Hu, K.; Zhao, B. Study of the depolymerization behavior of chitosan by hydrogen peroxide. Carbohydr. Polym. 2004, 57, 31–37. [CrossRef] 34. Lu, S.; Song, X.; Cao, D.; Chen, Y.; Yao, K. Preparation of Water-Soluble Chitosan. J. Appl. Polym. Sci. 2004, 685, 3497–3503. [CrossRef] 35. Yamaguchi, T.; Takamura, H.; Matoba, T.; Terao, J. HPLC method for evaluation of the free radical-scavenging activity of foods by using 1,1,-diphenyl-2-picrylhydrazyl. Biosci. Biotechnol. Biochem. 1998, 62, 1201–1204. [CrossRef] [PubMed] 36. Kim, K.W.; Thomas, R.L. Antioxidative activity of chitosan’s with varying molecular weight. Food Chem. 2007, 101, 308–313. [CrossRef] 37. Samar, M.M.; El-Kalyoubi, M.H.; Khalaf, M.M.; El-Razik, M.M.A. Physicochemical, functional, antioxidant and antibacterial properties of chitosan extracted from shrimp wastes by microwave technique. Ann. Agric. Sci. 2013, 58, 33–41. [CrossRef] 38. Xie, W.; Xu, P.; Liu, Q. Antioxidant activity of water-soluble chitosan derivatives. Bioorgan. Med. Chem. Lett. 2001, 11, 1699–1701. [CrossRef] 39. Je, J.Y.; Kim, S.K. Reactive oxygen species scavenging activity of aminoderivatized chitosan with different 651 degree of deacetylation. Bioorgan. Med. Chem. 2006, 14, 5989–5994. [CrossRef] 0. Chien, P.J.; Sheu, F.; Huang, W.T.; Su, M.S. Antioxidant Polymers: Synthesis, Properties, and Applicati Food Chem. 2007, 102, 1192. [CrossRef] 41. Yen, M.-T.; Yang, J.-H.; Mau, J.-L. Antioxidant properties of chitosan from crab shells. Carbohydr. Polym. 2008, 74, 840–844. [CrossRef] 42. Korycka-Dahl, M.; Richardson, T. References 2008, 28, 2659–2667. [CrossRef] 17. González-Gurriarán, E. El patexo, Polybius henslowii Leach (Decapoda, Brachyura), en las costas de Galicia (NW de Espanã): I. Distribución espacial y cambios temporales. Investig. Pesq. 1987, 51, 361–374. 18 Zariquey R Crustáceos decápodos ibéricos Investig Pesq 1968 32 83 18. Zariquey, R. Crustáceos decápodos ibéricos. Investig. Pesq. 1968, 32, 83. q y p g q 19. Abdou, E.S.; Nagy, K.S.A.; Elsabee, M.Z. Extraction and characterization of chitin and chitosan from local sources. Bioresour. Technol. 2008, 99, 1359–1367. [CrossRef] [PubMed] 20. Krzeczkowski, R.A.; Tenney, R.D.; Kelley, C. Alaska king crab: Fatty acid composition, carotenoid index and proximate analysis. J. Food Sci. 2006, 36, 604–606. [CrossRef] 21. King, I.; Childs, M.T.; Dorsett, C.; Ostrander, J.G.; Monsen, E.R. Shellfish: Proximate composition, minerals, fatty acids, and sterols. J. Am. Diet. Assoc. 1990, 90, 677–685. 13 of 15 13 of 15 Mar. Drugs 2019, 17, 239 22. Nalan, G.; Dlua, K.; Yerlikayaa, P. Determination of proximate and mineral contents of blue crab (Callinectes sapidus) and swim crab (Portunus pelagicus) caught of the Gulf of Antalya. Food Chem. 2003, 80, 495–498. [CrossRef] 23. Naczk, M.; Williams, J.; Brennan, K.; Liyanapathirana, C.; Shahidi, F. Compositional characteristics o crab (Carcinus maenas). Food Chem. 2004, 88, 429–434. [CrossRef] 24. Venugopal, V. Marine Polysaccharides: Food Applications; CRC Press Taylor & Francis Group: Boca Raton, FL, USA, 2011; Volume 3, pp. 61–84. 25. Youn, D.K.; No, H.K.; Prinyawiwatkul, W. Physicochemical and functional properties of chitosans prepared from shells of crabs harvested in three different years. Carbohydr. Polym. 2009, 78, 41–45. [CrossRef] 26. Mahmoud, N.S.; Ghaly, A.E.; Arab, F. Unconventional Approach for Demineralization of Deproteinized Crustacean Shells for Chitin Production. Am. J. Biochem. Biotechnol. 2007, 3, 1–9. [CrossRef] 27. No, H.K.; Meyers, S.P. Preparation and characterization of chitin and chitosan: A review. J. Aquat. Food Prod. Technol. 1995, 4, 27–52. [CrossRef] 28. Benhabile, M.S.; Salah, R.; Lounici, H.; Drouiche, N.; Goosen, M.F.A.; Mameri, N. Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste. Food Hydrol. 2012, 29, 48–56. [CrossRef] 28. Benhabile, M.S.; Salah, R.; Lounici, H.; Drouiche, N.; Goosen, M.F.A.; Mameri, N. Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste. Food Hydrol. 2012, 29, 48–56. [CrossRef] 29. Yen, M.-T.; Yang, J.-H.; Mau, J.-L. Physicochemical characterization of chitin and chitosan from crab shells. References Photogeneration of superoxide anion in serum of bovine milk and in model systems containing riboflavin and amino acids. J. Dairy Sci. 1978, 61, 400–407. [CrossRef] 43. Bloknina, O.; Virolainen, E.; Fagerstedt, K.V. Antioxidants, oxidative damage and oxygen deprivation stress: A review. Ann. Bot. 2003, 91, 179–194. [CrossRef] 44. Meyer, A.S.; Isaksen, A. Application of enzymes as food antioxidants. Trends Food Sci. Technol. 1995, 6, 300. [CrossRef] 45. Feng, T.; Du, Y.M.; Li, J.; Hu, Y.; Kennedy, J.F. Enhancement of antioxidant activity of chitosan by irradiation. Carbohydr. Polym. 2008, 73, 126–132. [CrossRef] 14 of 15 Mar. Drugs 2019, 17, 239 46. Xing, R.; Liu, S.; Guo, Z.; Yu, H.; Wang, P.; Li, C.; Li, Z.; Li, P. Relevance of molecular weight of chitosan and its derivatives and their antioxidant activities in vitro. Bioorgan. Med. Chem. 2005, 13, 1573–1577. [CrossRef] 47. Yang, Y.; Shu, R.; Shao, J.; Xu, G.; Gu, X. Radical scavenging activity of chitooligosaccharide with different molecular weights. Eur. Food Res. Technol. 2006, 222, 36–40. [CrossRef] Charernsriwilaiwat, N.; Opanasopit, P.; Rojanarata, T.; Ngawhirunpat, T. In Vitro Antioxidant Activity o 48. Charernsriwilaiwat, N.; Opanasopit, P.; Rojanarata, T.; Ngawhirunpat, T. In Vitro Antioxidant Activity of Chitosan Aqueous Solution: Effect of Salt Form. Trop. J. Pharm. Res. 2012, 11, 235–242. [CrossRef] Chitosan Aqueous Solution: Effect of Salt Form. Trop. J. Pharm. Res. 2012, 11, 235–242. [CrossRef] 49. Halliwell, B.; Aeschbach, R.; Löliger, J.; Aruoma, O.I. The characterization of antioxidants. Food Chem. Toxicol. 1995, 33, 601–617. [CrossRef] 50. Tikhonov, V.E.; Radigina, L.A.; Yamskov, Y.A. Metal-chelating chitin derivatives via reaction of chitosan with nitrilotriacetic acid. Carbohydr. Res. 1996, 290, 33–41. [CrossRef] 51. Lin, H.-Y.; Chou, C.-C. Antioxidative activities of water-soluble disaccharide chitosan derivatives. Food Res. Int. 2004, 37, 883–889. [CrossRef] 52. Milosavljevic, N.B.; Ristica, M.D.; Peric-Grujic, A.A.; Filipovic, J.M.; Strbacb, S.B.; Rakocevic, Z.L.; Krusic, M.T.K. Sorption of zinc by novel pH-sensitive hydrogels based on chitosan, itaconic acid and methacrylic acid. J. Hazard. Mater. 2011, 192, 846–854. [CrossRef] [PubMed] 53. Kurita, K. Chitin and Chitosan: Functional biopolymers from marine crustaceans. Mar. Biotechnol. 2006, 8, 203–226. [CrossRef] 54. Qin, Y. The chelating properties of chitosan fibers. J. Appl. Polym. Sci. 1993, 49, 727–731. [CrossRef] 55. Kong, M.; Chen, X.G.; Xing, K.; Park, H.J. Antimicrobial properties of chitosan and mode of action: A state of the art review. Int. J. Food Microbiol. 2010, 144, 51–63. [CrossRef] 56. Liu, J.; Tian, S.; Meng, X.; Xu, Y. References Effects of chitosan on control of postharvest diseases and physiological responses of tomato fruit. Postharvest Biol. Technol. 2007, 44, 300–306. [CrossRef] 57. Skjak-Braek, G.; Anthonsen, T.; Sandford, P.A. Chitin and Chitosan: Sources, Chemistry, Biochemistry, Physical Properties and Applications; Springer: Dordrecht, The Netherlands, 1989; ISBN 978-1-85166-395-8. 58. Jeon, Y. Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydr. Polym. 2001, 44, 71–76. [CrossRef] 59. No, H.K.; Young Park, N.; Ho Lee, S.; Meyers, S.P. Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. Int. J. Food Microbiol. 2002, 74, 65–72. [CrossRef] 60. Singh, T.; Vesentini, D.; Singh, A.P.; Daniel, G. Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi. Int. Biodeterior. Biodegrad. 2008, 62, 116–124. [CrossRef] 61. Ing, L.Y.; Zin, N.M.; Sarwar, A.; Katas, H. Antifungal Activity of Chitosan Nanoparticles and Correlation with Their Physical Properties. J. Appl. Polym. Sci. 2012, 2012, 632698. [CrossRef] [PubMed] 62. Vallapa, N.; Wiarachai, O.; Thongchul, N.; Pan, J.; Tangpasuthadol, V.; Kiatkamjornwong, S.; Hoven, V.P. Enhancing antibacterial activity of chitosan surface by heterogeneous quaternization. Carbohydr. Polym. 2011, 83, 868–875. [CrossRef] 63. Wiarachai, O.; Thongchul, N.; Kiatkamjornwong, S.; Hoven, V.P. Surface-quaternized chitosan particles as an alternative and effective organic antibacterial material. Colloids Surf. 2012, 92, 121–129. [CrossRef] 63. Wiarachai, O.; Thongchul, N.; Kiatkamjornwong, S.; Hoven, V.P. Surface-quaternized chitosan particles as an alternative and effective organic antibacterial material. Colloids Surf. 2012, 92, 121–129. [CrossRef] 64. Guo, Z.; Xing, R.; Liu, S.; Zhong, Z.; Ji, X.; Wang, L.; Li, P. Antifungal properties of Schiffbases of chitosan, 629 N substituted chitosan and quaternized chitosan Carbohydr Res 2007 342 1329 1332 [CrossRef] 63. Wiarachai, O.; Thongchul, N.; Kiatkamjornwong, S.; Hoven, V.P. Surface-quaternized chitosan particles as an alternative and effective organic antibacterial material. Colloids Surf. 2012, 92, 121–129. [CrossRef] 64. Guo, Z.; Xing, R.; Liu, S.; Zhong, Z.; Ji, X.; Wang, L.; Li, P. Antifungal properties of Schiffbases of chitosan, 629 N-substituted chitosan and quaternized chitosan. Carbohydr. Res. 2007, 342, 1329–1332. [CrossRef] 65. Galván Márquez, I.; Akuaku, J.; Cruz, I.; Cheetham, J.; Golshani, A.; Smith, M.L. Disruption of protein synthesis as antifungal mode of action by chitosan. Int. J. Food Microbiol. 2013, 164, 108–112. [CrossRef] 66. Johnson, M.K. Variable Sensitivity in Microbiuret Assay of Protein. Anal. Biochem. 1978, 86, 320–323. alternative and effective organic antibacterial material. Colloids Surf. 2012, 92, 121 129. [CrossRef] 64. Guo, Z.; Xing, R.; Liu, S.; Zhong, Z.; Ji, X.; Wang, L.; Li, P. © 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/). 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] References Antifungal properties of Schiffbases of chitosan, 629 N-substituted chitosan and quaternized chitosan. Carbohydr. Res. 2007, 342, 1329–1332. [CrossRef] 65. Galván Márquez, I.; Akuaku, J.; Cruz, I.; Cheetham, J.; Golshani, A.; Smith, M.L. Disruption of protein synthesis as antifungal mode of action by chitosan. Int. J. Food Microbiol. 2013, 164, 108–112. [CrossRef] 64. Guo, Z.; Xing, R.; Liu, S.; Zhong, Z.; Ji, X.; Wang, L.; Li, P. Antifungal properties of Schiffbases of chitosan, 629 N-substituted chitosan and quaternized chitosan. Carbohydr. Res. 2007, 342, 1329–1332. [CrossRef] 5. Galván Márquez, I.; Akuaku, J.; Cruz, I.; Cheetham, J.; Golshani, A.; Smith, M.L. Disruption of pro synthesis as antifungal mode of action by chitosan. Int. J. Food Microbiol. 2013, 164, 108–112. [CrossRef 6. Johnson, M.K. Variable Sensitivity in Microbiuret Assay of Protein. Anal. Biochem. 1978, 86, 320– [CrossRef] 67. ISO 1444 (1996); International Organisation for Standardisation: Geneva, Switzerland, 1996. 68. Haastert-Talini, K.; Geuna, S.; Dahlin, L.B.; Meyer, C.; Stenberg, L.; Freier, T.; Heimann, C.; Barwig, C.; Pinto, L.F.V.; Raimondo, S.; et al. Chitosan tubes of varying degrees of acetylation for bridging peripheral nerve defects. Biomaterials 2013, 34, 9886–9904. [CrossRef] 69. Brugnerotto, J.; Lizardi, J.; Goycoolea, F.M.; Argüelles-Monal, W.; Desbrières, J.; Rinaudo, M. An infrared investigation in relation with chitin and chitosan characterization. Polymer 2001, 42, 3569–3580. [CrossRef] 69. Brugnerotto, J.; Lizardi, J.; Goycoolea, F.M.; Argüelles-Monal, W.; Desbrières, J.; Rinaudo, M. An infrared investigation in relation with chitin and chitosan characterization. Polymer 2001, 42, 3569–3580. [CrossRef] 70. Shimada, K.; Fujikawa, K.; Yahara, K.; Nakamura, T. Antioxidative properties of xanthan on the autoxidation 69. Brugnerotto, J.; Lizardi, J.; Goycoolea, F.M.; Argüelles Monal, W.; Desbrières, J.; Rinaudo, M. An infrared investigation in relation with chitin and chitosan characterization. Polymer 2001, 42, 3569–3580. [CrossRef] 70. Shimada, K.; Fujikawa, K.; Yahara, K.; Nakamura, T. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 1992, 40, 945–948. [CrossRef] 70. Shimada, K.; Fujikawa, K.; Yahara, K.; Nakamura, T. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 1992, 40, 945–948. [CrossRef] 15 of 15 Mar. Drugs 2019, 17, 239 71. Nishikimi, M. Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system. Biochem. Biophys. Res. Commun. 1975, 63, 463–468. [CrossRef] 71. Nishikimi, M. Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase 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 618 pathogens of strawberry fruits. Phytopathology 1992, 82, 398–402. [CrossRef] © 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/).
https://openalex.org/W4210775871
https://hal.science/hal-03556308/document
English
null
Hydrogen steelmaking. Part 1: Physical chemistry and process metallurgy
Matériaux & techniques/Matériaux et techniques
2,021
cc-by
5,863
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 scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 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 new, low CO2 emissions, hydrogen steelmaking process, J. Clean. Prod. 46, 27–35 (2013) References 1. WorldSteel Association, Steel Statistical Yearbook 2020, 2020 https://www.worldsteel.org/steel-by-topic/statistics/ steel-statistical-yearbook.html (accessed on 25/10/2021) 14. N. Towhidi, J. Szekely, Reduction kinetics of commercial low-silica hematite pellets with CO-H2 mixtures over temperatures range 600-1234 °C, Ironmak Steelmak. 6, 237–249 (1981) 2. L. Holappa, A general vision for reduction of energy consumption and CO2 emissions from the steel industry, Metals 10, 1117 (2020) ( ) 15. A. Bonalde, A. Henriquez, M. Manrique, Kinetic analysis of the iron oxide reduction using hydrogen-carbon monoxide mixtures as reducing agent, ISIJ Int. 45(9), 155–1260 (2005). 3. J.-P. Birat, M. Antoine, A. Dubs, et al., Vers une sidérurgie sans carbone ?, in: Journées sidérurgiques 1992, 16 au 17 décembre, 1992 and Revue de métallurgie 90, 411 (1993) 16. MIDREX, https://www.midrex.com/technology/midrex- process/ (accessed on 26/10/2020) 4. CIRCORED Hydrogen-based reduction, METSO-OTTO- TEC, https://www.mogroup.com/portfolio/circored-hydro- gen-based-reduction / ( / / ) 17. HYL-ENERGIRON, https://www.tenova.com/product/ iron-reduction-technologies/ (accessed on 26/10/2020) 18. H. Hamadeh, Modélisation mathématique détaillée du procédé de réduction directe du minerai de fer, Ph.D. Thesis, Université de Lorraine, Nancy, France, 2017, Available from https://tel.archives-ouvertes.fr/tel-01740462 5. J.-P. Birat, J. Borlée, B. Korthas, et al., ULCOS program: A progress report in the spring of 2008, in: Scanmet III, 3rd International Conference on Process Development in Iron and Steelmaking, 8–11 June, 2008, Lulea, Sweden 25. K. Rechberger, et al., Green hydrogen-based direct reduction for low-carbon steelmaking, Steel Res. Int. 91, 2000110 (2020) 26. L. Shao et al., Computational analysis of hydrogen reduction of iron oxide pellets in a shaft furnace process, Renew. Energy 179, 1537–1547 (2021) 0 F. Patisson et al.: Matériaux & Techniques 109, 303 (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) https://tel.archives-ouvertes.fr/tel-01740462 19. A. Ranzani da Costa, La réduction du minerai de fer par l’hydrogène : étude cinétique, phénomène de collage et modélisation, Ph.D. Thesis, Institut national polytechnique de Lorraine, Nancy, France, 2011, Available from https://tel. archives-ouvertes.fr/tel-01204934/ 6. J.-P. Birat, CO2-lean steelmaking: ULCOS, other interna- tional programs and emerging concepts, in: ECCR Steel (METEC- 2011), 2011 7. J.-P. Birat, The progress and status of IISI’s CO2 Breakthrough Program and EU’s ULCOS, in: CO2 Reduc- tion Workshop, Kaohsiung, Taiwan, 1–2 November, 2007 / / 20. H.Y. Sohn, The law of additive reaction times in fluid-solid reactions, Metall. Trans. 9B, 89–96 (1978) 8. Breaking through the technology barriers: Steel producers are researching new production technologies that would radically reduce their environmental footprint, FACT SHEET – Breakthrough technologies, Worldsteel pamphlet, 10/2008 21. H. Hamadeh, O. Mirgaux, F. Patisson, Detailed modeling of the direct reduction of iron ore in a shaft furnace, Materials 11, 1865 (2018) 22. D.R. Parisi, M.A. Laborde, Modeling of counter current moving bed gas-solid reactor used in direct reduction of iron ore, Chem. Eng. J. 104, 35–43 (2004) 9. J.-P. Birat, F. Patisson, O. Mirgaux, Hydrogen Steelmaking, part 2: Competition with other zero-carbon steelmaking solutions – geopolitical issues, Matériaux & Techniques, (2021), in press 23. M.S. Valipour, Y. Saboohi, Numerical investigation of nonisothermal reduction of haematite using syngas: The shaft scale study, Model. Simul. Mater. Sci. Eng. 15, 487–507 (2007) 10. M. Hutson, The promise of carbon-neutral steel, The New Yorker, Sept. 18, 2021, https://www.newyorker.com/news/ annals-of-a-warming-planet/the-promise-of-carbon-neutral- steel (accessed on 26 October 2020) 24. A. Shams, F. Moazeni, Modeling and simulation of the MIDREX shaft furnace: reduction, transition and cooling zones, JOM 67, 2681–2689 (2015) 25. K. Rechberger, et al., Green hydrogen-based direct reduction for low-carbon steelmaking, Steel Res. Int. 91, 2000110 (2020) 26. L. Shao et al., Computational analysis of hydrogen reduction 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
https://openalex.org/W4382776607
https://translational-medicine.biomedcentral.com/counter/pdf/10.1186/s12967-023-04176-6
English
null
Alterations of gut microbiota are associated with blood pressure: a cross-sectional clinical trial in Northwestern China
Journal of translational medicine
2,023
cc-by
12,224
© 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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​ CCT​ACG​GGA​GGC​AGC​AG-3ʹ; 806R: 5ʹ-GGA​CTA​ CHVGGG​TWT​CTAAT-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.​genom​ics.​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, Geng B, et al. Gut microbiota dysbiosis contributes to the development of hypertension. Microbiome. 2017;5:19. Received: 13 March 2023 Accepted: 30 April 2023 21. Beale AL, Kaye DM, Marques FZ. The role of the gut microbiome in sex differences in arterial pressure. Biol Sex Diff. 2019;10:22. ff 22. Colafella KMM, Denton KM. Sex-specific differences in hypertension and associated cardiovascular disease. Nat Rev Nephrol. 2018;14:185–201. Availability of data and materials 13. Boulange CL, Neves AL, Chilloux J, Nicholson JK, Dumas M-E. Impact of the gut microbiota on inflammation, obesity, and metabolic disease. Genome Med. 2016;8:42. The data generated and analyzed during the current study are available from the corresponding authors on reasonable request. 14. Yan QL, Gu YF, Li XC, Yang W, Jia LQ, Chen CM, Han XY, Huang YK, Zhao LZ Li P, et al. Alterations of the gut microbiome in hypertension. Front Cell Infect Microbiol. 2017;7:9. Competing interests The authors declare that they have no competing interests. 18. Santos-Marcos JA, Haro C, Vega-Rojas A, Alcala-Diaz JF, Molina-Abril H, Leon-Acuña A, Lopez-Moreno J, Landa BB, Tena-Sempere M, Perez- Martinez P, et al. Sex differences in the gut microbiota as potential determinants of gender predisposition to disease. Mol Nutr Food Res. 2019;63:e1800870. Declarations 15. Kim S, Goel R, Kumar A, Qi YF, Lobaton G, Hosaka K, Mohammed M, Handberg EM, Richards EM, Pepine CJ, Raizada MK. Imbalance of gut microbiome and intestinal epithelial barrier dysfunction in patients with high blood pressure. Clin Sci. 2018;132:701–18. References 1. GBD 2019 Risk Factors Collaborators. Global burden of 87 risk factors in 204 countries and territories, 1990–2019: a systematic analysis for the global burden of disease study 2019. Lancet. 2020;396:1223–49. 1. GBD 2019 Risk Factors Collaborators. Global burden of 87 risk factors in 204 countries and territories, 1990–2019: a systematic analysis for the global burden of disease study 2019. Lancet. 2020;396:1223–49. 23. Lau E, Neves JS, Ferreira-Magalhaes M, Carvalho D, Freitas P. Probiotic ingestion, obesity, and metabolic-related disorders: results from NHANES, 1999–2014. Nutrients. 2019;11:1482. 2. Marques FZ, Jama HA, Tsyganov K, Gill PA, Rhys-Jones D, Muralitharan RR, Muir J, Holmes A, Mackay CR. Guidelines for transparency on gut micro- biome studies in essential and experimental hypertension. Hypertension. 2019;74:1279–93. 2. Marques FZ, Jama HA, Tsyganov K, Gill PA, Rhys-Jones D, Muralitharan RR, Muir J, Holmes A, Mackay CR. Guidelines for transparency on gut micro- biome studies in essential and experimental hypertension. Hypertension. 2019;74:1279–93. 24. Zhernakova A, Kurilshikov A, Bonder MJ, Tigchelaar EF, Schirmer M, Vatanen T, Mujagic Z, Vila AV, Falony G, Vieira-Silva S, et al. Population- based metagenomics analysis reveals markers for gut microbiome composition and diversity. Science. 2016;352:565–9. 3. Mills KT, Bundy JD, Kelly TN, Reed JE, Kearney PM, Reynolds K, Chen J, He J. Global disparities of hypertension prevalence and control: a system- atic analysis of population-based studies from 90 countries. Circulation. 2016;134:441–50. 3. Mills KT, Bundy JD, Kelly TN, Reed JE, Kearney PM, Reynolds K, Chen J, He J. Global disparities of hypertension prevalence and control: a system- atic analysis of population-based studies from 90 countries. Circulation. 2016;134:441–50. 25. Choi MS, Yu JS, Yoo HH, Kim D-H. The role of gut microbiota in the phar- macokinetics of antihypertensive drugs. Pharmacol Res. 2018;130:164–71 26. National Center for Cardiovascular Diseases, Chinese Medical Doctor Association, Hypertension Committee of the Chinese Medical Doctor Association. Chinese society of cardiology, chinese medical association, hypertension committee of cross-straits medicine exchange association. clinical practice guidelines for the management of hypertension in China. Chin J Cardiol. 2022;50:1050–95. 4. Mills KT, Stefanescu A, He J. The global epidemiology of hypertension. Nat Rev Nephrol. 2020;16:223–37. 4. Mills KT, Stefanescu A, He J. The global epidemiology of hypertension. Nat Rev Nephrol. 2020;16:223–37. 5. Evangelou E, Warren HR, Mosen-Ansorena D, Mifsu B, Pazoki R, Gao H, Ntritsos G, Dimou N, Cabrer CP, Karaman I, et al. Author details 1 1 Department of Clinical Laboratory, Honghui Hospital, Xi’an Jiaotong Univer- sity, Xi’an, China. 2 Institute of Medical Research, Northwestern Polytechnical University, Xi’an, China. 3 Department of Cardiology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China. 4 Department of Quality Control, Xi’an Mental Health Center, Xi’an, China. 5 Department of Spine Surgery, Honghui Hospital, Xi’an Jiaotong University, Xi’an, China. 19. Campaniello D, Corbo MR, Sinigaglia M, Speranza B, Racioppo A, Altieri C, Bevilacqua A. How diet and physical activity modulate gut microbiota: evidence, and perspectives. Nutrients. 2022;14:2456. 20. Liu X, Li Y, Guo Y, Li L, Yang K, Liu R, Mao Z, Bie R, Wang C. The burden, management rates and influencing factors of high blood pressure in a chinese rural population: the rural diabetes, obesity and lifestyle (Rural- Diab) study. J Hum Hypertens. 2018;32:236–46. Received: 13 March 2023 Accepted: 30 April 2023 Received: 13 March 2023 Accepted: 30 April 2023 Ethics approval and consent to participate This study was approved by the Ethics Committee of Honghui Hospital, Xi’an Jiaotong University (Protocol number 201801022) and was registered on the Chinese Clinical Trial Registry (http://​www.​chictr.​org.​cn/; registration number ChiCTR1800019191). Informed consent was provided by all recruited subjects prior to the study. 16. Palmu J, Salosensaari A, Havulinna AS, Cheng S, Inouye M, Jain M, Salido RA, Sanders K, Brennan C, Humphrey GC, et al. Association between the gut microbiota and blood pressure in a Population Cohort of 6953 individuals. J Am Heart Association. 2020;9:e016641. References Tong T, Wang YN, Zhang CM, Kang SG. In vitro and in vivo antihyperten- sive and antioxidant activities of fermented roots of Allium hookeri. Chin Herb Med. 2021;13:541–8. 45. Streppel MT, Arends LR, van’t Veer P, Grobbee DE, Geleijnse JM. Dietary fiber and blood pressure—a meta-analysis of randomized placebo- controlled trials. Arch Intern Med. 2005;165:150–6. 46. Whelton SP, Hyre AD, Pedersen B, Yi Y, Whelton PK, He J. Effect of dietary fiber intake on blood pressure: a meta-analysis of randomized, controlled clinical trials. J Hypertens. 2005;23:475–81. 68. Mörkl S, Lackner S, Meinitzer A, Mangge H, Lehofer M, Halwachs B, Gorkiewicz G, Kashofer K, Painold A, Holl AK, et al. Gut microbiota, dietary intakes and intestinal permeability reflected by serum zonulin in women. Eur J Nutr. 2018;57:2985–97. y 47. Chen L, He F, Dong YB, Huang Y, Wang CQ, Harshfield GA, Zhu HD. Modest Sodium reduction increases circulating short-chain fatty acids in untreated hypertensives a Randomized, Double-Blind, placebo-con- trolled trial. Hypertension. 2020;76:73–9. 69. Kang C, Wang B, Kaliannan K, Wang X, Lang H, Hui S, Huang L, Zhang Y, Zhou M, Chen M, Mi M. Gut microbiota mediates the protective effects of dietary capsaicin against chronic low-grade inflammation and associated obesity induced by high-fat diet. mBio. 2017;8:e00470-17. 48. Wilck N, Matus MG, Kearney SM, Olesen SW, Forslund K, Bartolomaeus H, Haase S, Mahler A, Balogh A, Marko L, et al. Salt-responsive gut commen- sal modulates T(H)17 axis and disease. Nature. 2017;551:585–9. 70. Leclercq S, Matamoros S, Cani PD, Neyrinck AM, Jamar F, Stärkel P, Windey K, Tremaroli V, Bäckhed F, Verbeke K, et al. Intestinal permeability, gut-bacterial dysbiosis, and behavioral markers of alcohol-dependence severity. Proc Natl Acad Sci U S A. 2014;111:E4485–93. 49. Marques FZ, Nelson E, Chu PY, Horlock D, Fiedler A, Ziemann M, Tan JK, Kuruppu S, Rajapakse NW, El-Osta A, et al. High-Fiber Diet and acetate supplementation change the gut microbiota and prevent the develop- ment of hypertension and heart failure in hypertensive mice. Circulation. 2017;135:964–77. 71. Yu Y, Mao G, Wang J, Zhu L, Lv X, Tong Q, Fang Y, Lv Y, Wang G. Gut dys- biosis is associated with the reduced exercise capacity of elderly patients with hypertension. Hypertens Res. 2018;41:1036–44. yp yp 72. Li J, Yang X, Zhou X, Cai J. The role and mechanism of intestinal flora in blood pressure regulation and hypertension development. Antioxid Redox Signal. 2021;34:811–30. 50. References Page 16 of 17 Page 16 of 17 Lv et al. Journal of Translational Medicine (2023) 21:429 Lv et al. Journal of Translational Medicine (2023) 21:429 31. Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007;73:5261–7. 54. Wolever TMS, Spadafora P, Eshuis H. Interaction between Colonic acetate and propionate in humans. Am J Clin Nutr. 1991;53:681–7. 55. Cummings JH, Pomare EW, Branch WJ, Naylor CPE, Macfarlane GT. Short chain fatty acids in human large intestine, portal, hepatic and venouse blood. Gut. 1987;28:1221–7. 32. Qin JJ, Li RQ, Raes J, Arumugam M, Burgdorf KS, Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T, et al. A human gut microbial gene cata- logue established by metagenomic sequencing. Nature. 2010;464:59–65. 56. Poll BG, Cheema MU, Pluznick JL. Gut microbial metabolites and blood pressure regulation: focus on SCFAsand TMAO. Physiology. 2020;35:275–84. 33. Ma J, Li H. The role of Gut microbiota in atherosclerosis and hypertension. Front Pharmacol. 2018;9:1082. 34. Qi Y, Kim S, Richards EM, Raizada MK, Pepine CJ. Gut microbiota potential for a unifying hypothesis for prevention and treatment of hypertension. Circul Res. 2017;120:1724–6. 57. Muralitharan RR, Jama HA, Xie L, Peh A, Snelson M, Marques FZ. Microbial peer pressure the role of the gut microbiota in hypertension and its complications. Hypertension. 2020;76:1674–87. 35. Howitt MR, Garrett WS. A complex microworld in the gut gut microbiota and cardiovascular disease connectivity. Nat Med. 2012;18:1188–9. 58. Avery EG, Bartolomaeus H, Maifeld A, Marko L, Wiig H, Wilck N, Rosshart SP, Forslund SK, Muller DN. The gut microbiome in hypertension recent advances and future perspectives. Circul Res. 2021;128:934–50. 36. Sekirov I, Russell SL, Antunes LC, Finlay BB. Gut microbiota in health and disease. Physiol Rev. 2010;90:859–904. 59. Gomez-Arango LF, Barrett HL, McIntyre HD, Callaway LK, Morrison M, Nitert MD, Grp ST. Increased systolic and diastolic blood pressure is Asso- ciated with altered gut microbiota composition and butyrate production in early pregnancy. Hypertension. 2016;68:974–81. 37. Yang T, Santisteban MM, Rodriguez V, Li E, Ahmari N, Carvajal JM, Zadeh M, Gong MH, Qi YF, Zubcevic J, et al. Gut dysbiosis is linked to hyperten- sion. Hypertension. 2015;65:1331–40. y 38. de La Cuesta-Zuluag J, Kelley ST, Chen Y, Escobar JS, Mueller NT, Ley RE, McDonald D, Huang S, Swafford AD, Knight R, Thackray VG. References Age- and sex-dependent patterns of gut microbial diversity in human adults. Msystems. 2019;4:e00261–00219. 60. Koh A, De Vadder F, Kovatcheva-Datchary P, Bäckhed F. From Dietary Fiber to host physiology: short-chain fatty acids as key bacterial metabolites. Cell. 2016;165:1332–45. 61. Miyamoto J, Kasubuchi M, Nakajima A, Irie J, Itoh H, Kimura I. The role of short-chain fatty acid on blood pressure regulation. Curr Opin Nephrol Hypertens. 2016;25:379–83. 39. Bowyer RCE, Jackson MA, Le Roy CI, Lochlainn MN, Spector TD, Dowd JB, Steves CJ. Socioeconomic status and the gut microbiome: a TwinsUK cohort study. Microorganisms. 2019;7:17. y 62. Canale MP, Noce A, Di Lauro M, Marrone G, Cantelmo M, Cardillo C, Federici M, Di Daniele N, Tesauro M. Gut dysbiosis and western diet in the pathogenesis of essential arterial hypertension: a narrative review. Nutrients. 2021;13:1162. 40. Valeri F, Endres K. How biological sex of the host shapes its gut micro- biota. Front Neuroendocr. 2021;61:15. 41. Takagi T, Naito Y, Inoue R, Kashiwagi S, Uchiyama K, Mizushima K, Tsuchiya S, Dohi O, Yoshida N, Kamada K, et al. Differences in gut microbiota asso- ciated with age, sex, and stool consistency in healthy japanese subjects. J Gastroenterol. 2019;54:53–63. 63. Gill PA, van Zelm MC, Muir JG, Gibson PR. Short chain fatty acids as potential therapeutic agents in human gastrointestinal and inflammatory disorders. Aliment Pharmacol Ther. 2018;48:15–34. 64. Macfarlane S, Macfarlane GT. Regulation of short-chain fatty acid produc- tion. Proc Nutr Soc. 2003;62:67–72.f 42. Bolnick DI, Snowberg LK, Hirsch PE, Lauber CL, Org E, Parks B, Lusis AJ, Knight R, Caporaso JG, Svanback R. Individual diet has sex-dependent effects on vertebrate gut microbiota. Nat Commun. 2014;5:13. 65. Li A, Wang N, Li N, Li B, Yan F, Song Y, Hou J, Huo G. Modulation effect of chenpi extract on gut microbiota in high-fat diet-induced obese C57BL/6 mice. J Food Biochem. 2021;45:e13541. 43. Richards EM, Pepine CJ, Raizada MK, Kim S. The Gut, its microbiome, and hypertension. Curr Hypertens Rep. 2017;19:36. 66. Piñero F, Vazquez M, Baré P, Rohr C, Mendizabal M, Sciara M, Alonso C, Fay F, Silva M. A different gut microbiome linked to inflammation found in cir- rhotic patients with and without hepatocellular carcinoma. Ann Hepatol. 2019;18:480–7. 44. Takase H, Sugiura T, Kimura G, Ohte N, Dohi Y. Dietary sodium con- sumption predicts future blood pressure and incident hypertension in the Japanese normotensive general population. J Am Heart Assoc. 2015;4:e001959. 67. References Genetic analysis of over 1 million people identifies 535 new loci associated with blood pressure traits. Nat Genet. 2018;50:1412–25. 5. Evangelou E, Warren HR, Mosen-Ansorena D, Mifsu B, Pazoki R, Gao H, Ntritsos G, Dimou N, Cabrer CP, Karaman I, et al. Genetic analysis of over 1 million people identifies 535 new loci associated with blood pressure traits. Nat Genet. 2018;50:1412–25. 27. Whelton PK, Carey RM, Aronow WS, Casey DE Jr, Collins KJ, Himmelfarb CD, DePalma SM, Gidding S, Jamerson KA, Jones DW, et al. ACC/AHA/ AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NM CNA Guideline for the prevention, detection, evaluation, and management of high blood pres- sure in adults a report of the American college of cardiology/american heart association task force on clinical practice guidelines. Hypertension. 2018;71:1269–324. 6. Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–20. 6. Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–20. 7. Jie ZY, Xia HH, Zhong SL, Feng Q, Li SH, Liang SS, Zhong HZ, Liu ZP, Gao Y, Zhao H, et al. The gut microbiome in atherosclerotic cardiovascular disease. Nat Commun. 2017;8:12. 8. Linz D, Wirth K, Linz W, Heuer HOO, Frick W, Hofmeister A, Heinelt U, Arndt P, Schwahn U, Boehm M, Ruetten H. Antihypertensive and laxative effects by pharmacological inhibition of sodium-proton-exchanger subtype 3-mediated sodium absorption in the gut. Hypertension. 2012;60:1560. 28. Franzosa EA, Morgan XC, Segata N, Waldron L, Reyes J, Earl AM, Gian- noukos G, Boylan MR, Ciulla D, Gevers D, et al. Relating the metatran- scriptome and metagenome of the human gut. Proc Natl Acad Sci USA. 2014;111:E2329–38. p g yp 9. Jama HA, Beale A, Shihata WA, Marques FZ. The effect of diet on hyper- tensive pathology: is there a link via gut microbiota-driven immunome- tabolism? Cardiovascular Res. 2019;115:1435–47. 29. Edgar RC. UPARSE: highly accurate OTU sequences from microbial ampli- con reads. Nat Methods. 2013;10:996–8. 30. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335–6. 10. Marques FZ, Mackay CR, Kaye DM. Beyond gut feelings: how the gut microbiota regulates blood pressure. Nat Reviews Cardiol. 2018;15:20–32. 11. Jama HA, Kaye DM, Marques FZ. The gut microbiota and blood pressure in experimental models. Curr Opin Nephrol Hypertens. 2019;28:97–104. Lv et al. Journal of Translational Medicine (2023) 21:429 Lv et al. Journal of Translational Medicine (2023) 21:429 76. Xie D, Zhang MS, Wang BL, Lin H, Wu EQ, Zhao HH, Li SC. Differential analysis of hypertension-associated intestinal microbiota. Int J Med Sci. 2019;16:872–81. 77. Fu JY, Bonder MJ, Cenit MC, Tigchelaar EF, Maatman A, Dekens JAM, Brandsma E, Marczynska J, Imhann F, Weersma RK, et al. The gut Micro- biome contributes to a substantial proportion of the variation in blood lipids. Circul Res. 2015;117:817–24. 78. Qin JJ, Li YR, Cai ZM, Li SH, Zhu JF, Zhang F, Liang SS, Zhang WW, Guan YL, Shen DQ, et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature. 2012;490:55–60. yp 79. Lambeth SM, Carson T, Lowe J, Ramaraj T, Leff JW, Luo L, Bell CJ, Shah VO. Composition, diversity and abundance of gut microbiome in prediabetes and type 2 diabetes. J diabetes Obes. 2015;2:1–7. 80. Org E, Blum Y, Kasela S, Mehrabian M, Kuusisto J, Kangas AJ, Soininen P, Wang Z, Ala-Korpela M, Hazen SL, et al. Relationships between gut micro- biota, plasma metabolites, and metabolic syndrome traits in the METSIM cohort. Genome Biol. 2017;18:70. 81. Ren Z, Li A, Jiang J, Zhou L, Yu Z, Lu H, Xie H, Chen X, Shao L, Zhang R, et al. Gut microbiome analysis as a tool towards targeted non-invasive biomarkers for early hepatocellular carcinoma. Gut. 2019;68:1014–23. 82. Liu B, Lin W, Chen S, Xiang T, Yang Y, Yin Y, Xu G, Liu Z, Liu L, Pan J, Xie L. Gut microbiota as a subjective measurement for auxiliary diagnosis of insomnia disorder. Front Microbiol. 2019;10:1770. 83. Liu H, Chen X, Hu X, Niu H, Tian R, Wang H, Pang H, Jiang L, Qiu B, Chen X, et al. Alterations in the gut microbiome and metabolism with coronary artery disease severity. Microbiome. 2019;7:68. 84. Zuo K, Li J, Li K, Hu C, Gao Y, Chen M, Hu R, Liu Y, Chi H, Wang H, et al. Disordered gut microbiota and alterations in metabolic patterns are associated with atrial fibrillation. Gigascience. 2019;8:giz058. i 85. Fernandes R, Viana SD, Nunes S, Reis F. Diabetic gut microbiota dysbiosis as an inflammaging and immunosenescence condition that fosters progression of retinopathy and nephropathy. Biochim Et Biophys Acta- Molecular Basis Disease. 2019;1865:1876–97. 86. Cho I, Blaser MJ. The human microbiome: at the interface of health and disease. Nat Rev Genet. 2012;13:260–70. 87. Lv et al. Journal of Translational Medicine (2023) 21:429 Aagaard K, Petrosino J, Keitel W, Watson M, Katancik J, Garcia N, Patel S, Cutting M, Madden T, Hamilton H, et al. The human Microbiome Project strategy for comprehensive sampling of the human microbiome and why it matters. FASEB J. 2013;27:1012–22. 88. Haro C, Rangel-Zuniga OA, Alcala-Diaz JF, Gomez-Delgado F, Perez-Mar- tinez P, Delgado-Lista J, Quintana-Navarro GM, Landa BB, Navas-Cortes JA, Tena-Sempere M, et al. Intestinal microbiota is influenced by gender and body Mass Index. PLoS ONE. 2016;11:16. y 89. Yoon K, Kim N. Roles of sex hormones and gender in the gut microbiota. J Neurogastroenterol Motil. 2021;27:314–25. y 89. Yoon K, Kim N. Roles of sex hormones and gender in the gut microbiota. J Neurogastroenterol Motil. 2021;27:314–25. g 90. Siddiqui R, Makhlouf Z, Alharbi AM, Alfahemi H, Khan NA. The gut micro- biome and female health. Biology. 2022;11:1683. 90. Siddiqui R, Makhlouf Z, Alharbi AM, Alfahemi H, Khan NA. The gut micro- biome and female health. Biology. 2022;11:1683. 91. Chen L, Liu Y, Tang Z, Song Z, Cao F, Shi X, Xie P, Wei P, Li M. Radix Angelica dahuricae extract ameliorates oestrogen deficiency-induced dyslipidae- mia in ovariectomized (OVX) rats by modulating the gut microbiota and bile acid signalling. Phytomedicine. 2022;107:154440. 91. Chen L, Liu Y, Tang Z, Song Z, Cao F, Shi X, Xie P, Wei P, Li M. Radix Angelica dahuricae extract ameliorates oestrogen deficiency-induced dyslipidae- mia in ovariectomized (OVX) rats by modulating the gut microbiota and bile acid signalling. Phytomedicine. 2022;107:154440. 92. Yano Y, Niiranen TJ. Gut microbiome over a lifetime and the association with hypertension. Curr Hypertens Rep. 2021;23:15. 92. Yano Y, Niiranen TJ. Gut microbiome over a lifetime and the association with hypertension. Curr Hypertens Rep. 2021;23:15. Lv et al. Journal of Translational Medicine (2023) 21:429 • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: References Bartolomaeus H, Balogh A, Yakoub M, Homann S, Marko L, Hoeges S, Tsvetkov D, Krannich A, Wundersitz S, Avery EG, et al. Short-chain fatty acid propionate protects from hypertensive cardiovascular damage. Circulation. 2019;139:1407–21. 73. Qin Y, Zhao J, Wang Y, Bai M, Sun S. Specific alterations of gut microbiota in chinese patients with hypertension: a systematic review and Meta- analysis. Kidney Blood Press Res. 2022;47:433–47. 51. Maier TV, Lucio M, Lee LH, VerBerkmoes NC, Brislawn CJ, Bernhardt J, Lamendella R, McDermott JE, Bergeron N, Heinzmann SS, et al. Impact of Dietary Resistant Starch on the human gut Microbiome, Metaproteome, and Metabolome. mBio. 2017;8:e01343–01317. 74. Santisteban MM, Kim S, Pepine CJ, Raizada MK. Brain-gut-bone Marrow Axis: implications for hypertension and related therapeutics. Circ Res. 2016;118:1327–36. 52. Yang F, Chen H, Gao Y, An N, Li X, Pan X, Yang X, Tian L, Sun J, Xiong X, Xing Y. Gut microbiota-derived short-chain fatty acids and hypertension: mechanism and treatment. Biomed Pharmacother. 2020;130:110503. 75. Hsu CN, Chan JYH, Wu KLH, Yu HR, Lee WC, Hou CY, Tain YL. Altered gut microbiota and its metabolites in hypertension of developmental origins: exploring differences between fructose and antibiotics exposure. Int J Mol Sci. 2021;22:2674. 53. Moore WEC, Cato EP, Holdeman LV. Some current concepts in intestinal bacteriology. Am J Clin Nutr. 1978;31:33-S42. Page 17 of 17 Lv et al. Journal of Translational Medicine (2023) 21:429 Publisher’s Note 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. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from:
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
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
https://openalex.org/W2244591382
https://europepmc.org/articles/pmc3470215?pdf=render
English
null
3-Methylamino-3-phenylpropan-1-ol
Acta crystallographica. Section E
2,012
cc-by
3,406
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
https://openalex.org/W4362378636
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
Greek, Modern
null
Figure S2 from Long Noncoding RNA DRAIC Inhibits Prostate Cancer Progression by Interacting with IKK to Inhibit NF-κB Activation
null
2,023
cc-by
525
WT KO1 KO1+FL KO1+E1-3 KO1+E4-5 701-1705 900-1705 1100-1705 701-1105 701-905 E DRAIC KO 0 0.2 0.4 0.6 0.8 1 1.2 EV FL E1-3 E4-5 NF-κB ReporterActivity * NS * A * * * * NS 0 1 2 3 4 5 6 7 EV FL E1-3 E4-5 EV Ca-IKKβ (S177E/S181E) - + + + + Normalized NF-κB Activity 0 20 40 60 80 DRAIC Expression EV FL E1-3 E4-5 * * * B C D 0 20 40 60 80 100 120 140 EV FL E1-3 E4-5 900-1705 1100-1705 701-1105 701-905 Relative DRAIC Expression F 293T 293T DRAIC KO DRAIC KO * WT KO1 KO1+FL KO1+E1-3 KO1+E4-5 701-1705 900-1705 1100-1705 701-1105 701-905 E DRAIC KO 0 0.2 0.4 0.6 0.8 1 1.2 EV FL E1-3 E4-5 NF-κB ReporterActivity * NS * A * * * * NS 0 1 2 3 4 5 6 7 EV FL E1-3 E4-5 EV Ca-IKKβ (S177E/S181E) - + + + + Normalized NF-κB Activity 0 20 40 60 80 DRAIC Expression EV FL E1-3 E4-5 * * * B C D 0 20 40 60 80 100 120 140 EV FL E1-3 E4-5 900-1705 1100-1705 701-1105 701-905 Relative DRAIC Expression F 293T 293T DRAIC KO DRAIC KO * DRAIC KO 0 0.2 0.4 0.6 0.8 1 1.2 EV FL E1-3 E4-5 NF-κB ReporterActivity * NS * A * * * * NS 0 1 2 3 4 5 6 7 EV FL E1-3 E4-5 EV Ca-IKKβ (S177E/S181E) - + + + + Normalized NF-κB Activity 0 20 40 60 80 DRAIC Expression EV FL E1-3 E4-5 * * * B C D 293T 293T DRAIC KO 0 20 40 60 80 DRAIC Expression EV FL 1-3 4-5 * * * C DRAIC KO WT KO1 KO1+FL KO1+E1-3 KO1+E4-5 701-1705 900-1705 1100-1705 701-1105 701-905 E DRAIC KO 0 0.2 0.4 0.6 0.8 1 1.2 EV FL E1-3 E4-5 NF-κB ReporterActivity * NS * A * * * * NS 0 1 2 3 4 5 6 7 EV FL E1-3 E4-5 EV Ca-IKKβ (S177E/S181E) - + + + + Normalized NF-κB Activity 0 20 40 60 80 DRAIC Expression EV FL E1-3 E4-5 * * * B C D n F 293T 293T DRAIC KO DRAIC KO C A WT KO1 KO1+FL KO1+E1-3 KO1+E4-5 701-1705 900-1705 1100-1705 701-1105 701-905 E DRAIC KO 0 0.2 EV FL E1-3 E4-5 NF-κB R 0 1 2 EV FL E1-3 E4-5 EV Ca-IKKβ (S177E/S181E) - + + + + Normal 0 20 DRAIC EV FL E1-3 E4-5 D Figure S4 0 20 40 60 80 100 120 140 EV FL E1-3 E4-5 900-1705 1100-1705 701-1105 701-905 Relative DRAIC Expression F DRAIC KO * WT KO1 KO1+FL KO1+E1-3 KO1+E4-5 701-1705 900-1705 1100-1705 701-1105 701-905 E DRAIC KO E E1 E4 N E E4 Ca-IKKβ (S177E/S181E) - + + + + N D E F E1- E4- D 701-1705 900-1705 1100-1705 701-1105 701-905 E DRAIC KO D D E WT KO1 KO1+FL KO1+E1-3 KO1+E4-5 ( ) D F 0 20 40 60 80 100 120 140 EV FL E1-3 E4-5 900-1705 1100-1705 701-1105 701-905 Relative DRAIC Expression F DRAIC KO * F Figure S4 Figure S4
https://openalex.org/W4311684228
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0278240&type=printable
English
null
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
2,022
cc-by
11,607
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:// doi.org/10.1016/j.jval.2022.03.019 PMID: 35527165 6. Thompson E., et al., COVID-19: A case for the collection of race data in Canada and abroad. Can Com- 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 Community-Based Participatory Focus Groups: Social Risks Assessment. Journal of health disparities research and practice, 2017. 10(1): p. 143–166. 15. Sampson R.J. and Laub J.H., Crime and Deviance in the Life Course. Annual Review of Sociology, 1992. 18(1): p. 63–84. 16. Hitlin S. and Elder G.H., Time, Self, and the Curiously Abstract Concept of Agency. Sociological Theory, 2007. 25(2): p. 170–191. 17. KFL&A Public Health Unit. Demographics. 2018 [cited 2022 September 14th]; Available from: https:// www.kflaph.ca/en/research-and-reports/demographics.aspx. 18. KFL&A Public Health Unit. Status of Cases in KFL&A. 2021 [cited 2021 April 1rst]; Available from: https://www.kflaph.ca/en/healthy-living/status-of-cases-in-kfla.aspx. 19. Spryng.io. Spryng io Home page. 2021; Available from: https://www.spryng.io/. 20. Information, C.I.o.H. COVID-19 Intervention Timeline in Canada. 2022 [cited 2022 September 14]; Available from: https://www.cihi.ca/en/covid-19-intervention-timeline-in-canada. 21. Government of Canada, Canada Emergency Response Benefit (CERB) with CRA. 2022 [cited 2022 September 14]; Available from: https://www.canada.ca/en/revenue-agency/services/benefits/apply-for- cerb-with-cra.html. 22. First Nations Information Governance Centre. First Nations Principles of OCAP. 2022 [cited 2022 Octo- ber 17]; Available from: https://fnigc.ca/ocap-training/. 23. Sevelius J.M., et al., Research with Marginalized Communities: Challenges to Continuity During the COVID-19 Pandemic. AIDS Behav, 2020. 24(7): p. 2009–2012. https://doi.org/10.1007/s10461-020- 02920-3 PMID: 32415617 24. Hyett S., Marjerrison S., and C., Improving health research among Indigenous Peoples in Canada. Cmaj, 2018. 190(20): p. E616–e621. https://doi.org/10.1503/cmaj.171538 PMID: 29789286 25. Assarroudi A., et al., Directed qualitative content analysis: the description and elaboration of its under- pinning methods and data analysis process. J Res Nurs, 2018. 23(1): p. 42–55. https://doi.org/10. 1177/1744987117741667 PMID: 34394406 26. Hsieh H.F. and Shannon S.E., Three approaches to qualitative content analysis. Qual Health Res, 2005. 15(9): p. 1277–88. https://doi.org/10.1177/1049732305276687 PMID: 16204405 27. Wei H. and Watson J., Healthcare interprofessional team members’ perspectives on human caring: A directed content analysis study. International Journal of Nursing Sciences, 2019. 6(1): p. 17–23. 28. Albert I. and Trommsdorff G., The Role of Culture in Social Development Over the Life Span: An Inter- personal Relations Approach. Online Readings in Psychology and Culture, 2014. 6: p. PLOS ONE | https://doi.org/10.1371/journal.pone.0278240 December 16, 2022 References 1. 29. Elder G.H., Families, Social Change, and Individual Lives. Marriage & Family Review, 2001. 31(1–2): p. 187–203. 19 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0278240 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- 19 with respect to perceived valence and self-efficacy. Curr Psychol, 2022: p. 1–17. 33. Bayoumi A.W.P.M.D.F.N.M.K.T., Indigenous Strength: Braiding Culture, Ceremony and Community as 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: Canada. 35. Heinz W.R. and Kru¨ger H., Life Course: Innovations and Challenges for Social Research. Current Soci- ology, 2001. 49(2): p. 29–45. 36. Macmillan R., The Structure of the Life Course: Classic Issues and Current Controversies. Advances in Life Course Research, 2005. 9: p. 3–24. 37. Pimental K., Mendoza N., Keeney A.J., Clarke L., Building community resilience toolkit series: Volume I. 2018, San Diego State University, Social Policy INstitute: San Diego, California. 38. Resilient Communities. [cited 2021 December 19]; Available from: https://publichealth.gwu.edu/ departments/redstone-center/resilient-communities. 39. Pritchard T., Kingston, Ont., area now has highest rate of COVID-19 cases in Canada, in CBC news. 2021. 20 / 20
https://openalex.org/W2305819264
https://europepmc.org/articles/pmc4800430?pdf=render
English
null
Collective atomic scattering and motional effects in a dense coherent medium
Nature communications
2,016
cc-by
10,220
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 (2014). 32. van Rossum, M. C. W. & Nieuwenhuizen, T. M. Multiple scattering of classical waves: microscopy, mesoscopy, and diffusion. Rev. Mod. Phys. 71, 313–371 (1999). 33. Walker, T., Sesko, D. & Wieman, C. Collective behaviour of optically trapped neutral atoms. Phys. Rev. Lett. 64, 408–411 (1990). 60. Mu¨ller, C. A. & Miniatura, C. Multiple scattering of light by atoms with internal degeneracy. J. Phys. A Math. Gen. 35, 10163–10188 (2002). y 34. Heidemann, R. et al. Evidence for coherent collective Rydberg excitation in the strong blockade regime. Phys. Rev. Lett. 99, 163601 (2007). 61. Allen, L. & Eberly, J. H. Dover Books on Physics (Dover Publications, 1987). g g 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 (2014). 36. Saffman, M., Walker, T. G. & Mølmer, K. Quantum information with Rydberg 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 59, 2427–2441 (1999). y 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 Vol 61 (eds Berman, P., Arimondo, E. & Lin, C.) Ch. 6, 295–329 (Academic Press, 2012). 38. Lo¨w, R. et al. An experimental and theoretical guide to strongly interacting Rydberg gases. J. Phys. B At. Mol. Opt. Phys. 45, 113001 (2012). 39. Peyronel, T. et al. Quantum nonlinear optics with single photons enabled by strongly interacting atoms. Nature 488, 57–60 (2012). 65. Friedberg, R., Hartmann, S. & Manassah, J. ARTICLE Frequency shifts in emission and absorption by resonant systems ot two-level atoms. Phys. Rep. 7, 101–179 (1973). 40. Ravets, S. et al. Coherent dipole-dipole coupling between two single Rydberg atoms at an electrically-tuned fo¨rster resonance. Nat. Phys. 10, 914–917 (2014). 66. Julienne, P. S. Cold binary atomic collisions in a light field. J. Res. Natl Inst. Stand. Technol. 101, 487–503 (1996). 41. Gu¨nter, G. et al. Observing the dynamics of dipole-mediated energy transport by interaction-enhanced imaging. Science 342, 954–956 (2013). 42. Chang, D. E., Ye, J. & Lukin, M. D. Controlling dipole-dipole frequency shifts in a lattice-based optical atomic clock. Phys. Rev. A 69, 023810 (2004). Acknowledgements 43. Kus´, M. & Wo´dkiewicz, K. Two-atom resonance fluorescence. Phys. Rev. A 23, 853–857 (1981). ˙ We are grateful to Paul Julienne, Chris Greene, John Cooper and Murray Holland for their important insights and stimulating discussions. This research is supported by NIST, NSF Physics Frontier Center at JILA, AFOSR, AFOSR-MURI, ARO, DARPA QuASAR, 44. Z˙akowicz, W. Superradiant decay of a small spherical system of harmonic oscillators. Phys. Rev. A 17, 343–352 (1978). NSF Center for Ultracold Atoms at Harvard-MIT, ITAMP and ANR-14-CE26-0032. 45. Sutherland, R. T. & Robicheaux, F. Coherent forward broadening in cold atom clouds. Phys. Rev. A 93, 023407 (2016). Author contributions 46. Nicholson, T. L. et al. Systematic evaluation of an atomic clock at 2  10  18 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
https://openalex.org/W2152887453
https://smw.ch/index.php/smw/article/download/1321/1526
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 Treated With Bevacizumab. J Clin Oncol. 2011; 29(6):632–9. Clinical observation study Evaluation of a Platelet Function Analyser (PFA-100) in patients with a bleeding tend- ency. Swiss Med Wkly. 2002;132(31-32):443–8. 15 Miles D, Chan A, Romieu G Dirix LY, Cortes J, Pivot X, et al. Ran- domized, double-blind, placebo-controlled, phase II study of bevaci- zumab with docetaxel or docetaxel with placebo as first-line therapy for patients with locally recurrent or metastatic breast cancer (mBC): AVADO. J Clin Oncol. 2008;26: ASCO Ann Met Sup LBA 1011. Funding / potential competing interests: The work has 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 competing financial interests. 16 Karger R, Donner-Banzhoff N, Mueller HH, Kretschmer V, Hunink M. Diagnostic performance of the platelet function analyzer (PFA-100) for the detection of disorders of the primary haemostasis in patients with a bleeding history – a systematic review and meta-analysis. Platelets. 2007;18(4):249–60. 17 Marshall PW, Williams AJ, Dixon RM, Growcott JW, Warburton S, Armstrong J, et al. A comparison of the effects of aspirin on bleeding time measured using the Simplate method and closure time measured using the PFA-100 in healthy volunteers. Br J Clin Pharmacol. 1997;44(2):151–5. 18 Franchini M. The platelet-function analyzer (PFA-100) for evaluating primary hemaostasis. Hematology. 2005;10(3):177–81. Correspondence: Dr. Roger von Moos, Division of Oncology, Department of Medicine, Kantonsspital Graubünden, Loéstrasse 170, CH-7000 Chur, Switzerland, Correspondence: Dr. Roger von Moos, Division of Oncology, Department of Medicine, Kantonsspital Graubünden, Loéstrasse 170, CH-7000 Chur, Switzerland, 19 Favaloro EJ. Clinical application of the PFA-100. Curr Opin Hematol. 2002;9(5):407–15. 20 Mammen EF, Comp PC, Gosselin R, Greenberg C, Hoots WK, Kessler CM, et al. PFA-100 system: a new method for assassement of platelet dysfunction. Semin Thromb Hemost. 1998;24(2):195–202. 1 Miller K, Wang M, Gralow J, Dickler M, Cobleigh M, Perez EA et al. Paclitaxel plus bevacizumab versus Paclitaxel alone for metastatic breast cancer. N Engl J Med. 2007;357(26):2666–6. References 21 Fressinaud E, Veyradier A, Truchund F, Martin I, Boyer-Neumann C, Trossaert M, et al. Screening for von Willebrand disease with a new analyzer using high shear stress: A study of 60 cases. Blood. 1998;91(4):1325–31. 1 Miller K, Wang M, Gralow J, Dickler M, Cobleigh M, Perez EA et al. Paclitaxel plus bevacizumab versus Paclitaxel alone for metastatic breast cancer. N Engl J Med. 2007;357(26):2666–6. 1 Miller K, Wang M, Gralow J, Dickler M, Cobleigh M, Perez EA et al. Paclitaxel plus bevacizumab versus Paclitaxel alone for metastatic breast cancer. N Engl J Med. 2007;357(26):2666–6. 22 Borzini P, Lazzaro A, Mazzucco L. Evaluation of the haemostatic func- tion of stored platelet concentrates using the platelet function analyzer (PFA-100). Haematologica. 1999;84(12):1104–9. 2 Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al. Paxlitaxel-Carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med. 2006;355(24):2542–50. 2 Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al. Paxlitaxel-Carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med. 2006;355(24):2542–50. 23 Galliard-Grigioni KS, Fehr M, Reinhart WH. Influence of combinations of acetylsalicylic acid, acetamino-phen and diclofenac on platelet ag- gregation. Eur J Pharmacol. 2008;595(1-3):65–8. 3 Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al. Bevacizumab plus irino-tecan, fluoruracil and leu- covorin for metastatic colorectal cancer. N Engl J Med. 2004;350(23):2335–42. 3 Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al. Bevacizumab plus irino-tecan, fluoruracil and leu- covorin for metastatic colorectal cancer. N Engl J Med. 2004;350(23):2335–42. Swiss Medical Weekly · PDF of the online version · www.smw.ch Page 5 of 6 Original article Swiss Med Wkly. 2011;141:w13243 24 Lehmann T, Mairbäurl H, Pleisch B, Maggiorini M, Bärtsch P, Reinhart WH. Platelet count and function at high altitude and in high-altitude pulmonary oedema. J Appl Physiol. 2006;100(2):690–4. 34 Wu G, Li F, Li P, Ruan C. Detection of plasma alpha-granule membrane protein GMP-140 using radio-labeled monoclonal antibodies in throm- botic diseases. Haemostasis. 1993;23(2):121–8. 25 Pontiggia L, Lassila R, Pederiva S, Schmid HR, Burger M, Beer JH. Increased platelet-collagen interaction associated with double homozy- gosity for receptor polymorphisms of platelet GPIa and GPIIIa. Arteri- oscler Thromb Vasc Biol. 2002;22(12):2093–8. 35 Blann AD, Dobrotova M, Kubisz P, McCollum CN. von Willebrand factor, soluble P-selectin, tissue plasminogen activator and plasmino- gen activator inhibitor in atherosclerosis. References Thromb Haemost. 1995;74(2):626–30. 36 Smith A, Quarnby JW, Collins M, Lockhart SM, Burnand KG. Changes in the levels of soluble adhesion molecules and coagulation factors in patients with deep vein thrombosis. Thromb Hae- most.1999;82(6):1593–9. 26 Lanza GA, Sestito A, Iacovella S, Morlacchi L, Romagnoli E, Schiavoni G, et al. Relation between platelet response to exercise and coronary angiographic findings in patients with effort angina. Circula- tion. 2003;107(10):1378–82. 27 Mimidis K, Papadopoulos V, Kartasis Z, Baka M, Tsatlidis V, Bourikas G, et al. Assessment of platelet adhesiveness and aggregation in mild acute pancreatitis using the PFA-100 system. JOP. 2004;5(3):132–7. 37 André P, Hartwell D, Hrachovinova I, Saffaripour S, Wagner DD. Pro- 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 ischemic 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- ol. 1996;93(2):432–6. 33 Chong BH, Murray B, Berndt MC, Dunlop LC, Brighton T, Chester- mann CN. Swiss Medical Weekly · PDF of the online version · www.smw.ch References Plasma P-selectin is increased in thrombotic consumptive platelet disorders. Blood. 1994;83(6):1535–41. Swiss Medical Weekly · PDF of the online version · www.smw.ch Page 6 of 6
https://openalex.org/W4281950537
https://www.nature.com/articles/s41467-022-30710-z.pdf
English
null
The androgen receptor is a therapeutic target in desmoplastic small round cell sarcoma
Nature communications
2,022
cc-by
14,963
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 breakpoint and chimeric transcripts in the EWS-WT1 gene fusion of desmoplastic small round cell tumor. Proc. Natl Acad. Sci. USA 92, 1028–1032 (1995). 4. Gerald, W. L. et al. Clinical, pathologic, and molecular spectrum of tumors associated with t(11;22)(p13;q12): desmoplastic small round-cell tumor and its variants. J. Clin. Oncol. 16, 3028–3036 (1998). ChIP-seq data processing. ChIP-seq data were quality controlled and processed 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 desmoplastic small round cell tumor: results from the surveillance, epidemiology, and end results database. J. cancer Epidemiol. 2014, 680126 (2014). 6. Gerald, W. L. & Rosai, J. Case 2. Desmoplastic small cell tumor with divergent differentiation. Pediatr. Pathol. 9, 177–183 (1989). 7. Shen, W. P., Towne, B. & Zadeh, T. M. Cytogenetic abnormalities in an intraabdominal desmoplastic small cell tumor. Cancer Genet Cytogenet 64, 189–191 (1992). 8. Ladanyi, M. & Gerald, W. Fusion of the EWS and WT1 genes in the desmoplastic small round cell tumor. Cancer Res. 54, 2837–2840 (1994). Differential peaks analysis. To identify variable AR or enhancer domains enriched 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 translocation t(11;22)(p13;q12) in an intraabdominal desmoplastic small 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 60. Michmerhuizen, A. R., Spratt, D. E., Pierce, L. J. & Speers, C. W. ARe we there yet? Understanding androgen receptor signaling in breast cancer. NPJ Breast Cancer 6, 47 (2020). 31. Zarif, J. C., Lamb, L. E., Schulz, V. V., Nollet, E. A. & Miranti, C. K. Androgen receptor non-nuclear regulation of prostate cancer cell invasion mediated by Src and matriptase. Oncotarget 6, 6862–6876 (2015). 61. Fong, E. L. S. et al. Modeling Ewing sarcoma tumors in vitro with 3D scaffolds. Proc. Natl Acad. Sci. USA 110, 6500–6505 (2013). g 32. Taylor, B. S. et al. Integrative genomic profiling of human prostate cancer. Cancer Cell 18, 11–22 (2010). 62. Lamhamedi-Cherradi, S. E. et al. IGF-1R and mTOR blockade: novel resistance mechanisms and synergistic drug combinations for Ewing sarcoma. J. Natl Cancer Inst. 108, djw182. https://doi.org/10.1093/jnci/djw182 (2016). 33. Dasgupta, S., Lonard, D. M. & O’Malley, B. W. Nuclear receptor coactivators: master regulators of human health and disease. Annu Rev. Med. 65, 279–292 (2014). 63. Puerto-Camacho, P. et al. Preclinical efficacy of endoglin-targeting antibody- drug conjugates for the treatment of Ewing sarcoma. Clin. Cancer Res. 25, 2228–2240 (2019). 34. Dasgupta, S. et al. Coactivator SRC-2-dependent metabolic reprogramming mediates prostate cancer survival and metastasis. J. Clin. Invest. 125, 1174–1188 (2015). 64. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013). 35. Foley, C. & Mitsiades, N. Moving beyond the androgen receptor (AR): targeting AR-interacting proteins to treat prostate cancer. Horm. Cancer 7, 84–103 (2016). 65. Anders, S., Pyl, P. T. & Huber, W. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015). 36. Agoulnik, I. U. et al. Role of SRC-1 in the promotion of prostate cancer cell growth and tumor progression. Cancer Res. 65, 7959–7967 (2005). 66. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). 37. Yan, J. et al. Steroid receptor coactivator-3/AIB1 promotes cell migration and invasiveness through focal adhesion turnover and matrix metalloproteinase expression. Cancer Res. 68, 5460–5468 (2008). f 67. Ritchie, M. E. et al. limma powers differential expression analyses for RNA- sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015). 38. Kemppainen, J. A., Lane, M. V., Sar, M. & Wilson, E. M. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J. ARTICLE Neoplasia 22, 111–119 (2020). 52. Shaffer, P. L., Jivan, A., Dollins, D. E., Claessens, F. & Gewirth, D. T. Structural basis of androgen receptor binding to selective androgen response elements. Proc. Natl Acad. Sci. USA 101, 4758–4763 (2004). 24. Holly, S. P., Larson, M. K. & Parise, L. V. Multiple roles of integrins in cell motility. Exp. Cell Res. 261, 69–74 (2000). 53. Pihlajamaa, P. et al. Tissue-specific pioneer factors associate with androgen receptor cistromes and transcription programs. EMBO J. 33, 312–326 (2014). y p 25. Kechagia, J. Z., Ivaska, J. & Roca-Cusachs, P. Integrins as biomechanical sensors of the microenvironment. Nat. Rev. Mol. Cell Biol. 20, 457–473 (2019). 54. Sahu, B. et al. FoxA1 specifies unique androgen and glucocorticoid receptor binding events in prostate cancer cells. Cancer Res. 73, 1570–1580 (2013). 26. Hornebeck, W., Emonard, H., Monboisse, J. C. & Bellon, G. Matrix-directed regulation of pericellular proteolysis and tumor progression. Semin Cancer Biol. 12, 231–241 (2002). 55. Sahu, B. et al. Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer. EMBO J. 30, 3962–3976 (2011). 27. Antonarakis, E. S. et al. Androgen receptor splice variant, AR-V7, and resistance to enzalutamide and abiraterone in men with metastatic castration- resistant prostate cancer (mCRPC). J. Clin. Oncol. 32, https://doi.org/10.1200/ jco.2014.32.15_suppl.5001 (2014). 56. Mirosevich, J. et al. Expression and role of Foxa proteins in prostate cancer. Prostate 66, 1013–1028 (2006). 57. Hingorani, P. et al. Transcriptome analysis of desmoplastic small round cell tumors identifies actionable therapeutic targets: a report from the Children’s Oncology Group. Sci. Rep. 10, 12318 (2020). j pp 28. Paschalis, A. et al. Alternative splicing in prostate cancer. Nat. Rev. Clin. Oncol. 15, 663–675 (2018). gy p p 58. Edlind, M. P. & Hsieh, A. C. PI3K-AKT-mTOR signaling in prostate cancer progression and androgen deprivation therapy resistance. Asian J. Androl. 16, 378–386 (2014). 29. Qu, Y. Y. et al. Constitutively active AR-V7 plays an essential role in the development and progression of castration-resistant prostate cancer. Sci. Rep. 5, 7654 (2015). 59. Qi, W. et al. Reciprocal feedback inhibition of the androgen receptor and PI3K as a novel therapy for castrate-sensitive and -resistant prostate cancer. Oncotarget 6, 41976–41987 (2015). 30. Lamb, L. E., Zarif, J. C. & Miranti, C. K. The androgen receptor induces integrin alpha6beta1 to promote prostate tumor cell survival via NF-kappaB and Bcl-xL Independently of PI3K signaling. Cancer Res. 71, 2739–2749 (2011). ARTICLE ARTICLE ARTICLE 16. Efstathiou, E. et al. Molecular characterization of enzalutamide-treated bone metastatic castration-resistant prostate cancer. Eur. Urol. 67, 53–60 (2015). 44. Takeda, D. Y. et al. A somatically acquired enhancer of the androgen receptor is a noncoding driver in advanced prostate cancer. Cell 174, 422–432.e413 (2018). p 17. Labrie, M. et al. Using reverse phase protein array (RPPA) to identify and target adaptive resistance. Adv. Exp. Med Biol. 1188, 251–266 (2019). 45. Jin, L. et al. Therapeutic targeting of the CBP/p300 bromodomain blocks the growth of castration-resistant prostate cancer. Cancer Res. 77, 5564–5575 (2017). 18. Akbani, R. et al. Realizing the promise of reverse phase protein arrays for clinical, translational, and basic research: a workshop report: the RPPA (Reverse Phase Protein Array) society. Mol. Cell Proteom. 13, 1625–1643 (2014). 46. Bradner, J. E., Hnisz, D. & Young, R. A. Transcriptional addiction in cancer. Cell 168, 629–643 (2017). 19. Mitra, R., Mueller, P., Ji, Y., Mills, G. & Lu, Y. Sparse Bayesian graphical models for RPPA time course data. IEEE Int Workshop Genom. Signal Process Stat. 2012, 113–117 (2012). 47. Huang, J. et al. Dissecting super-enhancer hierarchy based on chromatin interactions. Nat. Commun. 9, 943 (2018). 48. Khan, A. & Zhang, X. Integrative modeling reveals key chromatin and sequence signatures predicting super-enhancers. Sci. Rep. 9, 2877 (2019). 20. Carceles-Cordon, M. et al. Cellular rewiring in lethal prostate cancer: the architect of drug resistance. Nat. Rev. Urol. 17, 292–307. https://doi.org/10. 1038/s41585-020-0298-8 (2020). 49. Menegaz, B. A. et al. Clinical activity of pazopanib in patients with advanced desmoplastic small round cell tumor. Oncologist 23, 360–366 (2018). 21. Wang, G., Zhao, D., Spring, D. J. & DePinho, R. A. Genetics and biology of prostate cancer. Genes Dev. 32, 1105–1140 (2018). 50. Modak, S., Kramer, K., Gultekin, S. H., Guo, H. F. & Cheung, N. K. Monoclonal antibody 8H9 targets a novel cell surface antigen expressed by a wide spectrum of human solid tumors. Cancer Res. 61, 4048–4054 (2001). 22. Kregel, S. et al. Differential modulation of the androgen receptor for prostate cancer therapy depends on the DNA response element. Nucleic Acids Res. 48, 4741–4755. https://doi.org/10.1093/nar/gkaa178 (2020). p 51. Bulbul, A. et al. Desmoplastic small round blue cell tumor: a review of treatment and potential therapeutic genomic alterations. Sarcoma 2017, 1278268 (2017). p g g 23. Kregel, S. et al. Androgen receptor degraders overcome common resistance mechanisms developed during prostate cancer treatment. References 16, 411–416 (1992). g 10. Gedminas, J. M. et al. Desmoplastic small round cell tumor is dependent on the EWS-WT1 transcription factor. Oncogenesis 9, 41 (2020). Identification of AR and enhancer associated pathways. Differential AR binding sites and enhancers associated genes in each sample were imported into the ClusterProfiler80 for pathway analysis, restricted to GO, KEGG, Hallmark, and WiKi gene sets. The Enrichplot package81 was used to generate dot plot and bar plot for gene sets enriched with a false discovery rate cut-off of <0.05. 11. Thomas, C. et al. Synergistic targeting of PI3K/AKT pathway and androgen receptor axis significantly delays castration-resistant prostate cancer progression in vivo. Mol. Cancer Ther. 12, 2342–2355 (2013). p g 12. Chan, J. J., Tan, T. J. Y. & Dent, R. A. Novel therapeutic avenues in triple- negative breast cancer: PI3K/AKT inhibition, androgen receptor blockade, and beyond. Ther. Adv. Med Oncol. 11, 1758835919880429 (2019). Enrichment of motifs in AR-specific peaks. To identify the motifs over- represented within AR-specific peak sets, we used the HOMER motif database and the coordinates of AR-specific peak sets82. Enrichment of motifs in AR-specific peaks. To identify the motifs over- represented within AR-specific peak sets, we used the HOMER motif database and the coordinates of AR-specific peak sets82. 13. Subbiah, V. et al. Multimodality treatment of desmoplastic small round cell tumor: chemotherapy and complete cytoreductive surgery improve patient survival. Clin. Cancer Res. 24, 4865–4873 (2018). 14. Mora, J. et al. Desmoplastic small round cell tumor 20 years after its discovery. Future Oncol. 11, 1071–1081 (2015). Statistics and reproducibility. Bar and scatter dot plots are presented as mean values ± SD. Box plots indicate the median value and interquartile range; whiskers show minimum and maximum data points. All t-test p values are two-sided. To enhance reproducibility, no less than five animals were assigned per group. Three 15. Fine, R. L. et al. Androgen and c-Kit receptors in desmoplastic small round cell tumors resistant to chemotherapy: novel targets for therapy. Cancer Chemother. Pharmacol. 59, 429–437 (2007). 15. Fine, R. L. et al. Androgen and c-Kit receptors in desmoplastic small round cell tumors resistant to chemotherapy: novel targets for therapy. Cancer Chemother. Pharmacol. 59, 429–437 (2007). 14 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 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30710-z 75. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011). N.C.D., B.C., D.M., A.H.-J., R.R., J.A.Li., and J.A.Lu. helped identify the enrolled DSRCT patients in the study. B.U. assisted with confocal microscopy. W.-L.W. and A.J.L. pro- vided pathology interpretations. C.R.-A. assisted with preclinical in vivo experiments. M.T. performed the mass spectroscopy analysis. C.-C.W., H.C.B., and P.A.F. performed the transcriptomic analysis. N.E.A., M.M., and K.R. performed the ChIP-seq experiments and aided in their interpretation. All authors participated in data interpretation and manuscript editing. 76. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008). 77. Yu, G., Wang, L. G. & He, Q. Y. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 31, 2382–2383 (2015). 78. Loven, J. et al. Selective inhibition of tumor oncogenes by disruption of super- enhancers. Cell 153, 320–334 (2013). Competing interests 79. Khan, A. & Mathelier, A. Intervene: a tool for intersection and visualization of multiple gene or genomic region sets. BMC Bioinforma. 18, 287 (2017). A.R.M. is an employee and shareholder of Ionis Pharmaceuticals. The remaining authors declare no competing interests. 80. Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012). comparing biological themes among gene clusters. OMICS 16, 284–287 (2012). Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements Investigators at The University of Texas MD Anderson Cancer Center are supported by the National Institutes of Health through Cancer Center Support Grant CA016672. The Characterized Cell Line Core Facility is funded by the NIH [CA016672]. K.R. and M.M. were supported by grants from the NCI (CA222214, CA245395, CA226269), Cancer Prevention Research Institute of Texas (CPRIT; RP220410 and RP200390), and MDACC intramural funds. P.A.F. is supported by the Cancer Prevention Research Institute (R120501) and the Welch Foundation’s Robert A. Welch Distinguished University Chair Award (G-0040). We thank the Cory Monzingo Foundation and Blake Abercrombie Foundation for their generous philanthropy that supported this work. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. ARTICLE Biol. Chem. 267, 968–974 (1992). 68. Mootha, V. K. et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet 34, 267–273 (2003). 39. Efstathiou, E. et al. Enzalutamide in combination with abiraterone acetate in bone metastatic castration-resistant prostate cancer patients. Eur. Urol. Oncol. 3, 119–127 (2020). 69. Wang, K. et al. MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res. 38, e178 (2010). 70. Terranova, C. et al. An integrated platform for genome-wide mapping of chromatin states using high-throughput ChIP-sequencing in tumor tissues. J Vis. Exp. 56972. https://doi.org/10.3791/56972 (2018). 40. Maity, S. N. et al. Targeting of CYP17A1 Lyase by VT-464 inhibits adrenal and intratumoral androgen biosynthesis and tumor growth of castration resistant prostate cancer. Sci. Rep. 6, 35354 (2016). 71. Tang, M. pyflow-ChIPseq: a snakemake based ChIP-seq pipeline. Zenodo (2017). 41. Courtney, K. D., Corcoran, R. B. & Engelman, J. A. The PI3K pathway as drug target in human cancer. J. Clin. Oncol. 28, 1075–1083 (2010). 72. Koster, J. & Rahmann, S. Snakemake-a scalable bioinformatics workflow engine. Bioinformatics 28, 2520–2522 (2012). 42. Carver, B. S. et al. Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer. Cancer Cell 19, 575–586 (2011). 73. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory- efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009). 43. Gerhardt, J. et al. FOXA1 promotes tumor progression in prostate cancer and represents a novel hallmark of castration-resistant prostate cancer. Am. J. Pathol. 180, 848–861 (2012). 74. Ramirez, F. et al. deepTools2: a next generation web server for deep- sequencing data analysis. Nucleic acids Res. 44, W160–W165 (2016). 15 NATURE COMMUNICATIONS | (2022) 13:3057 | https://doi.org/10.1038/s41467-022-30710-z | www.nature.com/naturecommunications Additional information 81. Yu, G. enrichplot: visualization of functional enrichment result. R package version 1.6.1. https://github.com/GuangchuangYu/enrichplot (2019). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-022-30710-z. 82. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010). Correspondence and requests for materials should be addressed to Salah-Eddine Lamhamedi-Cherradi, Kunal Rai or Joseph A. Ludwig. Correspondence and requests for materials should be addressed to Salah-Eddine Lamhamedi-Cherradi, Kunal Rai or Joseph A. Ludwig. 83. Subudhi, S. K. et al. Neoantigen responses, immune correlates, and favorable outcomes after ipilimumab treatment of patients with prostate cancer. Sci. Transl. Med. 12, eaaz3577. https://doi.org/10.1126/scitranslmed.aaz3577 (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
https://openalex.org/W2043680195
https://www.hal.inserm.fr/inserm-01907371/file/journal.pgen.1003971.pdf
English
null
Roles of XRCC2, RAD51B and RAD51D in RAD51-Independent SSA Recombination
PLOS genetics
2,013
cc-by
8,810
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 recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- 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 mean. References 13. Shor E, Weinstein J, Rothstein R (2005) A genetic screen for top3 suppressors in Saccharomyces cerevisiae identifies SHU1, SHU2, PSY3 and CSM2: four genes involved in error-free DNA repair. Genetics 169: 1275–1289. 1. Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, Sandler SJ, et al. (2000) The importance of repairing stalled replication forks. Nature 404: 37–41. 1. Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, Sandler SJ, et al. (2000) The importance of repairing stalled replication forks. Nature 404: 37–41. 2. Whitaker SJ (1992) DNA damage by drugs and radiation: what is important and how is it measured? European journal of cancer 28: 273–276. 2. Whitaker SJ (1992) DNA damage by drugs and radiation: what is important and how is it measured? European journal of cancer 28: 273–276. p 14. Mankouri HW, Ngo HP, Hickson ID (2007) Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3. Mol Biol Cell 18: 4062–4073. 3. Heyer W-D, Ehmsen KT, Liu J (2010) Regulation of homologous recombina- tion in eukaryotes. Annual review of genetics 44: 113–139. g p 15. Godin S, Wier A, Kabbinavar F, Bratton-Palmer DS, Ghodke H, et al. (2013) The Shu complex interacts with Rad51 through the Rad51 paralogues Rad55- Rad57 to mediate error-free recombination. Nucleic acids research 41: 4525– 4534. 4. Waterworth WM, Drury GE, Bray CM, West CE (2011) Repairing breaks in the plant genome: the importance of keeping it together. The New phytologist 192: 805–822. 5. Masson JY, Tarsounas MC, Stasiak AZ, Stasiak A, Shah R, et al. (2001) Identification and purification of two distinct complexes containing the five RAD51 paralogs. Genes & development 15: 3296–3307. 16. She Z, Gao ZQ, Liu Y, Wang WJ, Liu GF, et al. (2012) Structural and SAXS analysis of the budding yeast SHU-complex proteins. FEBS Lett 586: 2306–2312. 17. Tao Y, Li X, Liu Y, Ruan J, Qi S, et al. (2012) Structural analysis of Shu proteins reveals a DNA binding role essential for resisting damage. J Biol Chem 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) Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA- catalyzed DNA strand exchange. Genes Dev 15: 3308–3318. y g 71. Tarsounas M, Munoz P, Claas A, Smiraldo PG, Pittman DL, et al. (2004) Telomere maintenance requires the RAD51D recombination/repair protein. Cell 117: 337–347. 44. Da Ines O, Degroote F, Amiard S, Goubely C, Gallego ME, et al. (2013) Effects of XRCC2 and RAD51B mutations on somatic and meiotic recombination in Arabidopsis thaliana. The Plant Journal 74: 959–970. p J 45. Liu Y, Masson J-Y, Shah R, O’Regan P, West SC (2004) RAD51C is required for Holliday junction processing in mammalian cells. Science 303: 243–246. 72. Lin FL, Sperle K, Sternberg N (1984) Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process. Molec Cell Biol 4: 1020–1034. 46. Liu Y, Tarsounas M, O’Regan P, West SC (2007) Role of RAD51C and XRCC3 in genetic recombination and DNA repair. The Journal of biological chemistry 282: 1973–1979. 73. Prado F, Aguilera A (1995) Role of reciprocal exchange, one-ended invasion crossover and single-strand annealing on inverted and direct repeat recombi- nation in yeast: different requirements for the RAD1, RAD10, and RAD52 genes. Genetics 139: 109–123. 47. Nagaraju G, Hartlerode A, Kwok A, Chandramouly G, Scully R (2009) XRCC2 and XRCC3 regulate the balance between short- and long-tract gene conversions between sister chromatids. Molecular and cellular biology 29: 4283–4294. g 74. Bleuyard J-Y, Gallego ME, Savigny F, White CI (2005) Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair. The Plant Journal 41: 533–545. 48. Rodrigue A, Coulombe Y, Jacquet K, Gagne JP, Roques C, et al. (2013) The RAD51 paralogs ensure cellular protection against mitotic defects and aneuploidy. J Cell Sci 126: 348–359. 75. Fauser F, Roth N, Pacher M, Ilg G, Sa´nchez-Ferna´ndez R, et al. (2012) In planta gene targeting. Proceedings of the National Academy of Sciences of the United States of America 109: 7535–7540. p y 49. Compton SA, Ozgur S, Griffith JD (2010) Ring-shaped Rad51 paralog protein complexes bind Holliday junctions and replication forks as visualized by electron microscopy. J Biol Chem 285: 13349–13356. 76. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high- throughput functional analysis of genes in planta. Plant physiology 133: 462– 469. 50. RAD51 Paralogues Act in SSA Recombination Orel N, Kyryk A, Puchta H (2003) Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome. The Plant Journal 35: 604–612. g 31. Shu Z, Smith S, Wang L, Rice MC, Kmiec EB (1999) Disruption of muREC2/ RAD51L1 in mice results in early embryonic lethality which can Be partially rescued in a p53(-/-) background. Molecular and cellular biology 19: 8686–8693. 59. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, et al. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic acids research 39: e82. p ( ) g gy 32. Bleuyard J-Y, White CI (2004) The Arabidopsis homologue of Xrcc3 plays an essential role in meiosis. The EMBO journal 23: 439–449. 60. Heacock M, Spangler E, Riha K, Puizina J, Shippen DE (2004) Molecular analysis of telomere fusions in Arabidopsis: multiple pathways for chromosome end-joining. The EMBO journal 23: 2304–2313. 33. Li W, Yang X, Lin Z, Timofejeva L, Xiao R, et al. (2005) The AtRAD51C gene is required for normal meiotic chromosome synapsis and double-stranded break repair in Arabidopsis. Plant physiology 138: 965–976. j g j 61. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, et al. (2011) A TALE nuclease architecture for efficient genome editing. Nature biotechnology 29: 143–148. p p p y gy 34. Liu N, Schild D, Thelen MP, Thompson LH (2002) Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells. Nucleic acids research 30: 1009–1015. 62. Puchta H (2005) The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. Journal of experimental botany 56: 1–14. 35. Miller KA, Sawicka D, Barsky D, Albala JS (2004) Domain mapping of the Rad51 paralog protein complexes. Nucleic acids research 32: 169–178. 63. Salomon S, Puchta H (1998) Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. The EMBO journal 17: 6086– 6095. 36. Miller KA, Yoshikawa DM, McConnell IR, Clark R, Schild D, et al. (2002) RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51. The Journal of biological chemistry 277: 8406–8411. 64. Malkova A, Ivanov EL, Haber JE (1996) Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. RAD51 Paralogues Act in SSA Recombination Proc Natl Acad Sci U S A 93: 7131–7136. J g y 37. Osakabe K, Abe K, Yamanouchi H, Takyuu T, Yoshioka T, et al. (2005) Arabidopsis Rad51B is important for double-strand DNA breaks repair in somatic cells. Plant molecular biology 57: 819–833. 65. Watanabe K, Pacher M, Dukowic S, Schubert V, Puchta H, et al. (2009) The Structural Maintenance of Chromosomes 5/6 complex promotes sister chromatid alignment and homologous recombination after DNA damage in Arabidopsis thaliana. Plant Cell 21: 2688–2699. gy 38. Osakabe K, Yoshioka T, Ichikawa H, Toki S (2002) Molecular cloning and characterization of RAD51-like genes from Arabidopsis thaliana. Plant molecular biology 50: 71–81. gy 39. Schild D, Lio YC, Collins DW, Tsomondo T, Chen DJ (2000) Evidence for simultaneous protein interactions between human Rad51 paralogs. The Journal of biological chemistry 275: 16443–16449. 66. Da Ines O, Degroote F, Goubely C, Amiard S, Gallego ME, et al. (2013) Meiotic Recombination in Arabidopsis Is Catalysed by DMC1, with RAD51 Playing a Supporting Role. PLoS genetics 9: e1003787. 40. Wiese C, Collins DW, Albala JS, Thompson LH, Kronenberg A, et al. (2002) Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells. Nucleic acids research 30: 1001–1008. 67. Yonetani Y, Hochegger H, Sonoda E, Shinya S, Yoshikawa H, et al. (2005) Differential and collaborative actions of Rad51 paralog proteins in cellular response to DNA damage. Nucleic acids research 33: 4544–4552. 41. Badie S, Liao C, Thanasoula M, Barber P, Hill MA, et al. (2009) RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation. The Journal of cell biology 185: 587–600. 68. Lio YC, Mazin AV, Kowalczykowski SC, Chen DJ (2003) Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro. J Biol Chem 278: 2469–2478. J 69. Kurumizaka H, Ikawa S, Nakada M, Enomoto R, Kagawa W, et al. (2002) Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex. J Biol Chem 277: 14315–14320. J gy 42. Brenneman MA, Wagener BM, Miller CA, Allen C, Nickoloff JA (2002) XRCC3 controls the fidelity of homologous recombination: roles for XRCC3 in late stages of recombination. Molecular cell 10: 387–395. g 43. Chun J, Buechelmaier ES, Powell SN (2013) Rad51 paralog complexes BCDX2 and CX3 act at different stages in the BRCA1-BRCA2-dependent homologous recombination pathway. Molecular and cellular biology 33: 387–395. 70. RAD51 Paralogues Act in SSA Recombination 24. Johnson RD, Liu N, Jasin M (1999) Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination. Nature 401: 397–399. 52. Mansour WY, Schumacher S, Rosskopf R, Rhein T, Schmidt-Petersen F, et al. (2008) Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks. Nucleic Acids Res 36: 4088–4098. 25. Pierce AJ, Johnson RD, Thompson LH, Jasin M (1999) XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes & development 13: 2633–2638. 53. Roth N, Klimesch J, Dukowic-Schulze S, Pacher M, Mannuss A, et al. (2012) The requirement for recombination factors differs considerably between different pathways of homologous double-strand break repair in somatic plant cells. The Plant journal 72: 781–790. p 26. Liu N, Lamerdin JE, Tebbs RS, Schild D, Tucker JD, et al. (1998) XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages. Molecular cell 1: 783–793. 27. Tebbs RS, Zhao Y, Tucker JD, Scheerer JB, Siciliano MJ, et al. (1995) Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene. Proceedings of the National Academy of Sciences of the United States of America 92: 6354–6358. 54. Stark JM, Pierce AJ, Oh J, Pastink A, Jasin M (2004) Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol Cell Biol 24: 9305–9316. cloned cDNA of the XRCC3 DNA repair gene. Proceedings of the National Academy of Sciences of the United States of America 92: 6354–6358. 55. Krogh BO, Symington LS (2004) Recombination proteins in yeast. Annual review of genetics 38: 233–271. 28. Deans B, Griffin CS, Maconochie M, Thacker J (2000) Xrcc2 is required for genetic stability, embryonic neurogenesis and viability in mice. The EMBO journal 19: 6675–6685. 56. McDonald JP, Rothstein R (1994) Unrepaired heteroduplex DNA in Saccharomyces cerevisiae is decreased in RAD1 RAD52-independent recom- bination. Genetics 137: 393–405. 29. Kuznetsov SG, Haines DC, Martin BK, Sharan SK (2009) Loss of Rad51c leads to embryonic lethality and modulation of Trp53-dependent tumorigenesis in mice. Cancer Res 69: 863–872. 57. Mozlin AM, Fung CW, Symington LS (2008) Role of the Saccharomyces cerevisiae Rad51 paralogs in sister chromatid recombination. Genetics 178: 113–126. 30. Pittman DL, Schimenti JC (2000) Midgestation lethality in mice deficient for the RecA-related gene, Rad51d/Rad51l3. Genesis 26: 167–173. 58. References Liu T, Wan L, Wu Y, Chen J, Huang J (2011) hSWS1.SWSAP1 is an evolutionarily conserved complex required for efficient homologous recombi- nation repair. J Biol Chem 286: 41758–41766. p gy 8. Thacker J (2005) The RAD51 gene family, genetic instability and cancer. Cancer letters 219: 125–135. p J 19. Bleuyard J-Y, Gallego ME, White CI (2006) Recent advances in understanding of the DNA double-strand break repair machinery of plants. DNA repair 5: 1–12. 9. Liu J, Renault L, Veaute X, Fabre F, Stahlberg H, et al. (2011) Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation. Nature 479: 245–248. nshif Y, Bernstein KA (2012) From yeast to mammals: recent advances 20. Karpenshif Y, Bernstein KA (2012) From yeast to mammals: recent advances in genetic control of homologous recombination. DNA Repair (Amst) 11: 781–788. 21. Godthelp BC, Wiegant WW, van Duijn-Goedhart A, Scha¨rer OD, van Buul PPW, et al. (2002) Mammalian Rad51C contributes to DNA cross-link resistance, sister chromatid cohesion and genomic stability. Nucleic acids research 30: 2172–2182. 10. Bernstein KA, Reid RJD, Sunjevaric I, Demuth K, Burgess RC, et al. (2011) The Shu complex, which contains Rad51 paralogues, promotes DNA repair through inhibition of the Srs2 anti-recombinase. Molecular biology of the cell 22: 1599–1607. 22. Takata M, Sasaki MS, Tachiiri S, Fukushima T, Sonoda E, et al. (2001) Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Molecular and cellular biology 21: 2858– 2866. 11. Martin V, Chahwan C, Gao H, Blais V, Wohlschlegel J, et al. (2006) Sws1 is a conserved regulator of homologous recombination in eukaryotic cells. EMBO J 25: 2564–2574. 12. Sasanuma H, Tawaramoto MS, Lao JP, Hosaka H, Sanda E, et al. (2013) A new protein complex promoting the assembly of Rad51 filaments. Nature communications 4: 1676. 23. Takata M, Sasaki MS, Sonoda E, Fukushima T, Morrison C, et al. (2000) The 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 the human Rad51B, Rad51C, Rad51D and Xrcc2 protein complex. Nucleic acids research 32: 2556–2565. 77. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. The Plant Journal 16: 735– 743. 78. Gallego ME, White CI (2001) RAD50 function is essential for telomere maintenance in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 98: 1711–1716. 51. Ivanov EL, Sugawara N, Fishman-Lobell J, Haber JE (1996) Genetic 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
https://openalex.org/W4387221564
https://ojs3.unpatti.ac.id/index.php/barekeng/article/download/8001/6546
Latin
null
THE DEVELOPMENT OF COVID-19 USING OUTBREAK THE SUSCEPTIBLE, INFECTED, AND RECOVERED (SIR) MODEL WITH VACCINATION
Barekeng
2,023
cc-by-sa
9,262
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"
W4244054441.txt
https://www.qeios.com/read/M12YRC/pdf
tl
Lupus Anticoagulant Antibody
Definitions
2,020
cc-by
111
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. Qeios ID: M12YRC · https://doi.org/10.32388/M12YRC 1/1
https://openalex.org/W3201159009
https://aip.scitation.org/doi/pdf/10.1063/5.0067197
English
null
Achieving high spatial resolution in a large field-of-view using lensless x-ray imaging
Applied physics letters
2,021
cc-by
5,846
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- graphic imaging of 1 mm2 integrated circuit and zoomed-in detail. 11M. Odstrcˇil, M. Lebugle, T. Lachat, J. Raabe, and M. Holler, “Fast positioning for X-ray scanning microscopy by a combined motion of sample and beam- defining optics,” J. Synchrotron Radiat. 26, 504–509 (2019). g p y 12P. M. Pelz, M. Guizar-Sicairos, P. Thibault, I. Johnson, M. Holler, and A. Menzel, “On-the-fly scans for x-ray ptychography,” Appl. Phys. Lett. 105, 251101 (2014). We would like to thank Manuel Guizar-Sicairos for sharing the PtychoShelves package and providing helpful insights about ptychographic reconstruction. REFERENCES 22M. Odstrcˇil, P. Baksh, S. A. Boden, R. Card, J. E. Chad, J. G. Frey, and W. S. Brocklesby, “Ptychographic coherent diffractive imaging with orthogonal probe relaxation,” Opt. Express 24, 8360–8369 (2016). 1M. Holler, M. Guizar-Sicairos, E. H. R. Tsai, R. Dinapoli, E. M€uller, O. Bunk, J. Raabe, and G. Aeppli, “High-resolution non-destructive three-dimensional imaging of integrated circuits,” Nature 543, 402–406 (2017). , p p , ( ) 23A. Maiden and J. Rodenburg, “An improved ptychographical phase retrieval algorithm for diffractive imaging,” Ultramicroscopy 109, 1256–1262 (2009). 24 g g g , , ( ) 2M. Holler, M. O. M. Guizar-Sicairos, M. Lebugle, E. M€uller, S. Finizio, G. Tinti, C. David, J. Zusman, W. Unglaub, O. Bunk, J. Raabe, A. F. J. Levi, and G. Aeppli, “Three-dimensional imaging of integrated circuits with macro- to nanoscale zoom,” Nat. Electron. 2, 464–470 (2019). g g g 2M. Holler, M. O. M. Guizar-Sicairos, M. Lebugle, E. M€uller, S. Finizio, G. Tinti, , , g , , , , C. David, J. Zusman, W. Unglaub, O. Bunk, J. Raabe, A. F. J. Levi, and G. Aeppli, “Three-dimensional imaging of integrated circuits with macro- to nanoscale zoom ” Nat Electron 2 464–470 (2019) C. David, J. Zusman, W. Unglaub, O. Bunk, J. Raabe, A. F. J. Levi, and G. Aeppli, “Three-dimensional imaging of integrated circuits with macro- to 24J. A. Klug, J. Deng, C. Preissner, C. Roehrig, S. T. Mashrafi, M. Wang, Z. Feng, M. Wojcik, M. Wyman, K. Lang, Z. Cai, B. Lai, and S. Vogt, “Performance and ongoing development of the velociprobe, a fast hard x-ray nanoprobe for high- resolution ptychographic imaging,” Microsc. Microanal. 24, 52–53 (2018). 25 C. David, J. Zusman, W. Unglaub, O. Bunk, J. Raabe, A. F. J. Levi, and G. Aeppli, “Three-dimensional imaging of integrated circuits with macro- to nanoscale zoom,” Nat. Electron. 2, 464–470 (2019). 3A. T. Kuan, J. S. Phelps, L. A. Thomas, T. M. Nguyen, J. Han, C.-L. Chen, A. W. Azevedo, J. C. Tuthill, J. Funke, P. Cloetens, A. Pacureanu, and W.-C. A. Lee, “Dense neuronal reconstruction through x-ray holographic nano- tomography,” Nat. Neurosci. 23, 1637–1643 (2020). 4 25M. van Heel and M. Schatz, “Fourier shell correlation threshold criteria,” J. Struct. Biol. 151, 250–262 (2005). 26 26K. Wakonig, H.-C. Stadler, M. Odstrcˇil, E. H. R. Tsai, A. Diaz, M. Holler, I. Usov, J. Raabe, A. Menzel, and M. REFERENCES Guizar-Sicairos, “PtychoShelves, a versatile high-level framework for high-performance analysis of ptychographic data,” J. Appl. Crystallogr. 53, 574–586 (2020). 2 4J. Rodenburg, A. Hurst, A. Cullis, B. Dobson, F. Pfeiffer, O. Bunk, C. David, K. Jefimovs, and I. Johnson, “Hard-x-ray lensless imaging of extended objects,” Phys. Rev. Lett. 98, 034801 (2007). y 5P. Thibault, M. Dierolf, A. Menzel, O. Bunk, C. David, and F. Pfeiffer, “High- resolution scanning x-ray diffraction microscopy,” Science 321, 379–382 (2008). pp y g 27H. T. Philipp, M. W. Tate, P. Purohit, K. S. Shanks, J. T. Weiss, and S. M. Gruner, “High-speed x-ray imaging pixel array detector for synchrotron bunch isolation,” J. Synchrotron Radiat. 23, 395–403 (2016). 6D. A. Shapiro, Y.-S. Yu, T. Tyliszczak, J. Cabana, R. Celestre, W. Chao, K. Kaznatcheev, A. L. D. Kilcoyne, F. Maia, S. Marchesini, Y. S. Meng, T. Warwick, L. L. Yang, and H. A. Padmore, “Chemical composition mapping with nanometre resolution by soft x-ray microscopy,” Nat. Photonics 8, 765–769 (2014). 28G. Blaj, A. Dragone, C. J. Kenney, F. Abu-Nimeh, P. Caragiulo, D. Doering, M. Kwiatkowski, B. Markovic, J. Pines, M. Weaver, S. Boutet, G. Carini, C.-E. Chang, P. Hart, J. Hasi, M. Hayes, R. Herbst, J. Koglin, K. Nakahara, J. Segal, and G. Haller, “Performance of ePix10k, a high dynamic range, gain auto- ranging pixel detector for FELs,” AIP Conf. Proc. 2054, 060062 (2019). 29 7H. €Ozt€urk, H. Yan, Y. He, M. Ge, Z. Dong, M. Lin, E. Nazaretski, I. K. Robinson, Y. S. Chu, and X. Huang, “Multi-slice ptychography with large numerical aperture multilayer laue lenses,” Optica 5, 601–607 (2018). 8 29Y. Nakaye, T. Sakumura, Y. Sakuma, S. Mikusu, A. Dawiec, F. Orsini, P. Grybos, R. Szczygiel, P. Maj, J. D. Ferrara, and T. Taguchi, “Characterization and performance evaluation of the XSPA-500k detector using synchrotron radiation,” J. Synchrotron Radiat. 28, 439–447 (2021). 30 8J. Spence, U. Weierstall, and M. Howells, “Coherence and sampling require- ments for diffractive imaging,” Ultramicroscopy 101, 149–152 (2004). 9 30M. Hammer, K. Yoshii, and A. Miceli, “Strategies for on-chip digital data com- pression for x-ray pixel detectors,” J. Instrum. 16, P01025 (2021). 31 9M. Guizar-Sicairos, I. Johnson, A. Diaz, M. Holler, P. Karvinen, H. C. Stadler, R. Dinapoli, O. Bunk, and A. Menze, “High-throughput ptychography using Eiger: Scanning x-ray nano-imaging of extended regions,” Opt. Express 22, 14859–14870 (2014). 31P. Huang, M. Du, M. Hammer, A. Miceli, and C. DATA AVAILABILITY 20M. Odstrcˇil, A. Menzel, and M. Guizar-Sicairos, “Iterative least-squares solver for generalized maximum-likelihood ptychography,” Opt. Express 26, 3108–3123 (2018). 2 The data that support the findings of this study are available from the corresponding author upon reasonable request. 21S. Preibisch, S. Saalfeld, and P. Tomancak, “Globally optimal stitching of tiled 3D microscopic image acquisitions,” Bioinformatics 25, 1463–1465 (2009). 22 ABSTRACT We also would like to thank Richard 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 policies or endorsements, either expressed or implied, of the ODNI, IARPA, or the U.S. Government. 13J. Deng, Y. S. G. Nashed, S. Chen, N. W. Phillips, T. Peterka, R. Ross, S. Vogt, C. Jacobsen, and D. J. Vine, “Continuous motion scan ptychography: Characterization for increased speed in coherent x-ray imaging,” Opt. Express 23, 5438–5451 (2015). 14X. Huang, K. Lauer, J. N. Clark, W. Xu, E. Nazaretski, R. Harder, I. K. Robinson, and Y. S. Chu, “Fly-scan ptychography,” Sci. Rep. 5, 9074 (2015). 15 15P. Thibault and A. Menzel, “Reconstructing state mixtures from diffraction measurements,” Nature 494, 68–74 (2013). 16J. N. Clark, X. Huang, R. J. Harder, and I. K. Robinson, “Continuous scanning mode for ptychography,” Opt. Lett. 39, 6066–6069 (2014). 17 17C. Jacobsen, J. Deng, and Y. Nashed, “Strategies for high-throughput focused- beam ptychography,” J. Synchrotron Radiat. 24, 1078–1081 (2017). 18 p y g p y y 18M. Odstrcˇil, M. Holler, and M. Guizar-Sicairos, “Arbitrary-path fly-scan ptychography,” Opt. Express 26, 12585–12593 (2018). 19 19J. Deng, C. Preissner, J. A. Klug, M. Sheikh, C. Roehrig, Y. Jiang, Y. Yao, M. Wojcik, M. Wyman, D. Vine, K. Yue, S. Chen, T. Mooney, M. Wang, Z. Feng, D. Jin, Z. Cai, B. Lai, and S. Vogt, “The velociprobe: An ultrafast hard x-ray nanoprobe for high-resolution ptychographic imaging,” Rev. Sci. Instrum. 90, 083701 (2019). REFERENCES Jacobsen, “Fast digital lossy compression for x-ray ptychographic data,” J. Synchrotron Radiat. 28, 292–300 (2021). 32R. N. Wilke, M. Vassholz, and T. Salditt, “Semi-transparent central stop in high-resolution x-ray ptychography using Kirkpatrick–Baez focusing,” Acta Crystallogr., Sect. A 69, 490–497 (2013). 10M. Stockmar, P. Cloetens, I. Zanette, B. Enders, M. Dierolf, F. Pfeiffer, and P. Thibault, “Near-field ptychography: Phase retrieval for inline holography using a structured illumination,” Sci. Rep. 3, 1927 (2013). 119, 124101-6 119, 124101-6 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 V C Author(s) 2021
https://openalex.org/W2148919163
https://europepmc.org/articles/pmc3866382?pdf=render
English
null
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
Frontiers in physiology
2,013
cc-by
9,611
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 spatial, temporal and energy constraints, and analysing efficiency, robustness and adaptability from molecular resolution to cellu- lar resolution. Biomolecular activities differ in timescales, which can range from microseconds, as observed in some of the most efficient enzymes, to minutes as observed in transcription and translation of functional products. Although these differences may not appear significant superficially, they have a significant impact on the self-organization of cellular processes. CONCLUSIONS AND PROSPECTS REFERENCES “Multiscale adaptive dynamics from molecules to cells,” in Foundations of Systems Biology in Engineering (Denver, CO: Omnipress), 105–108. Corning, P. A. (2002). Thermoeconomics: beyond the Second Law. J. Bioecon. 4, 57–88. doi: 10.1023/A:1020633317271 Plotkin, J. B. (2011). The lives of proteins. Science 331, 683–684. doi: 10.1126/sci- ence.1202010 Corrado, P. (2009). Algorithmic systems biology. Commun. ACM 52, 80–88. doi: 10.1145/1506409.1506427 Prigogine, I. (1984). Order Out of Chaos. New York, NY: Bantam. Regenmortel, M. H. V. V. (2004). Reductionism and complexity in molecular biology EMBO Rep 5, 1016–1020. doi: 10.1038/sj.embor. 7400284 Dada, J. O., and Mendes, P. (2011). Multi-scale modelling and simulation in systems biology. Integr. Biol. 3, 86–96. doi: 10.1039/c0ib00075b Dubitzky, W. (2006). Understanding the computational methodologies of systems biology. Brief. Bioinformatics 7, 315–317. doi: 10.1093/bib/ bbl044 Rosenfeld, J. S. (2002). Functional redundancy in ecology and conservation. Oikos 98, 156–162. doi: 10.1034/j.1600-0706.2002.980116.x Dusenbery, D. B. (1992). Sensory Ecology: How Organisms Acquire and Respond to Information. New York, NY: W H Freeman & Co. Saric, T., and Goldberg, A. L. (2006). Protein degradation and turnover. Encyclopedia Life Sci. doi: 10.1038/npg.els.0005722 Schnell, S., Grima, R., and Maini, P. K. (2007). Multiscale modeling in biology. Am. Sci. 95, 134. doi: 10.1511/2007.64.134 Eberhart, R. C., and Shi, Y. (2007). Computational Intelligence: Concepts to Implementations. Burlington: Morgan Kaufmann. Schut, M. C. (2007). Scientific Handbook for Simulation of Collective Intelligence. Creative Commons licence. Edelman, G. M., and Gally, J. A. (2001). Degeneracy and complexity in biological systems. Proc. Natl. Acad. Sci. U.S.A. 98, 13763–13768. doi: 10.1073/pnas.231499798 Shapiro, R. (2007). A simpler origin for life. Sci. Am. 296, 46–53. doi: 10.1038/ scientificamerican0607-46 Efroni, S., Harel, D., and Cohen, I. R. (2003). Toward rigorous compre- hension of biological complexity: modeling, execution, and visualization of thymic t-cell maturation. Genome Res. 13, 2485–2497. doi: 10.1101/gr. 1215303 Stearns, F. W. (2010). One hundred years of pleiotropy: a retrospective. Genetics 186, 767–773. doi: 10.1534/genetics.110.122549 Stelling, J., Sauer, U., Iii, F. J. D., and Doyle, J. (ed.). (2006). Complexity and Robustness of Cellular Systems. Cambridge, MA: The MIT Press. Felix, M. A., and Wagner, A. (2006). Robustness and evolution: concepts, insights and challenges from a developmental model system. Heredity 100, 132–140. doi: 10.1038/sj.hdy.6800915 Stefan, M. I., and Le Novère, N. (2013). Cooperative binding. PLoS Comput. Biol. 9:e1003106. doi: 10.1371/journal.pcbi.1003106 Stelling, J., Sauer, U., Szallasi, Z., Doyle, F. J., and Doyle, J. (2004). Robustness of cellular functions. Cell 118, 675–685. doi: 10.1016/j.cell.2004. 09.008 Fleischer, M. REFERENCES Kitano, H. (2007). Towards a theory of biological robustness. Mol. Syst. Biol. 3:137. doi: 10.1038/msb4100179 Abel, D. L. (ed.). (2011). The First Gene: The Birth of Programming, Messaging and Formal Control. New York, NY: LongView Press-Academic. Kitano, H. (2010). Violations of robustness trade-offs. Mol. Syst. Biol. 6:384. doi: 10.1038/msb.2010.40 Lobo, I. (2008). Pleiotropy: one gene can affect multiple traits. Nat. Educ. 1:10. Alberty, R. A. (2010). Biochemical thermodynamics and rapid-equilibrium enzyme kinetics. J. Phys. Chem. B 114, 17003–17012. doi: 10.1021/ jp107337g d, R. (2006). The Evolution of Cooperation. New York, NY: Basic Bo Michener, W. K., Baerwald, T. J., Firth, P., Palmer, M. A., Rosenberger, J. L., Sandlin, E. A., et al. (2001). Defining and unraveling biocomplex- ity. Bioscience 51, 1018–1023. doi: 10.1641/0006-3568(2001)051[1018:DAUB] 2.0.CO;2 Axelrod, R. (2006). The Evolution of Cooperation. New York, NY: Basic Books. A l d R d H il W ( ) Th l i f i S i Axelrod, R., and Hamilton, W. (1981). The evolution of cooperation. Science 211, 1390–1396. doi: 10.1126/science.7466396 Bachmair, A., Finley, D., and Varshavsky, A. (1986). In vivo half-life of a pro- tein is a function of its amino-terminal residue. Science 234, 179–186. doi: 10.1126/science.3018930 Ng, Y.-K. (2004). Welfare Economics: Towards a More Complete Analysis. New York, NY: Palgrave Macmillan. Noble, D. (2008). The Music of Life: Biology Beyond Genes. Oxford: Oxford University Press. Bassingthwaighte, J. B., Chizeck, H. J., and Atlas, L. E. (2006). Strategies and tactics in multiscale modeling of cell-to-organ systems. Proc. IEEE Inst. Electr. Electron. Eng. 94, 819–831. doi: 10.1109/JPROC.2006. 871775 Nowak, M. A. (2006). Five rules for the evolution of cooperation. Science 314, 1560–1563. doi: 10.1126/science.1133755 Pearson, H. (2008). Cell biology: the cellular hullabaloo. Nature 453, 150–153. doi: 10.1038/453150a Belle, A., Tanay, A., Bitincka, L., Shamir, R., and O’shea, E. K. (2006). Quantification of protein half-lives in the budding yeast proteome. Proc. Natl. Acad. Sci. U.S.A. 103, 13004–13009. doi: 10.1073/pnas.0605420103 Periyasamy, S., Gray, A., and Kille, P. (2008). “The epigenetic algorithm,” in IEEE Congress on Evolutionary Computation (IEEE World Congress on Computational Intelligence), (Hong Kong), 3228–3236. doi: 10.1109/CEC.2008. 4631235 Bruggeman, F. J., and Westerhoff, H. V. (2007). The nature of systems biology. Trends Microbiol. 15, 45–50. doi: 10.1016/j.tim.2006.11.003 Coello Coello, C. A. (2006). Evolutionary multi-objective optimization: a historical view of the field. Comput. Intell. Mag. IEEE 1, 28–36. doi: 10.1109/MCI.2006.1597059 Periyasamy, S., Gray, A., and Kille, P. (2009). CONCLUSIONS AND PROSPECTS Pareto optimality is an economic concept, which can be used to study the efficacy of biologi- cal cells and distribution of biomolecular activities (Ng, 2004). A Pareto efficient frontier is one, in which any change to enhance the performance of an objective is impossible without mak- ing the performance of another objective inferior. This is often the case, when there are conflicts among mutually dependent objectives. A mathematically oriented (quantitative) definition for self-organized behavior has been articulated as (Fleischer, 2005) “Self-organized behavior in a complex system involving multiple performance measures is a sequence of system states cor- responding to movement along a Pareto optimal frontier.” This defines the best global solution that can emerge based within the constraints. December 2013 | Volume 4 | Article 369 | 8 Frontiers in Physiology | Systems Biology The bottom–up approach to defining life Periyasamy et al. of functional changes driven by pathological or environmental changes derived from the re-organization of the underlying bio- logical components. Thus, this represents a paradigm shift to pro- mote a bottom–up approach to defining life allowing knowledge generated through reductionism to inform complex biological systems. Hartman, J. L. I. V., Garvik, B., and Hartwell, L. (2001). Principles for the Buffering of Genetic Variation. Science 291, 1001–1004. doi: 10.1126/sci- ence.291.5506.1001 Hodgkin, J. (1998). Seven types of pleiotropy. Int. J. Dev. Biol. 42, 501–505. Johnston, I. (2012). The chaos within: exploring noise in cellular biology. Significance 9, 17–21. doi: 10.1111/j.1740-9713.2012.00586.x Kauffman, S. (1993). The Origins of Order: Self-Organization and Selection in Evolution. New York, NY: Oxford University Press. Kitano, H. (2004). Biological Robustness. Nat. Rev. Genet. 5, 826–837. doi: 10.1038/ nrg1471 REFERENCES (2005). “Scale invariant pareto optimality: a meta–formalism for char- acterizing and modeling cooperativity in evolutionary systems,” in Proceedings of the 2005 Conference on Genetic and Evolutionary Computation, ed H. Beyer (New York, NY: ACM), 233–240. doi: 10.1145/1068009.1068044 L. (1988). Biochemistry. New York, NY: W.H.Freeman And Compa Stryer, L. (1988). Biochemistry. New York, NY: W.H.Freeman A (New York, NY: ACM), 233–240. doi: 10.1145/1068009.106804 Flintoft, L. (2007). Transcriptional memory remodelled. Nat. Rev. Genet. 8, 323–323. doi: 10.1038/nrg2110 Szallasi, Z., Periwal, V., and Stelling, J. (2006). “On modules and modularity,” in System Modeling in Cellular Biology: From Concepts to Nuts and Bolts, eds Z. Szallasi, J. Stelling and V. Periwal (Cambridge, MA: MIT Press), 41–50. doi: 10.7551/mitpress/9780262195485.003.0003 Fogel, D. B. (2006). Evolutionary Computation: Towards a New Philosophy of Machine Intelligence. Piscataway, NJ: Wiley-IEEE Press. Takahashi, K., Arjunan, S. N. V., and Tomita, M. (2005). Space in systems biology of signaling pathways - towards intracellular molecular crowding in silico. FEBS Lett. 579, 1783–1788. doi: 10.1016/j.febslet.2005.01.072 Garnier, S., Gautrais, J., and Theraulaz, G. (2007). The biological principles of swarm intelligence. Swarm Intell. 1, 3–31. doi: 10.1007/s11721-007-0004-y Gerstein, M. B., Bruce, C., Rozowsky, J. S., Zheng, D., Du, J., Korbel, J. O., et al. (2007). What is a gene, post-ENCODE? History and updated definition. Genome Res. 17, 669–681. doi: 10.1101/gr.6339607 Van Regenmortel, M. H. (2007). The rational design of biological complexity: a deceptive metaphor. Proteomics 7, 965–975. doi: 10.1002/pmic.200600407 December 2013 | Volume 4 | Article 369 | 9 www.frontiersin.org The bottom–up approach to defining life Periyasamy et al. Received: 29 August 2013; accepted: 26 November 2013; published online: 18 December 2013. Viney, M., and Reece, S. E. (2013). Adaptive noise. Proc. R. Soc. B Biol. Sci. 280:20131104. doi: 10.1098/rspb.2013.1104 Wagner, A. (2008). Robustness and evolvability: a paradox resolved. Proc. Biol. Sci. 275, 91–100. doi: 10.1098/rspb.2007.1137 Citation: Periyasamy S, Gray A and Kille P (2013) The bottom–up approach to defin- ing life: deciphering the functional organization of biological cells via multi-objective representation of biological complexity from molecules to cells. Front. Physiol. 4:369. doi: 10.3389/fphys.2013.00369 Westerhoff, H. V., and Palsson, B. O. (2004). The evolution of molecular biology into systems biology. Nat. Biotech. 22, 1249. doi: 10.1038/nbt1020 Wolfe, J. (2001). Cellular thermodynamics. Encyclopedia Life Sci. doi: 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., et al. (2013). A general framework of persistence strategies for biological systems 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
https://openalex.org/W2150751764
https://europepmc.org/articles/pmc4189975?pdf=render
English
null
Comparison of Relative Waist Circumference between Asian Indian and US Adults
Journal of obesity
2,014
cc-by
6,004
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, “Non-obese hyperlipidemic Asian northern Indian males have adverse anthropometric profile,” Nutrition, Metabolism and Cardiovascular Diseases, vol. 12, no. 4, pp. 178–183, 2002. [14] A. Ramachandran, C. Snehalatha, E. Latha, V. Vijay, and M. Viswanathan, “Rising prevalence of NIDDM in an urban population in India,” Diabetologia, vol. 40, no. 2, pp. 232–237, 1997. [30] A. M. Hodge, G. K. Dowse, V. R. Collins et al., “Abdominal fat distribution and insulin levels only partially explain adverse cardiovascular risk profile in Asian Indians,” Journal of Cardio- 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 Singapore, vol. 18, no. 6, pp. 642–645, 1989. [31] M. Deepa, R. Pradeepa, M. Rema et al., “The Chennai Urban Rural Epidemiology Study (CURES)—Study design and methodology (Urban Component) (CURES-1),” Journal of Association of Physicians of India, vol. 51, pp. 863–870, 2003. [16] P. M. McKeigue, G. J. Miller, and M. G. Marmot, “Coronary heart disease in South Asians overseas: a review,” Journal of Clinical Epidemiology, vol. 42, no. 7, pp. 597–609, 1989. [17] S. S. Anand, S. Yusuf, V. Vuksan et al., “Differences in risk factors, atherosclerosis, and cardiovascular disease between ethnic groups in Canada: the Study of Health Assessment and Risk in Ethnic groups (SHARE),” The Lancet, vol. 356, no. 9226, pp. 279–284, 2000. [32] H. Morgenstern, “Ecologic studies in epidemiology: concepts, principles, and methods,” Annual Review of Public Health, vol. 16, pp. 61–81, 1995. [33] “Third report of the National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III),” Circulation, vol. 106, no. 25, pp. 3143–3421, 2002. [18] J. Lee, D. Heng, K. S. Chia, S. K. Chew, B. Y. Tan, and K. Hughes, “Risk factors and incident coronary heart disease in Chinese, Malay and Asian Indian males: the Singapore cardiovascular cohort study,” International Journal of Epidemiology, vol. 30, no. 5, pp. 983–988, 2001. References 5366–5371, 2001. [7] M. Chandalia, N. Abate, A. Garg, J. Stray-Gundersen, and S. M. Grundy, “Relationship between generalized and upper body obesity to insulin resistance in Asian Indian men,” Journal of Clinical Endocrinology and Metabolism, vol. 84, no. 7, pp. 2329– 2335, 1999. [8] P. M. McKeigue, B. Shah, and M. G. Marmot, “Relation of central obesity and insulin resistance with high diabetes 10 Journal of Obesity Journal of Obesity [24] K. M. Shelgikar, T. D. R. Hockaday, and C. S. Yajnik, “Central rather than generalized obesity is related to hyperglycaemia in Asian Indian subjects,” Diabetic Medicine, vol. 8, no. 8, pp. 712– 717, 1991. prevalence and cardiovascular risk in South Asians,” The Lancet, vol. 337, no. 8738, pp. 382–386, 1991. [9] P. M. McKeigue, T. Pierpoint, J. E. Ferrie, and M. G. Marmot, “Relationship of glucose intolerance and hyperinsulinaemia to body fat pattern in South Asians and Europeans,” Diabetologia, vol. 35, no. 8, pp. 785–791, 1992. [25] I. Janssen, P. T. Katzmarzyk, and R. Ross, “Waist circumference and not body mass index explains obesity-related health risk,” The American Journal of Clinical Nutrition, vol. 79, no. 3, pp. 379–384, 2004. [10] H. M. Mather and H. Keen, “The Southall Diabetes Survey: prevalence of known diabetes in Asians and Europeans,” British Medical Journal, vol. 291, no. 6502, pp. 1081–1084, 1985. [26] P. S. Yusuf, S. Hawken, S. ˆOunpuu et al., “Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study,” The Lancet, vol. 364, no. 9438, pp. 937–952, 2004. [11] Q. Qiao, G. Hu, J. Tuomilehto et al., “Age- and sex-specific prevalence of diabetes and impaired glucose regulation in 11 Asian cohorts,” Diabetes Care, vol. 26, no. 6, pp. 1770–1780, 2003. [27] US Department of Health and Human Services and National Center for Health Statistics, NHANES III Refernce Manuals and Reports (CD-ROM), Center for Disease Control and Prevention, Hyattsville, Md, USA, 1996. [12] A. Ramachandran, C. Snehalatha, D. Dharmaraj, and M. Viswanathan, “Prevalence of glucose intolerance in Asian Indi- ans: urban-rural difference and significance of upper body adiposity,” Diabetes Care, vol. 15, no. 10, pp. 1348–1355, 1992. [28] R. Hakeem, J. Thomas, and S. H. Badruddin, “Urbanisation and coronary heart disease risk factors in South Asian children,” Journal of the Pakistan Medical Association, vol. 51, no. 1, pp. 22– 28, 2001. [13] A. Ramachandran, M. V. Jali, V. Mohan, C. Snehalatha, and M. References [34] “The IDF consensus worldwide definition of the metabolic syndrome,” International Diabetes Federation Avenue Emile De Mot 19 B-1000 Brussels, Belgium, 2006, http://www.idf.org/ webdata/docs/IDF Meta def final.pdf. [19] A. Laws, J. L. Jeppesen, P. C. Maheux, P. Schaaf, Y.-D. Chen, and G. M. Reaven, “Resistance to insulin-stimulated glucose uptake and dyslipidemia in Asian Indians,” Arteriosclerosis and Thrombosis, vol. 14, no. 6, pp. 917–922, 1994. [35] W. D. Ross, S. M. Crawford, D. A. Kerr, R. Ward, D. A. Bailey, and R. M. Mirwald, “Relationship of the body mass index with skinfolds, girths, and bone breadths in Canadian men and women aged 20–70 years,” American Journal of Physical Anthropology, vol. 77, no. 2, pp. 169–173, 1988.f [20] WHO Expert Consultation, “Appropriate body-mass index for Asian populations and its implications for policy and interven- tion strategies,” The Lancet, vol. 363, no. 9412, pp. 157–163, 2004. [36] W. D. Ross, “On combining samples that differ allometrically with size,” Revista Brasileira de Medicina do Esporte, vol. 3, no. 4, pp. 95–100, 1997. [21] A. Ramachandran, C. Snehalatha, and V. Vijay, “Temporal changes in prevalence of type 2 diabetes and impaired glucose tolerance in urban southern India,” Diabetes Research and Clinical Practice, vol. 58, no. 1, pp. 55–60, 2002. [22] F. Razak, S. Anand, V. Vuksan et al., “Ethnic differences in the relationships between obesity and glucose-metabolic abnormalities: a cross-sectional population-based study,” Inter- national Journal of Obesity, vol. 29, no. 6, pp. 656–667, 2005. [23] M. Anjana, S. Sandeep, R. Deepa, K. S. Vimaleswaran, S. Farooq, and V. Mohan, “Visceral and central abdominal fat and anthropometry in relation to diabetes in Asian Indians,” Diabetes Care, vol. 27, no. 12, pp. 2948–2953, 2004.
https://openalex.org/W2549580544
https://europepmc.org/articles/pmc5116174?pdf=render
English
null
Potential effects of samsum ant, Brachyponera sennaarensis, venom on TNF-α/NF-κB mediated inflammation in CCL4-toxicity in vivo
Lipids in health and disease
2,016
cc-by
8,244
© 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 References 1. Casewell NR, Wüster W, Vonk FJ, Harrison RA, Fry BG. Complex cocktails: the evolutionary novelty of venoms. Trends Ecol Evol. 2013;28:219–29. 25. Niki E, Yamamoto Y, Komuro E, Sato K. Membrane damage due to lipid peroxidation. Am J Clin Nutr. 1991;53:201S–5S. 26. Ebaid H, Al-Khalifa M, Isa AM, Gadoa S. Bioactivity of Samsum ant (Pachycondyla sennaarensis) venom against lipopolysaccharides through antioxidant and upregulation of Akt1 signaling in rats. Lipids Health Dis. 2012;11:93. Consent for publication The authors declare that there is no any individual person’s data in any form (including individual details, images or videos). 23. Shim JY, Kim MH, Kim HD, Ahn JY, Yun YS, Song JY. Protective action of the immunomodulator ginsan against carbon tetrachloride-induced liver injury via control of oxidative stress and the inflammatory response. Toxicol Appl Pharmacol. 2010;242:318–25. Abbreviations Ak P i ki Akt1: Protein kinase B; CAT: Catalase; DTNB: 5,5-dithiobis-2-nitrobenzoic acid; EDTA: Ethylenediaminetetraacetic acid; FAS: Cell surface death receptor; GSH: Glutathione; MDA: Malondialdehyde; SAV: Samsum ant venom; TBA: Thiobarbituric acid; TCA: Trichloroacetic acid 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. 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. Authors’ contributions JA prepared the introduction and conducted the venom extraction, animal care, analysed the biochemical data, prepared the results, figures, and the oxidative stress tests. HE designed this study, supervised the practical work and finalized the manuscript. AM dried the ant venom and worked on the antioxidant. IA provided the chemicals and contributed to the final revision. MA facilitated the ant collection and provided financial support. All authors read and approved the final manuscript. 18. Schmidt JO, Blum MS, Overal WL. Comparative enzymology of venoms from stinging Hymenoptera. Toxicon. 1986;24:907–21. 18. Schmidt JO, Blum MS, Overal WL. Comparative enzymology of venoms from stinging Hymenoptera. Toxicon. 1986;24:907–21. 19. King GF. Venoms as a platform for human drugs: translating toxins into therapeutics. Expert Opin Biol Ther. 2011;11:1469–84. 19. King GF. Venoms as a platform for human drugs: translating toxins into therapeutics. Expert Opin Biol Ther. 2011;11:1469–84. 20. Kou J, Ni Y, Li N, Wang J, Liu L, Jiang ZH. Analgesic and anti-inflammatory activities of total extract and individual fractions of Chinese medicinal ants Polyrhachis lamellidens. Biol Pharm Bull. 2005;28:176–80. 20. Kou J, Ni Y, Li N, Wang J, Liu L, Jiang ZH. Analgesic and anti-inflammatory activities of total extract and individual fractions of Chinese medicinal ants Polyrhachis lamellidens. Biol Pharm Bull. 2005;28:176–80. Competing interests 21. Wang CP, Wu YL. Study on mechanism underlying the treatment of rheumatoid arthritis by Keshiling. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China Chinese Mater medica. 2006;31:155–8. The authors declare that there is no conflict of interests regarding the publication of this paper. The authors declare that there is no conflict of interests regarding the publication of this paper. 22. Singh N, Kamath V, Narasimhamurthy K, Rajini PS. Protective effect of potato peel extract against carbon tetrachloride-induced liver injury in rats. Environ Toxicol Pharmacol. 2008;26:241–6. Funding We declare hereby the role of the Deanship of Scientific Research at king Saud University in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. 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. Acknowledgment h h ld l The authors would like to extend their sincere appreciation to the Deanship 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 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. 7. 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 the venom gland of the Samsum ant. Pachycondyla sennaarensis Toxicon. 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 evolutionary novelty of venoms. Trends Ecol Evol. 2013;28:219–29. Page 11 of 11 Al-Tamimi et al. Lipids in Health and Disease (2016) 15:198 27. Siomek A. NF-κB signaling pathway and free radical impact. Acta Biochim 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 • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries
https://openalex.org/W2713001338
https://europepmc.org/articles/pmc5787849?pdf=render
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 containing a 5-mm-wide and 5-mm-deep well filled with 3 mL of 401 Production of human vitronectin in plants 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 References Scholthof, HB (2007) Heterologous expression of viral RNA interference suppressors: RISC management. Plant Physiol. 145, 1110–1117. Azevedo, H, Lino-Neto, T and Tavares, RM (2003) An improved method for high-quality RNA isolation from needles of adult maritime pine trees. Plant Mol. Biol. Rep. 21, 333–338. Schvartz, I, Seger, D and Shaltiel, S (1999) Vitronectin. Int. J. Biochem. Cell Biol. 31, 539–544. Cacchioli, A, Ravanetti, F, Bagno, A, Dettin, M and Gabbi, C (2009) Human vitronectin-derived peptide covalently grafted onto titanium surface improves osteogenic activity: a pilot in vivo study on rabbits. Tissue Eng. Part A 15, 2917–2926. Shaffer, MC, Foley, TP and Barnes, DW (1984) Quantitation of spreading factor in human biologic fluids. J. Lab. Clin. Med. 103, 783–791. Streatfield, SJ, Lane, JR, Brooks, CA, Barker, DK, Poage, ML, Mayor, JM, Lamphear, BJ et al. (2003) Corn as a production system for human and animal vaccines. Vaccine, 21, 812–815. Chen, G, Gulbranson, DR, Hou, Z, Bolin, JM, Ruotti, V, Probasco, MD, Smuga-Otto, K et al. (2011) Chemically defined conditions for human iPsc derivation and culture. Nat Meth. 8, 424–431. Tollefsen, DM, Weigel, CJ and Kabeer, MH (1990) The presence of methionine or threonine at position 381 in vitronectin is correlated with proteolytic cleavage at arginine 379. J. Biol. Chem. 265, 9778–9781. Conlan, MG, Tomasini, BR, Schultz, RL and Mosher, DF (1988) Plasma vitronectin polymorphism in normal subjects and patients with disseminated intravascular coagulation. Blood, 72, 185–190. Underwood, PA and Bennett, FA (1989) A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin. J. Cell Sci. 93, 641–649. Deo, PC, Dugdale, B, Harding, RM, Kato, M and Dale, JL (2015) In vitro micro propagation of Nicotiana benthamiana via axillary shoots. S. Pac. J. Nat. Sci. 32, 55–60. Upton, Z, Cuttle, L, Noble, A, Kempf, M, Topping, G, Malda, J, Xie, Y et al. (2008) Vitronectin: growth factor complexes hold potential as a wound therapy approach. J. Invest. Dermatol. 128, 1535–1544. Dugdale, B, Mortimer, CL, Kato, M, James, TA, Harding, RM and Dale, JL (2013) In plant activation: an inducible, hyperexpression platform for recombinant protein production in plants. Plant Cell, 25, 2429–2443. Upton, Z, Wallace, HJ, Shooter, GK, van Lonkhuyzen, DR, Yeoh-Ellerton, S, Rayment, EA, Fleming, JM et al. (2011) Human pilot studies reveal the potential of a vitronectin: growth factor complex as a treatment for chronic wounds. Int. Wound J. 8, 522–532. Authors declare no conflict of interest. Saxena, P, Hsieh, YC, Alvarado, VY, Sainsbury, F, Saunders, K, Lomonossoff, GP and Scholthof, HB (2011) Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless. Plant Biotechnol. J. 9, 703–712. Statistical analysis Mortimer, CL, Dugdale, B and Dale, JL (2015) Updates in inducible transgene expression using viral vectors: from transient to stable expression. Curr. Opin. Biotechnol. 32, 85–92. One-way analysis of variance was performed to compare different batch yields or different cell binding activities (P ˂ 0.05 was considered significant). Data values were expressed as mean standard error for yield estimates and mean  standard deviation for cell attachment assays. Murashige, T and Skoog, F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. Nakasugi, K, Crowhurst, RN, Bally, J, Wood, CC, Hellens, RP and Waterhouse, PM (2013) De novo transcriptome sequence assembly and analysis of RNA silencing genes of Nicotiana benthamiana. PLoS ONE, 8, e59534. Acknowledgements Preissner, KT (1991) Structure and biological role of vitronectin. Annu. Rev. Cell Biol. 7, 275–310. The authors would like to thank the Queensland Government, Queensland University of Technology, and Leaf Energy Inc., for their financial support. The authors would also like to thank Dr Gary Shooter (IHBI, QUT) for supplying BALB-C/3T3 cells and technical advice. This work was facilitated by access to the Australian Proteome Analysis Facility supported under the Aus- tralian Government’s National Collaborative Research Infrastruc- ture Strategy (NCRIS). Sainsbury, F and Lomonossoff, GP (2008) Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol. 148, 1212–1218. Sainsbury, F, Thuenemann, EC and Lomonossoff, GP (2009) pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol. J. 7, 682–693. proteins in plants. Plant Biotechnol. J. 7, 682–693. Sanders, LC, Wang, C-S, Walling, LL and Lord, EM (1991) A homolog of the substrate adhesion molecule vitronectin occurs in four species of flowering plants. Plant Cell, 3, 629–635. Sano, K, Asanuma-Date, K, Arisaka, F, Hattori, S and Ogawa, H (2007) Changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration. Glycobiology, 17, 784–794. Cell adhesion assay The media were then aspirated and 200 lL of 100% (w/ v) DMSO added to each well in order to solubilize the precipitate. Absorbance was measured at 570 nm (compared to an absorbance at a reference wavelength of 630 nm) and plotted against hVN concentration to determine ED50, as above. Jin, H and Varner, J (2004) Integrins: roles in cancer development and as treatment targets. Br. J. Cancer, 90, 561–565. Li, J, Bardy, J, Yap, LY, Chen, A, Nurcombe, V, Cool, SM, Oh, SK et al. (2010) Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells. Biointerphases, 5, 132–142. Lico, C, Chen, Q and Santi, L (2008) Viral vectors for production of recombinant proteins in plants. J. Cell. Physiol. 216, 366–377. Cell adhesion assay The eluent from the Heparin Sepharose column was loaded directly onto the equilibrated TALON column. The 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- 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. 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 formed. Conflict of Interest during liver regeneration. Glycobiology, 17, 784–794. 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 References Dugdale, B, Mortimer, CL, Kato, M, James, TA, Harding, RM and Dale, JL (2014) Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants. Nat Protoc. 9, 1010–1027. Voinnet, O (2001) RNA silencing as a plant immune system against viruses. Trends Genet. 17, 449–459. Harrison, MD, Geijskes, RJ, Coleman, HD, Shand, K, Kinkema, M, Palupe, A, Hassall, R et al. (2011) Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane. Plant Biotechnol. J. 9, 884–896. Voinnet, O, Vain, P, Angell, S and Baulcombe, DC (1998) Systemic spread of sequence-specific transgene RNA degradation is initiated by localised introduction of ectopic promoterless DNA. Cell, 95, 177–187. Voinnet, O, Pinto, YM and Baulcombe, DC (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses. Proc. Natl Acad. Sci. USA, 96, 14147–14152. Hollier, B, Harkin, DG, Leavesley, D and Upton, Z (2005) Responses of keratinocytes to substrate-bound vitronectin:growth factor complexes. Exp. Cell Res. 305, 221–232. Wagner, VT and Matthysse, AG (1992) Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells. J. Bacteriol. 174, 5999–6003. Horsch, RB, Fry, JE, Hoffmann, NL, Eichholtz, D, Rogers, SG and Fraley, RT (1985) A simple and general method for transferring genes into plants. Science, 227, 1229–1231. t Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 16, 394–403 Production of human vitronectin in plants 403 Waterhouse, PM, Graham, MW and Wang, M-B (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. USA, 95, 13959–13964. 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 Supporting information and antisense RNA. Proc. Natl. Acad. Sci. USA, 95, 13959–13964. Additional Supporting Information may be found online in the supporting information tab for this article: Xie, Y, Rizzi, SC, Dawson, R, Lynam, E, Richards, S, Leavesley, DI and Upton, Z (2010) Development of a three-dimensional human skin equivalent wound model for investigating novel wound healing therapies. Tissue Eng. Part C, 16, 1111–1123. Appendix S1 Reverse Transcription (RT)-PCR to detect Rep/RepA 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 INPACT lines. Zhou, A, Huntington, JA, Pannu, NS, Carrell, RW and Read, RJ (2003) How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration. Nat. Struct. Biol. 10, 541–544. Appendix S4 Growth and development of transgenic plants in soil. Appendix S4 Growth and development of transgenic plants in soil. Zhu, J-K, Shi, J, Singh, U, Wyatt, SE, Bressan, RA, Hasegawa, PM and Carpita, NC (1993) Enrichment of vitronectin- and fibronectin-like proteins in NaCI- adapted plant cells and evidence for their involvement in plasma membrane- cell wall adhesion. Plant J. 3, 637–646. Appendix S5 Purification of recombinant hVN from N. ben- thamiana using a three- stage extraction protocol. cell wall adhesion. Plant J. 3, 637–646. Zhu, J-K, Damsz, B, Kononowicz, AK, Bressan, RA and Hasegawaai, PM (1994) A higher plant extracellular vitronectin-like adhesion protein is related to the translational elongation Factor-I a. Plant Cell, 6, 393–404.
https://openalex.org/W2905226814
https://europepmc.org/articles/pmc6334625?pdf=render
English
null
A Combined Ultrafiltration/Diafiltration Step Facilitates the Purification of Cyanovirin-N From Transgenic Tobacco Extracts
Frontiers in bioengineering and biotechnology
2,019
cc-by
8,316
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 experiments and performed the cost calculations. REFERENCES Buyel, J. F. (2015). Process development strategies in plant molecular farming. Curr. Pharm. Biotechnol. 16, 966–982. doi: 10.2174/138920101611150902115413 Amanda, A., and Mallapragada, S. K. (2001). Comparison of protein fouling on heat-treated poly(vinyl alcohol), poly(ether sulfone) and regenerated cellulose membranes using diffuse reflectance infrared fourier transform spectroscopy. Biotechnol. Prog. 17, 917–923. doi: 10.1021/bp0 100631 Buyel, J. F., and Fischer, R. (2012). Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs. Biotechnol. Bioeng. 109, 2575–2588. doi: 10.1002/bit.24523 Barros, G. O. F., Woodard, S. L., and Nikolov, Z. L. (2011). Phenolics removal from transgenic lemna minor extracts expressing mab and impact on mab production cost. Biotechnol. Prog. 27, 410–418. doi: 10.1002/btpr.543 Buyel, J. F., and Fischer, R. (2014a). Scale-down models to optimize a filter train for the downstream purification of recombinant pharmaceutical proteins produced in tobacco leaves. Biotechnol. J. 9, 415–425. doi: 10.1002/biot.201300369 Brink, L., Elbers, S., Robbertsen, T., and Both, P. (1993). The anti-fouling action of polymers preadsorbed on ultrafiltration and microfiltration membranes. J. Membr. Sci. 76, 281–291. doi: 10.1016/0376-7388(93)85225-L Buyel, J. F., and Fischer, R. (2014b). Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants. Bioengineered 5, 138–142. doi: 10.4161/bioe.28061 Burns, D. B., and Zydney, A. L. (1999). Effect of solution pH on protein transport through ultrafiltration membranes. Biotechnol. Bioeng. 64, 27–37. Buyel, J. F., and Fischer, R. (2014c). A juice extractor can simplify the downstream processing of plant-derived biopharmaceutical proteins Frontiers in Bioengineering and Biotechnology | www.frontiersin.org January 2019 | Volume 6 | Article 206 9 Opdensteinen et al. HCP Removal by UF/DF compared to blade-based homogenizers. Process Biochem. 50, 859–866. doi: 10.1016/j.procbio.2015.02.017 Koros, W., Ma, Y., and Shimidzu, T. (1996). Terminology for membranes and membrane processes. Sci. J. IUPAC 68, 1479–1490. Lightfoot, E. N., Root, T. W., and O’Dell, J. L. (2008). Emergence of ideal membrane cascades for downstream processing. Biotechnol. Prog. 24, 599–605. doi: 10.1021/bp070335l Buyel, J. F., and Fischer, R. (2014d). Generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants. Biotechnol. J. 9, 566–577. doi: 10.1002/biot.201300548 Loomis, W. D. (1974). Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods Enzymol. 31, 528–544. doi: 10.1016/0076-6879(74)31057-9 Buyel, J. F., and Fischer, R. (2014e). REFERENCES Characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study. J. Vis. Exp. 1:e51216. doi: 10.3791/51216 Menzel, S., Holland, T., Boes, A., Spiegel, H., Bolzenius, J., Fischer, R., et al. (2016). Optimized blanching reduces the host cell protein content and substantially enhances the recovery and stability of two plant-derived malaria vaccine candidates. Front. Plant Sci. 7, 1–15. doi: 10.3389/fpls.2016.00159 Buyel, J. F., Gruchow, H. M., Boes, A., and Fischer, R. (2014a). Rational design of a host cell protein heat precipitation step simplifies the subsequent purification of recombinant proteins from tobacco. Biochem. Eng. J. 88, 162–170. doi: 10.1016/j.bej.2014.04.015 Menzel, S., Holland, T., Boes, A., Spiegel, H., Fischer, R., and Buyel, J. F. (2018). Downstream processing of a plant-derived malaria transmission- blocking vaccine candidate. Protein Express. Purif. 152, 122–130. doi: 10.1016/j.pep.2018.07.012 Buyel, J. F., Opdensteinen, P., and Fischer, R. (2014b). Cellulose-based filter aids increase the capacity of depth filters during the downstream processing of plant-derived biopharmaceutical proteins. Biotechnol. J. 10, 584–591. doi: 10.1002/biot.201400611 Nestola, P., Martins, D. L., Peixoto, C., Roederstein, S., Schleuss, T., Alves, P. M., et al. (2014). Evaluation of novel large cut-offultrafiltration membranes for adenovirus serotype 5 (Ad5) concentration. PLoS ONE 9:e115802. doi: 10.1371/journal.pone.0115802 Buyel, J. F., Twyman, R. M., and Fischer, R. (2015). Extraction and downstream processing of plant-derived recombinant proteins. Biotechnol. Adv. 33, 902–913. doi: 10.1016/j.biotechadv.2015.04.010 Buyel, J. F., Twyman, R. M., and Fischer, R. (2017). Very-large-scale production of antibodies in plants: the biologization of manufacturing. Biotechnol. Adv. 35, 458–465. doi: 10.1016/j.biotechadv.2017.03.011 Palecek, S. P., and Zydney, A. L. (1994). Intermolecular electrostatic interactions and their effect on flux and protein deposition during protein filtration. Biotechnol. Prog. 10, 207–213. doi: 10.1021/bp00026a010 Buyel, J. F., Woo, J. A., Cramer, S. M., and Fischer, R. (2013). The use of quantitative structure-activity relationship models to develop optimized processes for the removal of tobacco host cell proteins during biopharmaceutical production. J. Chromatogr. A 1322, 18–28. doi: 10.1016/j.chroma.2013.10.076 Rangou, S., Buhr, K., Filiz, V., Clodt, J. I., Lademann, B., Hahn, J., et al. (2014). Self-organized isoporous membranes with tailored pore sizes. J. Membr. Sci. 451, 266–275. doi: 10.1016/j.memsci.2013.10.015 Sommerfeld, S., and Strube, J. (2005). Challenges in biotechnology production— generic processes and process optimization for monoclonal antibodies. Chem. Eng. Process. Process Intens. 44, 1123–1137. doi: 10.1016/j.cep.2005.03.006 Cromwell, M. E., Hilario, E., and Jacobson, F. (2006). Protein aggregation and bioprocessing. Am. Assoc. Pharmaceut. Sci. 8, E572–E579. REFERENCES doi: 10.1208/aapsj080366 Susanto, H., Franzka, S., and Ulbricht, M. (2007). Dextran fouling of polyethersulfone ultrafiltration membranes—Causes, extent and consequences. J. Membr. Sci. 296, 147–155. doi: 10.1016/j.memsci.2007.03.027 j Dosmar, M. (2005). Could membrane fouling be a thing of the past. BioProcess Int. 3 , 62–66. Truskey, G. A., Gabler, R., DiLeo, A., and Manter, T. (1987). The effect of membrane filtration upon protein conformation. PDA J. Pharmaceut. Sci. Technol. 41, 180–191. Drake, P. M. W., Barbi, T., Sexton, A., McGowan, E., Stadlmann, J., Navarre, C., et al. (2009). Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco. FASEB J. 23, 3581–3589. doi: 10.1096/fj.09-131771 Turpen, T. H. (1999). Tobacco mosaic virus and the virescence of biotechnology. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 354, 665–673. doi: 10.1098/rstb.1999.0419 Erickson, H. P. (2009). Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol. Proced. Online 11, 32–51. doi: 10.1007/s12575-009-9008-x Tuse, D. (2011). Safety of plant-made pharmaceuticals Product development and regulatory considerations based on case studies of two autologous human cancer vaccines. Hum. Vaccines 7, 322–330. doi: 10.4161/hv.7.3.14213 Fane, A., Fell, C., and Suki, A. (1983). The effect of pH and ionic environment on the ultrafiltration of protein solutions with retentive membranes. J. Membr. Sci. 16, 195–210. doi: 10.1016/S0376-7388(00)81310-1 van den Berg, G. B., and Smolders, C. A. (1989). Concentration polarization phenomena during dead-end ultrafiltration of protein mixtures. The influence of solute-solute interactions. J. Membr. Sci. 47, 1–24. doi: 10.1016/S0376-7388(00)80857-1 Hadidi, M., and Zydney, A. L. (2014). Fouling behavior of zwitterionic membranes: impact of electrostatic and hydrophobic interactions. J. Membr. Sci. 452, 97–103. doi: 10.1016/j.memsci.2013.09.062 van Eijndhoven, R. H., Saksena, S., and Zydney, A. L. (1995). Protein fractionation using electrostatic interactions in membranel filtration. Biotechnol. Bioeng. 48, 406–414. doi: 10.1002/bit.260480413 Hassan, S., van Dolleweerd, C. J., Ioakeimidis, F., Keshavarz-Moore, E., and Ma, J. K. (2008). Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants. Plant Biotechnol. J. 6, 733–748. doi: 10.1111/j.1467-7652.2008.00354.x Wilken, L. R., and Nikolov, Z. L. (2012). Recovery and purification of plant-made recombinant proteins. Biotechnol. Adv. 30, 419–433. doi: 10.1016/j.biotechadv.2011.07.020 Holler, C., Vaughan, D., and Zhang, C. (2007). Polyethyleneimine precipitation versus anion exchange chromatography in fractionating recombinant beta- glucuronidase from transgenic tobacco extract. J. Chromatogr. A 1142, 98–105. doi: 10.1016/j.chroma.2006.08.044 Zeman, L. J., Zydney, A. L., and Andrew, L (1996). REFERENCES Microfiltration and Ultrafiltration. New York, NY: Marcel Dekker. Hunter, R. J. (2013). “Zeta potential in colloid science,” in Colloid Science, R. H. Ottewill, eds R. L. Rowell (Cambridge, MA: Academic Press), 398. Zhang, D., Karkooti, A., Liu, L., Sadrzadeh, M., Thundat, T., Liu, Y., et al. (2018). Fabrication of antifouling and antibacterial polyethersulfone (PES)/cellulose nanocrystals (CNC) nanocomposite membranes. J. Membr. Sci. 549, 350–356. doi: 10.1016/j.memsci.2017.12.034 Jang, H., Kim, H.-S., Moon, S.-C., Lee, Y.-R., Yu, K. Y., Lee, B. K., et al. (2009). Effects of protein concentration and detergent on endotoxin reduction by ultrafiltration. Biochem. Mol. Biol. Rep. 42, 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
https://openalex.org/W2029854128
https://journals.assaf.org.za/index.php/tvl/article/download/2030/1749
French
null
Culturalisme et réflexion existentialiste— Une lecture de Retour de manivelle de Julien Kilanga Musinde
Tydskrif vir letterkunde
2,017
cc-by-sa
7,546
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
https://openalex.org/W2054879124
https://bmcneurosci.biomedcentral.com/counter/pdf/10.1186/1471-2202-10-S1-P144
English
null
Learning spike-timings based representations of sensory stimuli with leaky integrate-and-fire neurons
BMC neuroscience
2,009
cc-by
873
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 (page number not for citation purposes) (page number not for citation purposes) BMC Neuroscience 2009, 10(Suppl 1):P144 http://www.biomedcentral.com/1471-2202/10/S1/P144 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 Page 2 of 2 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge
https://openalex.org/W4391778246
https://juniperpublishers.com/oajggm/pdf/OAJGGM.MS.ID.555571.pdf
English
null
Thrombocytopenia and Physical Activity among Older Adults: The Tenuous Line between Bleeding Preventiona Physical Functional Decline
Open access journal of gerontology & geriatric medicine
2,017
cc-by
2,488
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: 730-739. 13. Janssen I, Heymsfield SB, Ross R (2002) Low relative skeletal muscle mass (sarcopenia) in older persons is associated with functional impairment and physical disability. Journal of the American Geriatric Society 50: 889-896. Conclusion 14. Kövari E, Gold G, Herrmann FR, Canuto A, Hof PR, et al. (2004) Cortical 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 will reach you the below assets • Quality Editorial service • Swift Peer Review • Reprints availability • E-prints Service • Manuscript Podcast for convenient understanding • Global attainment for your research • Manuscript accessibility in different formats ( Pdf, E-pub, Full Text, Audio) • Unceasing customer service Track the below URL for one-step submission https://juniperpublishers.com/online-submission.php This work is licensed under Creative Commons Attribution 4.0 Licens DOI: 10.19080/OAJGGM.2017.01.555571 This work is licensed under Creative Commons Attribution 4.0 Licens DOI: 10.19080/OAJGGM.2017.01.555571 003
https://openalex.org/W4360998424
https://www.qeios.com/read/ZKPXED/pdf
English
null
Review of: "Kirchhoff Coupling Generates ATP, the Chemical Energy of Life"
null
2,023
cc-by
169
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
https://openalex.org/W3116801266
https://www.frontiersin.org/articles/10.3389/fpsyg.2020.610156/pdf
English
null
Stay Mindful and Carry on: Mindfulness Neutralizes COVID-19 Stressors on Work Engagement via Sleep Duration
Frontiers in psychology
2,020
cc-by
11,872
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 et al., 2015). REFERENCES Consult. Clin. Psychol. 75, 447–455. doi: 10.1037/0022-006X.75.3.447 Bartlett, L., Martin, A., Neil, A. L., Memish, K., Otahal, P., Kilpatrick, M., et al. (2019). A systematic review and meta-analysis of workplace mindfulness training randomized controlled trials. J. Occup. Health Psychol. 24, 108–126. doi: 10.1037/ocp0000146 Garland, E. L., Hanley, A. W., Goldin, P. R., and Gross, J. J. (2017). Testing the mindfulness-to-meaning theory: evidence for mindful positive emotion regulation from a reanalysis of longitudinal data. PLoS One 12:e0187727. doi: 10.1371/journal.pone.0187727 Baruch, Y., Wordsworth, R., Mills, C., and Wright, S. (2016). Career and work attitudes of blue-collar workers, and the impact of a natural disaster chance event on the relationships between intention to quit and actual quit behaviour. Eur. J. Work Organ. Psy. 25, 459–473. doi: 10.1080/1359432X.2015.1113168h Gellis, L. A., and Lichstein, K. L. (2009). Sleep hygiene practices of good and poor sleepers in the United  States: an internet-based study. Behav. Ther. 40, 1–9. doi: 10.1016/j.beth.2008.02.001 Germain, A. (2013). Sleep disturbances as the hallmark of PTSD: where are we now? Am. J. Psychiatr. 170, 372–382. doi: 10.1176/appi.ajp.2012.12040432f Benca, R. M., Obermeyer, W. H., Thisted, R. A., and Gillin, J. C. (1992). Sleep and psychiatric disorders: a meta-analysis. Arch. Gen. Psychiatry 49, 651–668. doi: 10.1001/archpsyc.1992.01820080059010 Good, D. J., Lyddy, C. J., Glomb, T. M., Bono, J. E., Brown, K. W., Duffy, M. K., et al. (2016). Contemplating mindfulness at work: an integrative review. J. Manag. 42, 114–142. doi: 10.1177/0149206315617003 Berkman, L. F., Liu, S. Y., Hammer, L., Moen, P., Klein, L. C., Kelly, E., et al. (2015). Work–family conflict, cardiometabolic risk, and sleep duration in nursing employees. J. Occup. Health Psychol. 20, 420–433. doi: 10.1037/a0039143 Goodwin, R., Lemola, S., and Ben-Ezra, M. (2018). Media use and insomnia after terror attacks in France. J. Psychiatr. Res. 98, 47–50. doi: 10.1016/j. jpsychires.2017.12.006 Blau, G. (2011). Exploring the impact of sleep-related impairments on the perceived general health and retention intent of an emergency medical services (EMS) sample. Career Dev. Int. 16, 238–253. doi: 10.1108/13620431111140147 Hafenbrack, A. C., Cameron, L. D., Spreitzer, G. M., Zhang, C., Noval, L. J., and Shaffakat, S. (2020). Helping people by being in the present: mindfulness increases prosocial behavior. Organ. Behav. Hum. Decis. Process. 159, 21–38. doi: 10.1016/j.obhdp.2019.08.005 Bliese, P. D., Edwards, J. R., and Sonnentag, S. (2017). Stress and well-being at work: a century of empirical trends reflecting theoretical and societal influences. J. Appl. Psychol. 102, 389–402. doi: 10.1037/apl0000109hi Hochwarter, W. REFERENCES Christian, M. S., Garza, A. S., and Slaughter, J. E. (2011). Work engagement: a quantitative review and test of its relations with task and contextual performance. Pers. Psychol. 64, 89–136. doi: 10.1111/j.1744-6570.2010.01203.x Ashforth, B. E., and Humphrey, R. H. (1995). Emotion in the workplace: a reappraisal. Hum. Relat. 48, 97–125. Davidson, R. J. (2010). Empirical explorations of mindfulness: conceptual and methodological conundrums. Emotion 10, 8–11. doi: 10.1037/a0018480hf Bakker, A. B., Tims, M., and Derks, D. (2012). Proactive personality and job performance: the role of job crafting and work engagement. Hum. Relat. 65, 1359–1378. doi: 10.1177/0018726712453471 DeArmond, S., and Chen, P. Y. (2004). The effects of job stressors on workplace sleepiness: A meta-analysis. Unpublished master’s thesis, Colorado State University, Fort Collins, Colorado. Banks, S., Van Dongen, H. P., Maislin, G., and Dinges, D. F. (2010). Neurobehavioral dynamics following chronic sleep restriction: dose-response effects of one night for recovery. Sleep 33, 1013–1026. doi: 10.1093/sleep/33.8.1013 Dollard, M. F., Osborne, K., and Manning, I. (2013). Organization–environment adaptation: a macro-level shift in modeling work distress and morale. J. Organ. Behav. 34, 629–647. doi: 10.1002/job.1821 Barclay, N. L., Eley, T. C., Buysse, D. J., Tijsdijk, F. V., and Gregory, A. M. (2010). Genetic and environmental influences on different components of the Pittsburgh sleep quality index and their overlap. Sleep 33, 659–668. doi: 10.1093/sleep/33.5.659 Durmer, J. S., and Dinges, D. F. (2005). Neurocognitive consequences of sleep deprivation. In Seminars in neurology (Vol. 25, No. 01, pp. 117-129). Copyright© 2005 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New  York, NY 10001, USA. Barnes, C. M. (2012). Working in our sleep: sleep and self-regulation in organizations. Organ. Psychol. Rev. 2, 234–257. doi: 10.1177/2041386612450181 Eby, L. T., Allen, T. D., Conley, K. M., Williamson, R. L., Henderson, T. G., and Mancini, V. S. (2019). Mindfulness-based training interventions for employees: a qualitative review of the literature. Hum. Resour. Manag. Rev. 29, 156–178. doi: 10.1016/j.hrmr.2017.03.004 Barnes, C. M., Ghumman, S., and Scott, B. A. (2013). Sleep and organizational citizenship behavior: the mediating role of job satisfaction. J. Occup. Health Psychol. 18, 16–26. doi: 10.1037/a0030349 Barnes, C. M., Wagner, D. T., and Ghumman, S. (2012). Borrowing from sleep to pay work and family: expanding time-based conflict to the broader nonwork domain. Pers. Psychol. 65, 789–819. doi: 10.1111/peps.12002 Fresco, D. M., Segal, Z. V., Buis, T., and Kennedy, S. (2007). Relationship of 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 research, and applications. Cambridge: Cambridge University Press. James, K. (2011). The organizational science of disaster/terrorism prevention and response: theory-building toward the future of the field. J. Organ. Behav. 32, 1013–1032. doi: 10.1002/job.782 Rich, B. L., Lepine, J. A., and Crawford, E. R. (2010). Job engagement: antecedents and effects on job performance. Acad. Manag. J. 53, 617–635. doi: 10.5465/ amj.2010.51468988 j Jankowski, T., and Holas, P. (2014). Metacognitive model of mindfulness. Conscious. Cogn. 28, 64–80. doi: 10.1016/j.concog.2014.06.005 Schaufeli, W. B., and Bakker, A. B. (2004). Job demands, job resources, and their relationship with burnout and engagement: a multi-sample study. J. Organ. Behav. 25, 293–315. doi: 10.1002/job.248h Kahn, W. A. (1990). Psychological conditions of personal engagement and disengagement at work. Acad. Manag. J. 33, 692–724. Kay, A., Masters-Waage, T., and Skarlicki, D. (2019). “Mindfulness at work” in Oxford bibliographies in management. ed. R. W. Griffin (New York, NY: Oxford University Press). Schaufeli, W. B., Bakker, A. B., and Salanova, M. (2006). The measurement of work engagement with a short questionnaire: a cross-national study. Educ. Psychol. Meas. 66, 701–716. doi: 10.1177/0013164405282471 Kay, A. A., and Skarlicki, D. P. (2020). Cultivating a conflict-positive workplace: how mindfulness facilitates constructive conflict management. Organ. Behav. Hum. Decis. Process. 159, 8–20. doi: 10.1016/j.obhdp.2020.02.005 Schaufeli, W. B., Salanova, M., González-Romá, V., and Bakker, A. B. (2002). The measurement of engagement and burnout: a two sample confirmatory factor analytic approach. J. Happiness Stud. 3, 71–92. doi: 10.1023/A:101 5630930326 Keith, M. G., Tay, L., and Harms, P. D. (2017). Systems perspective of Amazon Mechanical Turk for organizational research: review and recommendations. Front. Psychol. 8:1359. doi: 10.3389/fpsyg.2017.01359 Schaufeli, W. B., Taris, T. W., and Van Rhenen, W. (2008). Workaholism, burnout, and work engagement: three of a kind or three different kinds of employee well-being? Appl. Psychol. 57, 173–203. doi: 10.1111/j.1464-0597.2007.00285.xh Kessler, R. C., Berglund, P. A., Coulouvrat, C., Hajak, G., Roth, T., Shahly, V., et al. (2011). Insomnia and the performance of US workers: results from the America insomnia survey. Sleep 34, 1161–1171. doi: 10.5665/SLEEP.1230 Schuh, S. C., Zheng, M. X., Xin, K. R., and Fernandez, J. A. (2019). REFERENCES A., Laird, M. D., and Brouer, R. L. (2008). Board up the windows: the interactive effects of hurricane-induced job stress and perceived resources on work outcomes. J. Manag. 34, 263–289. doi: 10.1177/01492 06307309264 l Brown, K. W., and Ryan, R. M. (2003). The benefits of being present: mindfulness and its role in psychological well-being. J. Pers. Soc. Psychol. 84, 822–848. doi: 10.1037/0022-3514.84.4.822 Byron, K., and Peterson, S. (2002). The impact of a large-scale traumatic event on individual and organizational outcomes: exploring employee and company reactions to September 11, 2001. J. Organ. Behav. 23, 895–910. doi: 10.1002/job.176 Hofmann, D. A., Griffin, M. A., and Gavin, M. B. (2000). “The application of hierarchical linear modeling to organizational research” in Multilevel theory, research, and methods in organizations: Foundations, extensions, and new directions. eds. K. J. Klein and S. W. J. Kozlowski (San Francisco: Jossey-Bass), 467–511. Carvalho, A., and Areal, N. (2016). Great places to work®: resilience in times of crisis. Hum. Resour. Manag. 55, 479–498. doi: 10.1002/hrm.21676 December 2020 | Volume 11 | Article 610156 Frontiers in Psychology | www.frontiersin.org 11 Zheng et al. Mindfulness and COVID-19 Hülsheger, U. R., Feinholdt, A., and Nübold, A. (2015). A low-dose mindfulness intervention and recovery from work: effects on psychological detachment, sleep quality, and sleep duration. J. Occup. Organ. Psychol. 88, 464–489. doi: 10.1111/joop.12115 Preacher, K. J., Zyphur, M. J., and Zhang, Z. (2010). A general multilevel SEM framework for assessing multilevel mediation. Psychol. Methods 15, 209–233. doi: 10.1037/a0020141 Ragins, B. R., Lyness, K. S., Williams, L. J., and Winkel, D. (2014). Life spillovers: the spillover of fear of home foreclosure to the workplace. Pers. Psychol. 67, 763–800. doi: 10.1111/peps.12065 Hülsheger, U. R., Lang, J. W., Depenbrock, F., Fehrmann, C., Zijlstra, F. R., and Alberts, H. J. (2014). The power of presence: the role of mindfulness at work for daily levels and change trajectories of psychological detachment and sleep quality. J. Appl. Psychol. 99, 1113–1128. doi: 10.1037/a0 037702 Raudenbush, S. W., and Bryk, A. S. (2002). Hierarchical linear models: Applications and data analysis methods. 2nd Edn. Vol. 1. University of Chicago, USA: SAGE Publications, Inc. Reb, J., Allen, T., and Vogus, T. J. (2020). Mindfulness arrives at work: deepening our understanding of mindfulness in organizations. Organ. Behav. Hum. Decis. Process. 159, 1–7. doi: 10.1016/j.obhdp.2020.04.001 Itani, O., Jike, M., Watanabe, N., and Kaneita, Y. (2017). Short sleep duration and health outcomes: a systematic review, meta-analysis, and meta-regression. REFERENCES The interpersonal benefits of leader mindfulness: a serial mediation model linking leader mindfulness, leader procedural justice enactment, and employee exhaustion and performance. J. Bus. Ethics 156, 1007–1025. doi: 10.1007/s10551-017-3610-7 Kiken, L. G., and Shook, N. J. (2011). Looking up: mindfulness increases positive judgments and reduces negativity bias. Soc. Psychol. Personal. Sci. 2, 425–431. doi: 10.1177/1948550610396585 Silver, R. C. (2020). Surviving the trauma of COVID-19. Science 369:11. doi: 10.1126/science.abd5396 Kniffin, K. M., Narayanan, J., Anseel, F., Antonakis, J., Ashford, S. J., Bakker, A. B., et al. (In press). COVID-19 and the workplace: implications, issues, and insights for future research and action. Am. Psychol. doi: 10.1037/amp0000716 Sinha, S. S. (2016). Trauma-induced insomnia: a novel model for trauma and sleep research. Sleep Med. Rev. 25, 74–83. doi: 10.1016/j.smrv.2015.01.008 Lanaj, K., Johnson, R. E., and Barnes, C. M. (2014). Beginning the workday yet already depleted? Consequences of late-night smartphone use and sleep. Organ. Behav. Hum. Decis. Process. 124, 11–23. doi: 10.1016/j.obhdp.2014.01.001 Sonnentag, S. (2003). Recovery, work engagement, and proactive behavior: a new look at the interface between nonwork and work. J. Appl. Psychol. 88, 518–528. doi: 10.1037/0021-9010.88.3.518 Lavie, P. (2001). Sleep disturbances in the wake of traumatic events. N. Engl. J. Med. 345, 1825–1832. doi: 10.1056/NEJMra012893 Steed, L. B., Swider, B. W., Keem, S., and Liu, J. T. (2019). Leaving work at work: a meta-analysis on employee recovery from work. J. Manag. [Epub ahead of print].f Liang, L. H., Brown, D. J., Ferris, D. L., Hanig, S., Lian, H., and Keeping, L. M. (2018). The dimensions and mechanisms of mindfulness in regulating aggressive behaviors. J. Appl. Psychol. 103, 281–299. doi: 10.1037/apl0000283 Sutcliffe, K. M., Vogus, T. J., and Dane, E. (2016). Mindfulness in organizations: a cross-level review. Annu. Rev. Organ. Psych. Organ. Behav. 3, 55–81. doi: 10.1146/annurev-orgpsych-041015-062531 Lim, J., and Dinges, D. F. (2010). A meta-analysis of the impact of short-term sleep deprivation on cognitive variables. Psychol. Bull. 136, 375–389. doi: 10.1037/a0018883 Tedeschi, R. G., and Blevins, C. L. (2015). From mindfulness to meaning: implications for the theory of posttraumatic growth. Psychol. Inq. 26, 373–376. doi: 10.1080/1047840X.2015.1075354 Lindsay, E. K., Young, S., Brown, K. W., Smyth, J. M., and Creswell, J. D. (2019). Mindfulness training reduces loneliness and increases social contact in a randomized controlled trial. Proc. Natl. Acad. Sci. 116, 3488–3493. doi: 10.1073/pnas.1813588116 Tuckey, M. R., Sonnentag, S., and Bryan, J. (2018). Are state mindfulness and state work engagement related during the workday? REFERENCES Work Stress. 32, 33–48. doi: 10.1080/02678373.2017.1420707 Litwiller, B., Snyder, L. A., Taylor, W. D., and Steele, L. M. (2017). The relationship between sleep and work: a meta-analysis. J. Appl. Psychol. 102, 682–699. doi: 10.1037/apl0000169 United Nations (2020). Coronavirus: Greatest test since World War Two, says UN chief. Available at: https://www.bbc.com/news/world-52114829 p Long, E. C., and Christian, M. S. (2015). Mindfulness buffers retaliatory responses to injustice: a regulatory approach. J. Appl. Psychol. 100, 1409–1422. doi: 10.1037/apl0000019 Van Bavel, J. J., Baicker, K., Boggio, P. S., Capraro, V., Cichocka, A., Cikara, M., et al. (2020). Using social and behavioural science to support COVID-19 pandemic response. Nat. Hum. Behav. 4, 460–471. doi: 10.1038/s41562- 020-0884-z Mrazek, A. J., Mrazek, M. D., Calcagnotto, L. A., Cloughesy, J. N., Holman, A. M., Masters-Waage, T. C., et al. (2020). Familiarity, attitudes, and self-regulatory challenges related to mindfulness. Mindfulness 11, 1218–1225. doi: 10.1007/ s12671-020-01332-7 Van Wingerden, J., Derks, D., and Bakker, A. B. (2017). The impact of personal resources and job crafting interventions on work engagement and performance. Hum Resour Manag 56 51 67 doi: 10 1002/hrm 21758 Van Wingerden, J., Derks, D., and Bakker, A. B. (2017). The impac resources and job crafting interventions on work engagement and p Hum. Resour. Manag. 56, 51–67. doi: 10.1002/hrm.21758 resources and job crafting interventions on work engagement and performan Hum. Resour. Manag. 56, 51–67. doi: 10.1002/hrm.21758l Hum. Resour. Manag. 56, 51–67. doi: 10.1002/hrm.21758 Ong, J. C., and Smith, C. E. (2017). Using mindfulness for the treatment of insomnia. Curr. Sleep Med. Rep. 3, 57–65. doi: 10.1007/s40675-017-0068-1 Wang, M., Liu, S., Zhan, Y., and Shi, J. (2010). Daily work–family conflict and alcohol use: testing the cross-level moderation effects of peer drinking norms and social support. J. Appl. Psychol. 95, 377–386. doi: 10.1037/a0018138 Ong, J. C., Ulmer, C. S., and Manber, R. (2012). Improving sleep with mindfulness and acceptance: a metacognitive model of insomnia. Behav. Res. Ther. 50, 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
https://openalex.org/W3191105665
https://lirias.kuleuven.be/bitstream/123456789/653711/2/Gitelman%20syndrome.pdf
English
null
Gitelman syndrome
Orphanet journal of rare diseases
2,008
cc-by
4,084
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 "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 Page 6 of 6 (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.
https://openalex.org/W2056141790
https://www.revistas.usp.br/geousp/article/download/74016/77675
Portuguese
null
BREVES NOTAS SOBRE O VIII COLÓQUIO DE GEOCRÍTICA.
GEOUSP
2,006
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.
https://openalex.org/W2900564207
https://www.surgonco.ru/jour/article/download/316/290
Russian
null
Technical Features of Robot-Assisted Prostatectomy in Patients with Very Enlarged Prostates
Kreativnaâ hirurgiâ i onkologiâ
2,018
cc-by
5,822
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 у пациентов с более крупными простатами. Список литературы DOI: 10.1186/s12894-015- 0006-9 23 Rocco B., Cozzi G., Spinelli M.G. Coelho R.F., Patel V.R., Tewari A., et al. Posterior musculofascial reconstruction after radical prostatecto- my: a systematic review of the literature. Eur Urol. 2012;62(5):779–90. DOI: 10.1016/j.eururo.2012.05.041 6 Ficarra V., Novara G., Ahlering T.E., Costello A., Eastham J.A., Grae- fen M., et al. Systematic review andmeta-analysis of studies reporting potency rates after robotassisted radical prostatectomy. Eur Urol. 2012;62(3):418–30. DOI: 10.1016/j.eururo.2012.05.046 24 Grasso A.A., Mistretta F.A., Sandri M., Cozzi G., De Lorenzis E., Rosso M., et al. Posterior musculofascial reconstruction after radical prostatectomy: an updated systematic review and a meta-analysis. BJU Int. 2016;118(1):20–34. DOI: 10.1111/bju.13480 7 Albisinni S., Aoun F., Le Dinh D., Limani K., Hawaux E., Peltier A., et al. Adapting the robotic platform to small operating theaters: our experience with the side-docking technique for robotic-assisted laparo- scopic prostatectomy. Surg Endosc. 2016;30(10):4464–8. DOI: 10.1007/ s00464-016-4777-1 25 Tillier C., van Muilekom H.A.M., Bloos-van der Hulst J., Grivas N., van der Poel H.G. Vesico-urethral anastomosis (VUA) evaluation of short- and long-term outcome after robot-assisted laparoscopic radical prostatectomy (RARP): selective cystogram to improve outcome. J Ro- bot Surg. 2017;11(4):441–6. DOI: 10.1007/s11701-017-0677-1 8 Graefen M., Beyer B., Schlomm T. Outcome of radical prostatectomy: is it the approach or the surgical expertise?. Eur Urol. 2014;66(3):457– 8. DOI: 10.1016/j.eururo.2013.12.010 26 Liu Z., Li Y.W., Wu W.R., Lu Q. Long-term clinical efficacy and safety profile of transurethral resection of prostate versus plasmakinetic resection of the prostate for benign prostatic hyperplasia. Urology. 2017;103:198–203. DOI: 10.1016/j.urology.2017.02.006 9 Tugcu V., Atar A., Sahin S., Kargi T., Gokhan Seker K., IlkerComez Y., et al. Robot-assisted radical prostatectomy after previous prostate sur- gery. JSLS. 2015;19(4):e2015.00080. DOI: 10.4293/JSLS.2015.00080 10 Mustafa M., Pettaway C.A., Davis J.W., Pisters L. Robotic or open radical prostatectomy after previous open surgery in the pelvic region. Korean J Urol. 2015;56(2):131–7. DOI: 10.4111/kju.2015.56.2.131 27 Dai X., Fang X., Ma Y., Xianyu J. Benign prostatic hyperplasia and the risk of prostate cancer and bladder cancer: a meta-analysis of observa- tional studies. Medicine (Baltimore). 2016;95(18):e3493. DOI: 10.1097/ MD.0000000000003493 11 Schiavina R., Borghesi M., Dababneh H., Rossi M.S., Pultrone C.V., Vagnoni V., et al. The impact of a structured intensive modular training in the learning curve of robot assisted radical prostatectomy. Arch Ital Urol Androl. 2018;90(1):1–7. DOI: 10.4081/aiua.2018.1.1 28 Зырянов А.В., Пономарев А.В., Суриков А.С., Коваленко Р.Ю., Попов И.Б. Робот-ассистированная радикальная простатэктомия у пациентов с увеличенным объемом предстательной железы. Уральский медицинский журнал. 2017;(2):65–8. Список литературы a complete intrafascial approach. Clin Anat. 2015;28(7):896–902. DOI: 10.1002/ca.22576 1 Зырянов А.В., Кельн А.А., Пономарев А.В., Попов И.Б., Сури- ков А.С., Сальников М.А. и др. Таргетная МРТ-УЗ FUSION би- опсия предстательной железы: новые возможности диагностики РПЖ. Уральский медицинский журнал. 2017;(2):45–51. 1 Зырянов А.В., Кельн А.А., Пономарев А.В., Попов И.Б., Сури- ков А.С., Сальников М.А. и др. Таргетная МРТ-УЗ FUSION би- опсия предстательной железы: новые возможности диагностики РПЖ. Уральский медицинский журнал. 2017;(2):45–51. 19 Antonelli A., Palumbo C., Veccia A., Fisogni S., Zamboni S., Furlan M., et al. Standard vs delayed ligature of the dorsal vascular complex dur- ing robot-assisted radical prostatectomy: results from a randomized controlled trial. J Robot Surg. 2018 Jul 13. [Epub ahead of print]. DOI: 10.1007/s11701-018-0847-9 2 Porcaro A.B., Novella G., Molinari A., Terrin A., Minja A., De Mar- co V., et al. Prostate volume index and chronic inflammation of the prostate type IV with respect to the risk of prostate cancer. Urol Int. 2015;94(3):270–85. DOI: 10.1159/000362176.3 20 Li H., Liu C., Zhang H., Xu W., Liu J., Chen Y., et al. The use of unidi- rectional barbed suture for urethrovesical anastomosis during robot- assisted radical prostatectomy: a systematic review and meta-analysis of efficacy and safety. PLoS One. 2015;10(7):e0131167. DOI: 10.1371/ journal.pone.0131167 3 Yahya N., Ebert M.A., Bulsara M., Haworth A., Kennedy A., Jo- seph D.J., Denham J.W. Dosimetry, clinical factors and medication intake influencing urinary symptoms after prostate radiotherapy: An analysis of data from the RADAR prostate radiotherapy trial. Ra- diother Oncol. 2015;116(1):112–8. DOI: 10.1016/j.radonc.2015.06.011 21 Molinari A.L., Simonelli G., De Concilio B., Porcaro A.B., Del Biondo D., Zeccolini G., Celia A. Is ureteral stent placement by the transurethral approach during robot-assisted radical prostatectomy an effective option to preoperative technique?. J Endourol. 2014 Aug;28(8):896–8. DOI: 10.1089/end.2014.0061 4 Cho S.Y., Ro Y.K., Kim H., Son H. Preoperative urinary retention increased the risk of urinary retention after photoselective vaporization of the prostate. World J Mens Health. 2015;33(3):182–7. DOI: 10.5534/ wjmh.2015.33.3.182 22 Alessandro S., Alessandro G., Susanna C., Michele I., Francesca D.Q., Andrea F., et al. Laparoscopic versus open radical prostatectomy in high prostate volume cases: impact on oncological and functional results. Int Braz J Urol. 2016;42(2):223–33. DOI: 10.1590/S1677-5538. IBJU.2015.0385 5 Bove P., Iacovelli V., Celestino F., De Carlo F., Vespasiani G., Finazzi Agrò E. 3D vs 2D laparoscopic radical prostatectomy in organ-confined prostate cancer: comparison of operative data and pentafecta rates: a single cohort study. BMC Urol. 2015;15:12. Список литературы 12 Аляев Ю.Г., Пшихачев А.М., Шпоть Е.В., Сорокин Н.И., Ды- мов А.М. Камни мочевого пузыря у больных раком простаты: осо- бенности хирургического лечения. В кн.: V Российский конгресс по эндоурологии и новым технологиям: материалы конференции. Ростов-на-Дону, 2016. 29 Yasui T., Tozawa K., Kurokawa S., Okada A., Mizuno K., Umemoto Y., et al. Impact of prostate weight on perioperative outcomes of robot- assisted laparoscopic prostatectomy with a posterior approach to the seminal vesicle. BMC Urol. 2014;14:6. DOI: 10.1186/1471-2490-14-6 13 Guillonneau B., Vallancien G. Laparoscopic radical prostatectomy: the Montsouris technique. J Urol. 2000;163(6):1643–9. PMID: 10799152. 30 Galfano A., Panarello D., Secco S., Di Trapani D., Barbieri M., Napoli G., et al. Does prostate volume have an impact on the functional and oncological results of Retzius-sparing robot-assisted radical pros- tatectomy?. Minerva Urol Nefrol. 2018;70(4):408–13. DOI: 10.23736/ S0393-2249.18.03069-2 14 Shah A.A., Gahan J.C., Sorokin I. Comparison of robot-assisted versus open simple prostatectomy for benign prostatic hyperplasia. Curr Urol Rep. 2018;19(9):71. DOI: 10.1007/s11934-018-0820-1 15 Yasui T., Tozawa K., Okada A., Kurokawa S., Kubota H., Mizuno K. Outcomes of robot-assisted laparoscopic prostatectomy with a posterior approach to the seminal vesicle in 300 patients. Int Sch Res Notices. 2014;2014:565737. DOI: 10.1155/2014/565737 31 Kasivisvanathan V., Challacombe B. (eds) The big prostate. London: Springer, 2017. 32 Пушкарь Д.Ю., Дьяков В.В., Васильев А.О., Котенко Д.В. Сравне- ние функциональных результатов после радикальной позадилон- ной и робот-ассистированной простатэктомий, выполненных по нервосберегающей методике хирургами с опытом более 1000 операций. Урология. 2017;(1):50–3. DOI: 10.18565/urol.2017.1.50-53 16 Ja Yoon Ku, Hong Koo Ha. Learning curve of robot-assisted laparo- scopic radical prostatectomy for a single experienced surgeon: com- parison with simultaneous laparoscopic radical prostatectomy. World J Mens Health. 2015;33(1):30–5. DOI: 10.5534/wjmh.2015.33.1.30 33 Tang K., Jiang K., Chen H., Chen Z., Xu H., Ye Z. Robotic vs. Retro- pubic radical prostatectomy in prostate cancer: A systematic review and an meta-analysis update. Oncotarget. 2017;8(19):32237–57. DOI: 10.18632/oncotarget.13332 17 Bartoletti R., Mogorovich A., Francesca F., Pomara G., Selli C. Com- bined bladder neck preservation and posterior musculofascial recon- struction during robotic assisted radical prostatectomy: effects on early and long-term urinary continence recovery. BMC Urol. 2017;17(1):119. DOI: 10.1186/s12894-017-0308-1 34 Gandaglia G., Sammon J.D., Chang S.L., Choueiri T.K., Hu J.C., Karakiewicz P.I., et al. Comparative effectiveness of robot-assisted and open radical prostatectomy in the postdissemination era. J Clin Oncol. 2014;32(14):1419–26. DOI: 10.1200/JCO.2013.53.5096 18 Asimakopoulos A.D., Miano R., Galfano A., Bocciardi A.M., Vespa- siani G., Spera E., et al. Информация о конфликте интересов. Конфликт интересов отсутствует. Информация о конфликте интересов. Конфликт интересов отсутствует. Информация о спонсорстве. Данная работа не финансировалась. Необходимо более длительное наблюдение, чтобы под- твердить гипотезу, согласно которой более низкая ча- 122 Creative Surgery and Oncology, Volume 8, No. 2, 2018 Оригинальные исследования References a complete intrafascial approach. Clin Anat. 2015;28(7):896–902. DOI: 10.1002/ca.22576 1 Zyryanov A.V., Keln A.A., Ponomarev A.V., Popov I.B., Surikov A.S., Salnykov M.A., et al. Targeted MRi-US fusion prostate biopsy: new possibilities of diagnosis of prostate cancer. Ural Medical Journal. 2017;(2):45–51. (in Russ.) 1 Zyryanov A.V., Keln A.A., Ponomarev A.V., Popov I.B., Surikov A.S., Salnykov M.A., et al. Targeted MRi-US fusion prostate biopsy: new possibilities of diagnosis of prostate cancer. Ural Medical Journal. 2017;(2):45–51. (in Russ.) 19 19 Antonelli A., Palumbo C., Veccia A., Fisogni S., Zamboni S., Furlan M., et al. Standard vs delayed ligature of the dorsal vascular complex dur- ing robot-assisted radical prostatectomy: results from a randomized controlled trial. J Robot Surg. 2018 Jul 13. [Epub ahead of print]. DOI: 10.1007/s11701-018-0847-9 2 Porcaro A.B., Novella G., Molinari A., Terrin A., Minja A., De Marco V., et al. Prostate volume index and chronic inflammation of the prostate type IV with respect to the risk of prostate cancer. Urol Int. 2015;94(3):270–85. DOI: 10.1159/000362176 20 Li H., Liu C., Zhang H., Xu W., Liu J., Chen Y., et al. The use of unidi- rectional barbed suture for urethrovesical anastomosis during robot- assisted radical prostatectomy: a systematic review and meta-analysis of efficacy and safety. PLoS One. 2015;10(7):e0131167. DOI: 10.1371/ journal.pone.0131167 3 Yahya N., Ebert M.A., Bulsara M., Haworth A., Kennedy A., Jo- seph D.J., Denham J.W. Dosimetry, clinical factors and medication intake influencing urinary symptoms after prostate radiotherapy: An analysis of data from the RADAR prostate radiotherapy trial. Radiother Oncol. 2015;116(1):112–8. DOI: 10.1016/j.radonc.2015.06.011 21 21 Molinari A.L., Simonelli G., De Concilio B., Porcaro A.B., Del Biondo D., Zeccolini G., Celia A. Is ureteral stent placement by the transurethral approach during robot-assisted radical prostatectomy an effective option to preoperative technique?. J Endourol. 2014 Aug;28(8):896–8. DOI: 10.1089/end.2014.0061 4 Cho S.Y., Ro Y.K., Kim H., Son H. Preoperative urinary retention increased the risk of urinary retention after photoselective vaporization of the prostate. World J Mens Health. 2015;33(3):182–7. DOI: 10.5534/ wjmh.2015.33.3.182 22 Alessandro S., Alessandro G., Susanna C., Michele I., Francesca D.Q., Andrea F., et al. Laparoscopic versus open radical prostatectomy in high prostate volume cases: impact on oncological and functional results. Int Braz J Urol. 2016;42(2):223–33. DOI: 10.1590/S1677-5538. IBJU.2015.0385 5 Bove P., Iacovelli V., Celestino F., De Carlo F., Vespasiani G., Finazzi Agrò E. 3D vs 2D laparoscopic radical prostatectomy in organ-confined prostate cancer: comparison of operative data and pentafecta rates: a single cohort study. References BMC Urol. 2015;15:12. DOI: 10.1186/s12894-015- 0006-9 23 Rocco B., Cozzi G., Spinelli M.G. Coelho R.F., Patel V.R., Tewari A., et al. Posterior musculofascial reconstruction after radical prostatecto- my: a systematic review of the literature. Eur Urol. 2012;62(5):779–90. DOI: 10.1016/j.eururo.2012.05.041 6 Ficarra V., Novara G., Ahlering T.E., Costello A., Eastham J.A., Grae- fen M., et al. Systematic review andmeta-analysis of studies reporting potency rates after robotassisted radical prostatectomy. Eur Urol. 2012;62(3):418–30. DOI: 10.1016/j.eururo.2012.05.046 24 Grasso A.A., Mistretta F.A., Sandri M., Cozzi G., De Lorenzis E., Rosso M., et al. Posterior musculofascial reconstruction after radical prostatectomy: an updated systematic review and a meta-analysis. BJU Int. 2016;118(1):20–34. DOI: 10.1111/bju.13480 7 Albisinni S., Aoun F., Le Dinh D., Limani K., Hawaux E., Peltier A., et al. Adapting the robotic platform to small operating theaters: our experience with the side-docking technique for robotic-assisted laparo- scopic prostatectomy. Surg Endosc. 2016;30(10):4464–8. DOI: 10.1007/ s00464-016-4777-1 25 Tillier C., van Muilekom H.A.M., Bloos-van der Hulst J., Grivas N., van der Poel H.G. Vesico-urethral anastomosis (VUA) evaluation of short- and long-term outcome after robot-assisted laparoscopic radical prostatectomy (RARP): selective cystogram to improve outcome. J Ro- bot Surg. 2017;11(4):441–6. DOI: 10.1007/s11701-017-0677-1 8 Graefen M., Beyer B., Schlomm T. Outcome of radical prostatectomy: is it the approach or the surgical expertise?. Eur Urol. 2014;66(3):457– 8. DOI: 10.1016/j.eururo.2013.12.010 26 Liu Z., Li Y.W., Wu W.R., Lu Q. Long-term clinical efficacy and safety profile of transurethral resection of prostate versus plasmakinetic resection of the prostate for benign prostatic hyperplasia. Urology. 2017;103:198–203. DOI: 10.1016/j.urology.2017.02.006 9 Tugcu V., Atar A., Sahin S., Kargi T., Gokhan Seker K., IlkerComez Y., et al. Robot-assisted radical prostatectomy after previous prostate sur- gery. JSLS. 2015;19(4):e2015.00080. DOI: 10.4293/JSLS.2015.00080 10 Mustafa M., Pettaway C.A., Davis J.W., Pisters L. Robotic or open radical prostatectomy after previous open surgery in the pelvic region. Korean J Urol. 2015;56(2):131–7. DOI: 10.4111/kju.2015.56.2.131 27 Dai X., Fang X., Ma Y., Xianyu J. Benign prostatic hyperplasia and the risk of prostate cancer and bladder cancer: a meta-analysis of observa- tional studies. Medicine (Baltimore). 2016;95(18):e3493. DOI: 10.1097/ MD.0000000000003493 11 Schiavina R., Borghesi M., Dababneh H., Rossi M.S., Pultrone C.V., Vagnoni V., et al. The impact of a structured intensive modular training in the learning curve of robot assisted radical prostatectomy. Arch Ital Urol Androl. 2018;90(1):1–7. DOI: 10.4081/aiua.2018.1.1 28 Zyryanov A.V., Ponomarev A.V., Surikov A.S., Kovalenko R.Y., Po- pov I.B. Robot-assisted radical prostatectomy in patients with enlarged prostate. Ural Medical Journal. References 2017;(2):65–8. (in Russ.) 12 Alyaev Yu.G., Pshikhachev A.M., Shpot E.V., Sorokin N.I., Dymov A.M. Bladder stones in patients with prostate cancer: features of surgical treatment. In: Proceedings of the 5th Russian Congress on Endourology and New Technologies. Rostov-na-Donu, 2016. (in Russ.) 29 Yasui T., Tozawa K., Kurokawa S., Okada A., Mizuno K., Umemoto Y., et al. Impact of prostate weight on perioperative outcomes of robot- assisted laparoscopic prostatectomy with a posterior approach to the seminal vesicle. BMC Urol. 2014;14:6. DOI: 10.1186/1471-2490-14-6 13 Guillonneau B., Vallancien G. Laparoscopic radical prostatectomy: the Montsouris technique. J Urol. 2000;163(6):1643–9. PMID: 10799152. 30 Galfano A., Panarello D., Secco S., Di Trapani D., Barbieri M., Napoli G., et al. Does prostate volume have an impact on the functional and oncological results of Retzius-sparing robot-assisted radical pros- tatectomy?. Minerva Urol Nefrol. 2018;70(4):408–13. DOI: 10.23736/ S0393-2249.18.03069-2 14 Shah A.A., Gahan J.C., Sorokin I. Comparison of robot-assisted versus open simple prostatectomy for benign prostatic hyperplasia. Curr Urol Rep. 2018;19(9):71. DOI: 10.1007/s11934-018-0820-1 15 Yasui T., Tozawa K., Okada A., Kurokawa S., Kubota H., Mizuno K. Outcomes of robot-assisted laparoscopic prostatectomy with a posterior approach to the seminal vesicle in 300 patients. Int Sch Res Notices. 2014;2014:565737. DOI: 10.1155/2014/565737 31 Kasivisvanathan V., Challacombe B. (eds) The big prostate. London: Springer, 2017. 32 Pushkar D.Yu., Dyakov V.V., Vasilyev A.O., Kotenko D.V. Comparison of functional outcomes after retropubic and robot-assisted radi- cal nerve-sparing prostatectomy conducted by surgeons with total caseloads of over 1000 prostatectomies. Urologiia. 2017;(1):50–3. DOI: 10.18565/urol.2017.1.50-53 (in Russ.) 16 Ja Yoon Ku, Hong Koo Ha. Learning curve of robot-assisted laparo- scopic radical prostatectomy for a single experienced surgeon: com- parison with simultaneous laparoscopic radical prostatectomy. World J Mens Health. 2015;33(1):30–5. DOI: 10.5534/wjmh.2015.33.1.30 33 Tang K., Jiang K., Chen H., Chen Z., Xu H., Ye Z. Robotic vs. Retro- pubic radical prostatectomy in prostate cancer: A systematic review and an meta-analysis update. Oncotarget. 2017;8(19):32237–57. DOI: 10.18632/oncotarget.13332 17 Bartoletti R., Mogorovich A., Francesca F., Pomara G., Selli C. Com- bined bladder neck preservation and posterior musculofascial recon- struction during robotic assisted radical prostatectomy: effects on early and long-term urinary continence recovery. BMC Urol. 2017;17(1):119. DOI: 10.1186/s12894-017-0308-1 34 Gandaglia G., Sammon J.D., Chang S.L., Choueiri T.K., Hu J.C., Karakiewicz P.I., et al. Comparative effectiveness of robot-assisted and open radical prostatectomy in the postdissemination era. J Clin Oncol. 2014;32(14):1419–26. Список литературы Retzius-sparing robot-assisted laparoscopic 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 Asimakopoulos A.D., Miano R., Galfano A., Bocciardi A.M., Vespa- siani G., Spera E., et al. Retzius-sparing robot-assisted laparoscopic radical prostatectomy: Critical appraisal of the anatomic landmarks for 124 Creative Surgery and Oncology, Volume 8, No. 2, 2018
https://openalex.org/W4391695085
https://pjmd.zu.edu.pk/wp-content/uploads/2024/01/PJMD-13.1-OA_PFO-January-March-2024-Final.pdf
English
null
Proximal Fibular Osteotomy Vs Proximal Fibular Osteotomy with Intra-Articular Hyaluronic Acid: Difference in Terms of Pain Relief and Functional Outcome
Pakistan journal of medicine and dentistry
2,024
cc-by
4,164
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 97/md.0000000000016609 REFERENCES 1. Hsu H, Siwiec RM. Knee Osteoarthritis. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2023. 1. Hsu H, Siwiec RM. Knee Osteoarthritis. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2023. 2. Primorac D, Molnar V, Rod E, Jeleč Ž, Čukelj F, Matišić V, et al. Knee Osteoarthritis: A Review of Pathogenesis and State-Of-The-Art Non-Operative Therapeutic Considerations. Genes. 2020;11(8):854 3. Cui A, Li H, Wang D, Zhong J, Chen Y, Lu H. Global, regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies. eClinicalMedi- cine. 2020;29 DOI: 10.1016/j.eclinm.2020.100587 4. Ravalli S, Pulici C, Binetti S, Aglieco A, Vecchio M, 1. Hsu H, Siwiec RM. Knee Osteoarthritis. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2023. 2. Primorac D, Molnar V, Rod E, Jeleč Ž, Čukelj F, Matišić V, et al. Knee Osteoarthritis: A Review of Pathogenesis and State-Of-The-Art Non-Operative Therapeutic Considerations. Genes. 2020;11(8):854 3. Cui A, Li H, Wang D, Zhong J, Chen Y, Lu H. Global, regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies. eClinicalMedi- cine. 2020;29 DOI: 10.1016/j.eclinm.2020.100587 4 R lli S P li i C Bi tti S A li A V hi M 14. Yang Z-Y, Chen W, Li C-X, Wang J, Shao D-C, Hou Z-Y, et al. Medial compartment decompression by fibular osteotomy to treat medial compartment knee osteoarthritis: a pilot study. Orthopedics. 2015;38(12):e1110-e4 15. Zheng Z, Sun Y, Zhang X, Chen W, Li S, Zhang Y. The pathogenesis and clinical imageology research of the knee osteoarthritis. J Hebei Med Univ. 2014;35(5):599-600 4. Ravalli S, Pulici C, Binetti S, Aglieco A, Vecchio M, 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 Edition). 2013;57(1):6-14 ment patients with osteoarthritis. Revista Española de Cirugía Ortopédica y Traumatología (English Edition). 2013;57(1):6-14 Knee Surgery & Related Research. 2019;31(1):1-7 Knee Surgery & Related Research. 2019;31(1):1-7 17. Emami A, Tepper J, Short B, Yaksh TL, Bendele AM, Ramani T, et al. Toxicology evaluation of drugs admin- istered via uncommon routes: intranasal, intraocular, intrathecal/intraspinal, and intra-articular. Internation- al journal of toxicology. 2018;37(1):4-27 Knee Surgery & Related Research. 2019;31(1):1 7 17. REFERENCES Emami A, Tepper J, Short B, Yaksh TL, Bendele AM, Ramani T, et al. Toxicology evaluation of drugs admin- istered via uncommon routes: intranasal, intraocular, intrathecal/intraspinal, and intra-articular. Internation- al journal of toxicology. 2018;37(1):4-27 22. Delbarre A, Amor B, Bardoulat I, Tetafort A, Pelletier-Fleury N. Do intra-articular hyaluronic acid injections delay total knee replacement in patients with osteoarthritis–a Cox model analysis. PLoS One. 2017;12(11):e0187227 18. Cao Y, Ma Y, Tao Y, Lin W, Wang P. Intra-Articular Drug Delivery for Osteoarthritis Treatment. Pharmaceu- tics. 2021;13(12) DOI: 10.3390/pharmaceutics13122166 19. Huang H, Lou Z, Zheng S, Wu J, Yao Q, Chen R, et al. Intra-articular drug delivery systems for osteoar- thritis therapy: shifting from sustained release to enhancing penetration into cartilage. Drug Deliv. 2022;29(1):767-791 DOI: 10.1080/10717544.2022.2048130 20. McIntyre LF, Bhattacharyya S, Yadalam S, Bisson B, Kim M. Impact of hyaluronic acid injections on utilization of pain management medications. Am J Pharm Benefits. 2017;9(6):195-199 23. Altman RD, Bedi A, Karlsson J, Sancheti P, Schemitsch E. Product differences in intra-articular hyaluronic acids for osteoarthritis of the knee. The American journal of sports medicine. 2016;44(8):215821-65 24. Zou G, Lan W, Zeng Y, Xie J, Chen S, Qiu Y. Early clinical effect of proximal fibular osteotomy on knee osteoarthritis. Biomedical Research (0970-938X). 2017;28(21) 23. Altman RD, Bedi A, Karlsson J, Sancheti P, Schemitsch E. Product differences in intra-articular hyaluronic acids for osteoarthritis of the knee. The American journal of sports medicine. 2016;44(8):215821-65 24. Zou G, Lan W, Zeng Y, Xie J, Chen S, Qiu Y. Early clinical effect of proximal fibular osteotomy on knee osteoarthritis. Biomedical Research (0970-938X). 2017;28(21) 25. Yang ZY, Chen W, Li CX, Wang J, Shao DC, Hou ZY, et al. Medial Compartment Decompression by Fibular Osteoto- my to Treat Medial Compartment Knee Osteoarthritis: A Pilot Study. Orthopedics. 2015;38(12):e1110-1114 DOI: 10.3928/01477447-20151120-08 21. Mar J, Jurado MR, Arrospide A, Fidalgo AE, Lopez BS. Cost-analysis of viscosupplementation treatment with hyaluronic acid in candidate knee replace- 21. Mar J, Jurado MR, Arrospide A, Fidalgo AE, Lopez BS. Cost-analysis of viscosupplementation treatment with hyaluronic acid in candidate knee replace- PAKISTAN JOURNAL OF MEDICINE AND DENTISTRY 2024, VOL. 13 (01) 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
https://openalex.org/W3047119975
https://jbaar.journals.ekb.eg/article_105884_fc9091b31209ea115888ef82f26a9628.pdf
English
null
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
cc-by-sa
3,640
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 ameliorated effect on MTX-induced hepatic and 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 expressions in the liver. Abouzeinab, N.S. (2013). Cytoprotective effect and antioxidant properties of silymarin on cisplatin induced hepatotoxicity in rats: A biochemical and histochemical study. International Journal of Cancer Research, 9(1):9-23. El-Atrash, A., Zaki, S., Tousson, E., Shoir, M.A. (2017). Protective potential of grape seed extract against monosodium glutamate induced liver toxicity and oxidative stress in young rats. Journal of Advanced Trends in Basic and Applied Science, 1(3):257-262. 5. References Abd Eldaim, M.A., Tousson, E., El Sayed, I.E.T., Awd, W.M. (2018). Ameliorative effects of Saussurea lappa root aqueous extract against Ethephon‐induced reproductive toxicity in male rats. Environmental toxicology, 2018:1-10. Akela, Akela, M.A., El-Atrash, A., El-Kilany, M.I., Tousson, E. (2018). Qualitative and quantitative characterization of biologically active compounds of Rosemary (Rosmarinus officinalis) Leaf Extract. Journal of Journal of Bioscience and Applied Research, 2019, Vol.5, No. 1, P.1 -7 pISSN: 2356 - 9174, eISSN: 2356 –9182 6 Advanced Trends in Basic and Applied Science, 2(1):59-64. Hafez, E., El-Atrash, A., El-Basuoney, H.A., Tousson, E. (2017). Protective role of silymarin against anticancer drug Glivec- induced testicular damage in adult male rats. Advanced Trends in Basic and Applied Science, 1(2):128-134. Bancroft, J.D., Stevens, A., Turner, D. (1990). Theory and practice of histological technique 3rd ed. Churchill, Livingstone, Edinburgh, London. Habtemariam, S. (2016). The therapeutic potential of rosemary (Rosmarinus officinalis) diterpenes for Alzheimer’s disease. Evidence-Based Complementary and Alternative Medicine, 1- 14. Basuony, M., Hafez, E., Tousson, E., Massoud, A., Elsomkhraty, S., Eldakamawy, S. (2015). Beneficial role of Panax ginseng root aqueous extract against Cisplatin induced blood toxicity in rats. American Journal of Biological Chemistry, 3(1): 1-7. Juma, F.D. (1984). Effect of liver failure on the pharmacokinetics of cyclophosphamide. European journal of clinical pharmacology, 26(5):591-593. Doumas, B. T., Watson, W. A., Biggs, H. G. (1971). Albumin standards and the measurement of serum albumin with bromcresol green. Clinica Chimica Acta, 31(1): 87-96. Kamboj, S.S., Sandhir, R. (2011). Protective effect of N-acetylcysteine supplementation on mitochondrial oxidative stress and mitochondrial enzymes in cerebral cortex of streptozotocin-treated diabetic rats. Mitochondrion, 11(1):214-222. Tousson, E., Elgharabawy, R.M., Elmasry, T.A. (2018a). Grape Seed Proanthocyanidin Ameliorates Cardiac Toxicity Induced by Boldenone Undecylenate through Inhibition of NADPH Oxidase and Reduction in the Expression of NOX2 and NOX4. Oxidative Medicine and Cellular Longevity, 1-12. McDonald, G.B., Slattery, J.T., Bouvier, M.E., Ren, S., Batchelder, A.L., Kalhorn, T.F., Schoch, H.G., Anasetti, C., Gooley, T. (2003). Cyclophosphamide metabolism, liver toxicity, and mortality following hematopoietic stem cell transplantation. Blood, 101(5):2043-2048. Tousson, E., Hafez, E., Zaki, S., Gad, A. (2016). The cardioprotective effects of L-carnitine on rat cardiac injury, apoptosis, and oxidative stress caused by amethopterin. Environmental Science and Pollution Research, 23(20): 20600-20608. Bayomy, M.F., Tousson, E., Ahmed, A.A. (2017). Protective role of rosemary against anticancer drug Etoposide-induced testicular toxicity and oxidative stress in rats. Journal of Advanced Trends in Basic and Applied Science, 1(2):1-5 Tousson, E., Bayomy, M.F., Ahmed, A.A. (2018b). 5. References Rosemary extract modulates fertility potential, DNA fragmentation, injury, KI67 and P53 alterations induced by etoposide in rat testes. Biomedicine & Pharmacotherapy., 98: 769-774. Moss, D.M., Henderson, A.R. 1999. Tietz Textbook of Clinical Enzymology. WB Saunders Co: Philadelphia, PA, USA, 617-721. Elmasry, T. A., Al-Shaalan, N. H., Tousson, E., El- Morshedy, K., Al-Ghadeer, A. (2018). Star anise extracts modulation of reproductive parameters, fertility potential and DNA fragmentation induced by growth promoter Equigan in rat testes. Brazilian Journal of Pharmaceutical Sciences, 54(1):1-10. Nasr, A.Y. (2013). Morphological, biochemical, histological, and ultrastructural protective effects of misoprostol on cisplatin induced- hepatotoxicity in adult male rats. Saudi Medical Journal, 34(12):1237-1247. El-Moghazy, M., Zedan, N.S., El-Atrsh, A.M., El- Gogary, M., Tousson, E. (2014). The possible effect of diets containing fish oil (omega-3) on hematological, biochemical and histopathogical alterations of rabbit liver and kidney. Biomedicine & Preventive Nutrition, 4(3): 371-377. Al-Rasheed, N.M., El-Masry, T.A., Tousson, E., Hassan, H.M., Al-Ghadeer, A. (2018). Hepatic protective effect of grape seed proanthocyanidin extract against Gleevec- induced apoptosis, liver Injury and Ki67 alterations in rats. Brazilian Journal of Pharmaceutical Sciences, 54(2):1-6. pISSN: 2356 - 9174, eISSN: 2356 –9182 Journal of Bioscience and Applied Research, 2019, Vol.5, No. 1, P.1 -7 7 by methotrexate in rats. Journal of Cancer Research and Treatment, 2(3): 44-51. Al-Rasheed, N.M., El-Masry, T.A., Tousson, E., Hassan, H. M., Al-Ghadeer, A. (2017). Protective Potential of Grape Seed Proanthocyandins Extract against Glivec (Imatinib Mesylate) Induced Liver Toxicity and Oxidative Stress in Male Rats. Annual Research & Review in Biology, 20(6): 1-9. Tousson, E., Hafez, E., Zaki, S., Gad, A. (2014d). P53, Bcl-2 and CD68 expression in response to amethopterin-induced lung injury and ameliorating role of l-carnitine. Biomedicine & Pharmacotherapy, 68(5): 631-639. Oyouni, A.A.A., Saggu, S., Tousson, E., Rehman, H. (2018). Immunosuppressant drug tacrolimus induced mitochondrial nephrotoxicity, modified PCNA and Bcl-2 expression attenuated by Ocimum basilicum L. in CD1 mice. Toxicology Reports, 5:687-694. Tousson, E., Hegazy, M., Hafez, E., Ahmed, E.A. (2014c). The effect of L-carnitine on amethopterin-induced toxicity in rat large intestine. Journal of Cancer Research and Treatment, 2(3):55-63. Tousson, E., Ibrahim, W., Barakat, L., & Abd El- Hakeem, A. (2015). Role of Proplis administration in boldenone-induced oxidative stress, Ki-67 protein alterations and toxicity in rat liver and kidney. International Journal of Scientific & Engineering Research, 6 (8):660-664. Saggu, S., Sakeran, M.I., Zidan, N., Tousson, E., Mohan, A., Rehman, H. (2014). 5. References Ameliorating effect of chicory (Chichorium intybus L.) fruit extract against 4-tert-octylphenol induced liver injury and oxidative stress in male rats. Food and chemical toxicology, 72:138-146. Saleh, R.M., Awadin, W.F., Elseady, Y.Y., Waheish, F. E. (2014). Renal and cardiovascular damage induced by cisplatin in rats. Life Science Journal, 11(2):191-203. Tousson, E., Zaki, Z.T., Abu-Shaeir, W.A., Hassan, H. (2014b). Methotrexate-induced hepatic and renal toxicity: role of L-carnitine in treatment. Biomed Biotechnol, 2(4):85-92. Schumann, G., Klauke, R. (2003). New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects. Clinica chimica acta, 327(1-2):69-79. Valentovic, M.A., Ball, J.G., Brown, J.M., Terneus, M.V., McQuade, E., Van Meter, S., Hedrick, H.M., Roy, A.A., Williams, T. (2014). Resveratrol attenuates cisplatin renal cortical cytotoxicity by modifying oxidative stress. Toxicology in Vitro, 28(2):248-257. Shin, H.J., Kwon, H.K., Lee, J.H., Anwar, M.A., Choi, S. (2016). Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells. Scientific reports, 6:1-13. Elmasry, T. A., Al-Shaalan, N. H., Tousson, E., El- Morshedy, K., & Al-Ghadeer, A. (2017). P53 expression in response to equigan induced testicular injury and oxidative stress in male rat and the possible prophylactic effect of star anise extracts. Annu Res Rev Biol, 14(1):1-8. Tousson, E., Ali, E.M., Ibrahim, W., Mansour, M.A. (2011). Proliferating cell nuclear antigen as a molecular biomarker for spermatogenesis in PTU-induced hypothyroidism of rats. Reproductive sciences, 18(7):679-686. Tousson, E., Atteya, E., El-Atrash, A., Jeweely, O.I. (2014a). Abrogation by Ginkgo Byloba leaf extract on hepatic and renal toxicity induced
https://openalex.org/W3119595308
https://periodicos.feevale.br/seer/index.php/revistapraksis/article/download/2396/2722
Portuguese
null
POR UMA HISTÓRIA QUE NÃO SEJA ÚNICA: COLONIZAÇÃO E IDENTIDADE NO CONTO “A HISTORIADORA OBSTINADA”, DE CHIMAMANDA NGOZI ADICHIE
Revista Prâksis
2,021
cc-by
6,375
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
https://openalex.org/W4246135731
https://arts.units.it/bitstream/11368/2972783/1/s12933-020-01116-2.pdf
English
null
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
Research Square (Research Square)
2,020
cc-by
7,926
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://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ 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://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ 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 vs the TAXUS Liberté paclitaxel-eluting stent in diabetic patients with de 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 everolimus-eluting stents versus first-generation sirolimus- and paclitaxel-eluting stents in humans. Circulation. 2014;129:211–23. 10. Iqbal J, Serruys PW, Silber S, et al. Comparison of zotarolimus- and everolimus-eluting coronary stents: final 5-year report of the RESOLUTE all-comers trial. Circ Cardiovasc Interv. 2015;8:e002230. 11. Kereiakes DJ, Onuma Y, Serruys PW, Stone GW. Bioresorbable vascular 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 contemporary all-comer stent trials: balancing sensitivity and specificity Addendum to the historical MI definitions used in stent studies. Euro 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 revascularization. 1. Ritsinger V, Saleh N, Lagerqvist B, Norhammer A. High event rate after a first percutaneous coronary intervention in patients with diabetes mellitus: results from the Swedish coronary angiography and angioplasty registry. Circ Cardiovasc Interv. 2015;8:e002328. 1. Ritsinger V, Saleh N, Lagerqvist B, Norhammer A. High event rate after a first percutaneous coronary intervention in patients with diabetes mellitus: results from the Swedish coronary angiography and angioplasty registry. Circ Cardiovasc Interv. 2015;8:e002328. g y 2. Singh M, Gersh BJ, McClelland RL, et al. Clinical and angiographic predic- tors of restenosis after percutaneous coronary intervention: insights from the prevention of restenosis with tranilast and its outcomes (PRESTO) trial. Circulation. 2004;109:2727–31. g y 2. Singh M, Gersh BJ, McClelland RL, et al. Clinical and angiographic predic- tors of restenosis after percutaneous coronary intervention: insights from the prevention of restenosis with tranilast and its outcomes (PRESTO) trial. Circulation. 2004;109:2727–31. 2. Singh M, Gersh BJ, McClelland RL, et al. Clinical and angiographic predic- tors of restenosis after percutaneous coronary intervention: insights from the prevention of restenosis with tranilast and its outcomes (PRESTO) 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 revascularization in diabetic patients. J Am Coll Cardiol. 2002;40:410–7. Consent for publication 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 read and approved the final manuscript. 5. Jensen LO, Maeng M, Thayssen P, et al. Influence of diabetes mellitus on clinical outcomes following primary percutaneous coronary intervention in patients with ST-segment elevation myocardial infarction. Am J Cardiol. 2012;109:629–35. 5. Jensen LO, Maeng M, Thayssen P, et al. Influence of diabetes mellitus on clinical outcomes following primary percutaneous coronary intervention in patients with ST-segment elevation myocardial infarction. Am J Cardiol. 2012;109:629–35. 6. Stone GW, Kedhi E, Kereiakes DJ, et al. Differential clinical responses to everolimus-eluting and paclitaxel-eluting coronary stents in patients with and without diabetes mellitus. Circulation. 2011;124:893–900. further prospective long-term follow-up research with 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 is widely utilized since 2007 and the Promus Element EES since 2010. These clinical trials do not involve a medicinal product and are therefore not subject to The European Clinical Trials Directive (2001/20/EC). 12. Tenekecioglu E, Farooq V, Bourantes CV, et al. Bioresorbable scaffolds: a new paradigm in percutaneous coronary intervention. BMC Cardiovasc Disord. 2016;16:38. 13. Hommels TM, Hermanides RS, Rasoul S, et al. The 1-year safety and effi- cacy outcomes of Absorb bioresorbable vascular scaffolds for coronary artery disease treatment in diabetes mellitus patients: the ABSORB DM Benelux study. Neth Heart J. 2019;27:541–9. 14. Hommels TM, Hermanides RS, Rasoul S, et al. Everolimus-eluting biore- sorbable scaffolds for treatment of coronary artery disease in patients with diabetes mellitus: the midterm follow-up of the prospective ABSORB DM Benelux study. Cardiovasc Diabetol. 2019;18:25. 15. von Birgelen C, Basalus MW, Tandjung K, et al. A randomized controlled trial in second-generation zotarolimus-eluting Resolute stents versus everolimus-eluting Xience V stents in real-world patients: the TWENTE trial. J Am Coll Cardiol. 2012;59:1350–61. Author details 1 1 Isala Hospital, Zwolle, The Netherlands. 2 Cardiovascular Department, Uni- versity of Trieste, Trieste, Italy. 3 AOU Maggiore della Carità, Eastern Piedmont University, Novara, Italy. 4 Medisch Spectrum Twente, Thoraxcentrum & Univer- sity of Twente, Thoraxcentrum, The Netherlands. 5 Department of Cardiology, Hôpital Erasme Université Libre de Bruxelles, Brussels, Belgium. 19. Thygesen K, Alpert JS, Jaffe AS, et al. Third universal definition of myocar- dial infarction. J Am Coll Cardiol. 2012;60:1581–98. 20. Hicks KA, Tcheng JE, Bozkurt B, et al. 2014 ACC/AHA key data elements and definitions for cardiovascular endpoint events in clinical trials: a Hommels et al. Cardiovasc Diabetol (2020) 19:165 Page 10 of 10 Hommels et al. Cardiovasc Diabetol (2020) 19:165 Page 10 of 10 report of the American College of Cardiology/American Heart Associa- tion task force on clinical data standards (writing committee to develop cardiovascular endpoints data standards). J Nucl Cardiol. 2015;22:1041–4 randomised, controlled, single-blind, multicentre clinical trial. Lancet. 2016;388:2479–91. 32. Tijssen RYG, Kraak RP, Hofma SH, et al. Complete two-year follow-up with formal non-inferiority testing on primary outcomes of the AIDA trial com- paring the Absorb bioresorbable scaffold with the XIENCE drug-eluting metallic stent in routine PCI. Euro Intervention. 2018;14:e426–33. 21. Konigstein M, Ben-Yehuda O, Smits PC, et al. Outcomes among diabetic patients undergoing percutaneous coronary intervention with contem- porary drug-eluting stents: analysis from the BIONICS randomized trial. JACC Cardiovasc Interv. 2018;11:2467–76. 33. Ortega-Paz L, Capodanno D, Gori T, et al. Predilation, sizing and post- dilation scoring in patients undergoing everolimus-eluting bioresorb- able scaffold implantation for prediction of cardiac adverse events: development and internal validation of the PSP score. Euro Intervention. 2017;12:2110–7.f 22. Milojevic M, Serruys PW, Sabik JF 3rd, et al. Bypass surgery or stenting for left main coronary artery disease in patients with diabetes. J Am Coll Cardiol. 2019;73:1616–28. 23. Konishi A, Mitsutake Y, Ho M, et al. Patient and lesion characteristics in late/very late stent thrombosis with everolimus-eluting stents from real- world adverse event reporting. J Cardiol. 2020;75:255–60. 34. Stone GW, Abizaid A, Onuma Y, et al. Effect of technique on outcomes following bioresorbable vascular scaffold implantation: analysis from the ABSORB trials. J Am Coll Cardiol. 2017;70:2863–74. 24. Kereiakes DJ, Ellis SG, Kimura T, et al. Efficacy and safety of the Absorb everolimus-eluting bioresorbable scaffold for treatment of patients with diabetes mellitus: results of the Absorb diabetic substudy. JACC Cardio- vasc Interv. 2017;10:42–9. 35. Caixeta A, Campos CM, Felix C, et al. Author details 1 Predictors of long-term adverse events after Absorb bioresorbable vascular scaffold implantation: a 1,933-patient pooled analysis from international registries. EuroInterven- tion. 2019;15:623–30. 25. Tang XF, Ma YL, Song Y, et al. Biodegradable polymer drug-eluting stents versus second-generation drug-eluting stents in patients with and without diabetes mellitus: a single-center study. Cardiovasc Diabetol. 2018;17:114. 36. Chevalier B, Cequier A, Dudek D, et al. Four-year follow-up of the ran- 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, • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? Publisher’s Note Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? Choose BMC and benefit from:
https://openalex.org/W4230919329
https://figshare.com/articles/journal_contribution/Geometric_Calibration_and_Radiometric_Correction_of_LiDAR_Data_and_Their_Impact_on_the_Quality_of_Derived_Products/14639838/1/files/28116216.pdf
Latin
null
Geometric Calibration and Radiometric Correction of LiDAR Data and Their Impact on the Quality of Derived Products
null
2,024
cc-by
15,213
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= ܺԦ௢൅ܴఠ,ఝ,఑ܲሬԦீ൅ܴఠ,ఝ,఑ܴ௱ఠ,௱ఝ,௱఑ܴௌഀఈௌഁఉ൥ 0 0 െሺݎ൅߂ݎ) 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 ܺԦீ= ܺԦ௢൅ܴఠ,ఝ,఑ܲሬԦீ൅ܴఠ,ఝ,఑ܴ௱ఠ,௱ఝ,௱఑ܴௌഀఈௌഁఉ൥ 0 0 െሺݎ൅߂ݎ) ൩ (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൥ െሺݎ൅Δݎ)ݏ݅݊ሺܵߚ) 0 െሺݎ൅Δݎ)ܿ݋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 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 ܺԦீ= ܺԦ௢൅ܴఠ,ఝ,఑ܲሬԦீ൅ܴఠ,ఝ,఑ܴ௱ఠ,௱ఝ,௱఑ܴௌഀఈௌഁఉ൥ 0 0 െሺݎ൅߂ݎ) 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) = ݂ሺݔ௕െߜݔ, ݈௡െ݁) (5) (3) ܺԦீሺܾ/݊) = ݂ሺݔ௕, ݈௡) (4) ܺԦீሺܶݎݑ݁) = ݂ሺݔ௕െߜݔ, ݈௡െ݁) (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) ൅ܣߜݔ൅ܤ݁= ܺԦீሺܶݎݑ݁) ൅ߜܺԦீ൅ܤ݁ (7) ܺԦீ ሺܶݎݑ݁) ൎ݂ሺݔ௕, ݈௡) െ ߲݂߲ݔ ⁄ |௫್,௟೙ߜݔെ߲݂߲݈ ⁄ |௫್,௟೙݁= ܺԦீሺܾ/݊) െ ܣߜݔെܤ݁ (6) ܺԦீሺܾ/݊) = ܺԦீ ሺܶݎݑ݁) ൅ܣߜݔ൅ܤ݁= ܺԦீሺܶݎݑ݁) ൅ߜܺԦீ൅ܤ݁ (7) ߜܺԦீ= ൥ ܿ݋ݏߢ ߜ߂ܺെݏ݅݊݇ߜ߂ܻ൅ݏ݅݊ߢݖߜ߂߱൅ܿ݋ݏߢݖߜ߂߮ ݏ݅݊ߢ ߜ߂ܺ൅ܿ݋ݏ݇ߜ߂ܻെܿ݋ݏߢݖߜ߂߱൅ݏ݅݊ߢݖߜ߂߮ ߜ߂ܼെݔ ߜ߂߮ ൩ ൅቎ െݏ݅݊ߢ ݔ ߜ߂ߢെܿ݋ݏ݇ݏ݅݊ሺܵߚ) ߜ߂ݎ൅ܿ݋ݏߢ ݖ ߚ ߜܵ ܿ݋ݏ݇ ݔ ߜ߂ߢെݏ݅݊ߢݏ݅݊ሺܵߚ) ߜ߂ݎ൅ݏ݅݊ߢݖߚߜܵ െܿ݋ݏሺܵߚ) ߜ߂ݎെݔߚߜܵ ቏ (8) ܺԦீ ሺܶݎݑ݁) ൎ݂ሺݔ௕, ݈௡) െ ߲݂߲ݔ ⁄ |௫್,௟೙ߜݔെ߲݂߲݈ ⁄ |௫್,௟೙݁= ܺԦீሺܾ/݊) െ ܣߜݔെܤ݁ (6) ܺԦீሺܾ/݊) = ܺԦீ ሺܶݎݑ݁) ൅ܣߜݔ൅ܤ݁= ܺԦீሺܶݎݑ݁) ൅ߜܺԦீ൅ܤ݁ (7) ߜܺԦீ= ൥ ܿ݋ݏߢ ߜ߂ܺെݏ݅݊݇ߜ߂ܻ൅ݏ݅݊ߢݖߜ߂߱൅ܿ݋ݏߢݖߜ߂߮ ݏ݅݊ߢ ߜ߂ܺ൅ܿ݋ݏ݇ߜ߂ܻെܿ݋ݏߢݖߜ߂߱൅ݏ݅݊ߢݖߜ߂߮ ߜ߂ܼെݔ ߜ߂߮ ൩ ൅቎ െݏ݅݊ߢ ݔ ߜ߂ߢെܿ݋ݏ݇ݏ݅݊ሺܵߚ) ߜ߂ݎ൅ܿ݋ݏߢ ݖ ߚ ߜܵ ܿ݋ݏ݇ ݔ ߜ߂ߢെݏ݅݊ߢݏ݅݊ሺܵߚ) ߜ߂ݎ൅ݏ݅݊ߢݖߚߜܵ െܿ݋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൥ െሺݎ൅Δݎ)ݏ݅݊ሺܵߚ) 0 െሺݎ൅Δݎ)ܿ݋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) ൅ߜܺԦீ஺൅݁஺െܺԦீಳሺܶݎݑ݁) െߜܺԦீ஻െ݁஻ (9) ܺԦீಲሺܾ/݊) െܺԦீಳሺܾ/݊) = ߜܺԦீ஺െߜܺԦீ஻൅݁஺െ݁஻= ߜܺԦீ஺െߜܺԦீ஻൅݁ (10) ܺԦீಲሺܾ/݊) െܺԦீಳሺܾ/݊) = ൥ ሺܿ݋ݏߢ஺െܿ݋ݏߢ஻)ߜ߂ܺെሺݏ݅݊ߢ஺െݏ݅݊ߢ஻)ߜ߂ܻ ሺݏ݅݊ߢ஺െݏ݅݊ߢ஻)ߜ߂ܺ൅ሺܿ݋ݏߢ஺െܿ݋ݏߢ஻)ߜ߂ܻ 0 ൩ (9) (10) ൅቎ ሺݏ݅݊ߢ஺ݖ஺െݏ݅݊ߢ஻ݖ஻)ߜ߂߱൅ሺܿ݋ݏߢ஺ݖ஺െܿ݋ݏߢ஻ݖ஻)ߜ߂߮െሺݏ݅݊ߢ஺ݔ஺െݏ݅݊ߢ஻ݔ஻)ߜ߂ߢ െሺܿ݋ݏߢ஺ݖ஺െܿ݋ݏߢ஻ݖ஻)ߜ߂߱൅ሺݏ݅݊ߢ஺ݖ஺െݏ݅݊ߢ஻ݖ஻)ߜ߂߮൅ሺܿ݋ݏߢ஺ݔ஺െܿ݋ݏߢ஻ݔ஻)ߜ߂ߢ െሺݔ஺െݔ஻)ߜ߂߮ ቏ (11) ൅቎ ሺݏ݅݊ߢ஺ݖ஺െݏ݅݊ߢ஻ݖ஻)ߜ߂߱൅ሺܿ݋ݏߢ஺ݖ஺െܿ݋ݏߢ஻ݖ஻)ߜ߂߮െሺݏ݅݊ߢ஺ݔ஺െݏ݅݊ߢ஻ݔ஻)ߜ߂ߢ െሺܿ݋ݏߢ஺ݖ஺െܿ݋ݏߢ஻ݖ஻)ߜ߂߱൅ሺݏ݅݊ߢ஺ݖ஺െݏ݅݊ߢ஻ݖ஻)ߜ߂߮൅ሺܿ݋ݏߢ஺ݔ஺െܿ݋ݏߢ஻ݔ஻)ߜ߂ߢ െሺݔ஺െݔ஻)ߜ߂߮ ቏ (11) (11) ቎ሺ ஺஺ ஻஻) ሺ ஺஺ ஻஻) ߮ ሺ ஺஺ ஻ ஻) െሺݔ஺െݔ஻)ߜ߂߮ ቏ ൅቎ െሾܿ݋ݏ݇஺ ݏ݅݊ሺܵߚ஺) െܿ݋ݏߢ஻ ݏ݅݊ሺܵߚ஻)ሿߜ߂ݎ൅ሺܿ݋ݏߢ஺ݖ஺ߚ஺െܿ݋ݏߢ஻ݖ஻ߚ஻)ߜܵ െሾݏ݅݊݇஺ ݏ݅݊ሺܵߚ஺) െݏ݅݊ߢ஻ ݏ݅݊ሺܵߚ஻)ሿߜ߂ݎ൅ሺݏ݅݊ߢ஺ݖ஺ߚ஺െݏ݅݊ߢ஻ݖ஻ߚ஻)ߜܵ െሾܿ݋ݏሺܵߚ஺) െܿ݋ݏሺܵߚ஻)ሿߜ߂ݎെሺݔ஺ߚ஺െݔ஻ߚ஻)ߜܵ ቏൅݁ (11) ܺԦீ಴೚೙೟ೝ೚೗െܺԦீಳሺܾ/݊) = ൥ െܿ݋ݏߢ஻ߜ߂ܺ൅ݏ݅݊ߢ஻ߜ߂ܻ െݏ݅݊ߢ஻ߜ߂ܺെܿ݋ݏߢ஻ߜ߂ܻ െߜ߂ܼ ൩ ൅൥ െݏ݅݊ߢ஻ݖ஻ ߜ߂߱െܿ݋ݏߢ஻ݖ஻ ߜ߂߮൅ݏ݅݊ߢ஻ݔ஻ߜ߂ߢ ܿ݋ݏߢ஻ݖ஻ ߜ߂߱െݏ݅݊ߢ஻ݖ஻ ߜ߂߮െܿ݋ݏߢ஻ݔ஻ ߜ߂ߢ ݔ஻ ߜ߂߮ ൩ ൅቎ ܿ݋ݏߢ஻ ݏ݅݊ሺܵߚ஻)ߜ߂ݎെܿ݋ݏߢ஻ݖ஻ߚ஻ ߜܵ ݏ݅݊ߢ஻ݏ݅݊ሺܵߚ஻) ߜ߂ݎെݏ݅݊ߢ஻ݖ஻ߚ஻ߜܵ ܿ݋ݏሺܵߚ஻) ߜ߂ݎ൅ݔ஻ߚ஻ߜܵ ቏൅ሺ݁஼௢௡௧௥௢௟െ݁஻) (12) ൥ ܺ෨ ܻ෨ ܼ෨ ൩ ஼௢௥௥௘௖௧௘ௗ = ൥ ܺ ܻ ܼ ൩ ஻௜௔௦௘ௗ െ቎ ܿ݋ݏߢߜ߂መܺെݏ݅݊ߢߜ߂መܻ൅ݏ݅݊ߢݖߜ߂መ߱൅ܿ݋ݏߢ ݖ ߜ߂መ߮ ݏ݅݊ߢ ߜ߂መܺ൅ܿ݋ݏߢߜ߂መܻെܿ݋ݏߢݖߜ߂መ߱൅ݏ݅݊ߢ ݖ ߜ߂መ߮ ߜ߂መܼെݔ ߜ߂መ߮ ቏ െ቎ െݏ݅݊ߢ ݔ ߜ߂መߢെܿ݋ݏߢ ݏ݅݊ሺܵߚ)ߜ߂መݎ൅ܿ݋ݏߢ ݖ ߚ ߜܵመ ܿ݋ݏߢ ݔ ߜ߂መߢെݏ݅݊ߢݏ݅݊ሺܵߚ)ߜ߂መݎ൅ݏ݅݊ߢݖߚߜܵመ െܿ݋ݏሺܵߚ) ߜ߂መݎെݔߚߜܵመ ቏ (13) ൅቎ െሾܿ݋ݏ݇஺ ݏ݅݊ሺܵߚ஺) െܿ݋ݏߢ஻ ݏ݅݊ሺܵߚ஻)ሿߜ߂ݎ൅ሺܿ݋ݏߢ஺ݖ஺ߚ஺െܿ݋ݏߢ஻ݖ஻ߚ஻)ߜܵ െሾݏ݅݊݇஺ ݏ݅݊ሺܵߚ஺) െݏ݅݊ߢ஻ ݏ݅݊ሺܵߚ஻)ሿߜ߂ݎ൅ሺݏ݅݊ߢ஺ݖ஺ߚ஺െݏ݅݊ߢ஻ݖ஻ߚ஻)ߜܵ െሾܿ݋ݏሺܵߚ஺) െܿ݋ݏሺܵߚ஻)ሿߜ߂ݎെሺݔ஺ߚ஺െݔ஻ߚ஻)ߜܵ ቏൅݁ ܺԦீ಴೚೙೟ೝ೚೗െܺԦீಳሺܾ/݊) = ൥ െܿ݋ݏߢ஻ߜ߂ܺ൅ݏ݅݊ߢ஻ߜ߂ܻ െݏ݅݊ߢ஻ߜ߂ܺെܿ݋ݏߢ஻ߜ߂ܻ െߜ߂ܼ ൩ ൅൥ െݏ݅݊ߢ஻ݖ஻ ߜ߂߱െܿ݋ݏߢ஻ݖ஻ ߜ߂߮൅ݏ݅݊ߢ஻ݔ஻ߜ߂ߢ ܿ݋ݏߢ஻ݖ஻ ߜ߂߱െݏ݅݊ߢ஻ݖ஻ ߜ߂߮െܿ݋ݏߢ஻ݔ஻ ߜ߂ߢ ݔ஻ ߜ߂߮ ൩ (12) ൅቎ െሾܿ݋ݏ݇஺ ݏ݅݊ሺܵߚ஺) െܿ݋ݏߢ஻ ݏ݅݊ሺܵߚ஻)ሿߜ߂ݎ൅ሺܿ݋ݏߢ஺ݖ஺ߚ஺െܿ݋ݏߢ஻ݖ஻ߚ஻)ߜܵ െሾݏ݅݊݇஺ ݏ݅݊ሺܵߚ஺) െݏ݅݊ߢ஻ ݏ݅݊ሺܵߚ஻)ሿߜ߂ݎ൅ሺݏ݅݊ߢ஺ݖ஺ߚ஺െݏ݅݊ߢ஻ݖ஻ߚ஻)ߜܵ െሾܿ݋ݏሺܵߚ஺) െܿ݋ݏሺܵߚ஻)ሿߜ߂ݎെሺݔ஺ߚ஺െݔ஻ߚ஻)ߜܵ ቏൅݁ ቎ െሾܿ݋ݏ݇஺ ݏ݅݊ሺܵߚ஺) െܿ݋ݏߢ஻ ݏ݅݊ሺܵߚ஻)ሿߜ߂ݎ൅ሺܿ݋ݏߢ஺ݖ஺ߚ஺െܿ݋ݏߢ஻ݖ஻ߚ஻)ߜܵ െሾݏ݅݊݇஺ ݏ݅݊ሺܵߚ஺) െݏ݅݊ߢ஻ ݏ݅݊ሺܵߚ஻)ሿߜ߂ݎ൅ሺݏ݅݊ߢ஺ݖ஺ߚ஺െݏ݅݊ߢ஻ݖ஻ߚ஻)ߜܵ െሾܿ݋ݏሺܵߚ஺) െܿ݋ݏሺܵߚ஻)ሿߜ߂ݎെሺݔ஺ߚ஺െݔ஻ߚ஻)ߜܵ ቏൅݁ ܺԦீ಴೚೙೟ೝ೚೗െܺԦீಳሺܾ/݊) = ൥ െܿ݋ݏߢ஻ߜ߂ܺ൅ݏ݅݊ߢ஻ߜ߂ܻ െݏ݅݊ߢ஻ߜ߂ܺെܿ݋ݏߢ஻ߜ߂ܻ െߜ߂ܼ ൩ ൅൥ െݏ݅݊ߢ஻ݖ஻ ߜ߂߱െܿ݋ݏߢ஻ݖ஻ ߜ߂߮൅ݏ݅݊ߢ஻ݔ஻ߜ߂ߢ ܿ݋ݏߢ஻ݖ஻ ߜ߂߱െݏ݅݊ߢ஻ݖ஻ ߜ߂߮െܿ݋ݏߢ஻ݔ஻ ߜ߂ߢ ݔ஻ ߜ߂߮ ൩ (12) ൅൥ െݏ݅݊ߢ஻ݖ஻ ߜ߂߱െܿ݋ݏߢ஻ݖ஻ ߜ߂߮൅ݏ݅݊ߢ஻ݔ஻ߜ߂ߢ ܿ݋ݏߢ஻ݖ஻ ߜ߂߱െݏ݅݊ߢ஻ݖ஻ ߜ߂߮െܿ݋ݏߢ஻ݔ஻ ߜ߂ߢ ݔ஻ ߜ߂߮ ൩ (12) ൅൥ܿ݋ݏߢ஻ݖ஻ ߜ߂߱െݏ݅݊ߢ஻ݖ஻ ߜ߂߮െܿ݋ݏߢ஻ݔ஻ ߜ߂ߢ ݔ஻ ߜ߂߮ ൩ (12) (12) ൅቎ ܿ݋ݏߢ஻ ݏ݅݊ሺܵߚ஻)ߜ߂ݎെܿ݋ݏߢ஻ݖ஻ߚ஻ ߜܵ ݏ݅݊ߢ஻ݏ݅݊ሺܵߚ஻) ߜ߂ݎെݏ݅݊ߢ஻ݖ஻ߚ஻ߜܵ ܿ݋ݏሺܵߚ஻) ߜ߂ݎ൅ݔ஻ߚ஻ߜܵ ቏൅ሺ݁஼௢௡௧௥௢௟െ݁஻) ൥ ܺ෨ ܻ෨ ܼ෨ ൩ ஼௢௥௥௘௖௧௘ௗ = ൥ ܺ ܻ ܼ ൩ ஻௜௔௦௘ௗ െ቎ ܿ݋ݏߢߜ߂መܺെݏ݅݊ߢߜ߂መܻ൅ݏ݅݊ߢݖߜ߂መ߱൅ܿ݋ݏߢ ݖ ߜ߂መ߮ ݏ݅݊ߢ ߜ߂መܺ൅ܿ݋ݏߢߜ߂መܻെܿ݋ݏߢݖߜ߂መ߱൅ݏ݅݊ߢ ݖ ߜ߂መ߮ ߜ߂መܼെݔ ߜ߂መ߮ ቏ െ቎ െݏ݅݊ߢ ݔ ߜ߂መߢെܿ݋ݏߢ ݏ݅݊ሺܵߚ)ߜ߂መݎ൅ܿ݋ݏߢ ݖ ߚ ߜܵመ ܿ݋ݏߢ ݔ ߜ߂መߢെݏ݅݊ߢݏ݅݊ሺܵߚ)ߜ߂መݎ൅ݏ݅݊ߢݖߚߜܵመ െܿ݋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, ݐ൅Δݐ); (I) For a LiDAR point mapped at time ݐ, we identify trajectory positions within a certain time interval ሺݐെΔݐ, ݐ൅Δݐ); (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: ƒ 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 ƒ 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; ƒ 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 ƒ 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 ƒ 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. ƒ 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, ݔ஺െݔ஻ = 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, ݔ஺െݔ஻ = 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= ܣݔ൅݁ ݁~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) ൅ߜܺԦீ஺െܺԦீಳሺܶݎݑ݁) െߜܺԦீ஻൅݁ = ߜܺԦீ஺െߜܺԦீ஻൅ܦ൅݁ (20) where: – ܦ= ܺԦீಲሺܶݎݑ݁) െܺԦீಳሺܶݎݑ݁) ݕ= ܣݔ൅ܦ൅݁ ݁~ሺ0, ߑ) ݓ݄݁ݎ݁ߑ= ߪ௢ ଶܲିଵ (21) – ܦ= ܺԦீಲሺܶݎݑ݁) െܺԦீಳሺܶݎݑ݁) ) െܺԦீಳሺܶݎݑ݁) ݕ= ܣݔ൅ܦ൅݁ ݁~ሺ0, ߑ) ݓ݄݁ݎ݁ߑ= ߪ௢ ଶܲିଵ (21) (21) ݕ= ܣݔ൅ܦ൅݁ ݁~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= ܣݔ൅ܦ൅݁ ݁~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= ܯܲ௑௒௓ܯ்= ൥ ܲ௎ ܲ௎௏ ܲ௎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: ܲ௑௒௓ ` = ܯ்ܲ௎௏ௐ ` ܯ (33) ܲ௑௒௓ ` ܦ= ܯ் ܲ௎௏ௐ ` ܯ൥ ݀ܺ ܻ݀ ܼ݀ ൩= ܯ்ܲ௎௏ௐ ` ൥ ܷ݀ ܸ݀ 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 ଶߟ௦௬௦ߟ௔௧௠ߪ (35) ܲ௥= ܲ௧ܦ௥ ଶ 4ߨݎସߚ௧ ଶߟ௦௬௦ߟ௔௧௠ߪ (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 biomass estimation of meadows in rugged terrain. In Proceedings of SPIE, EUROPTO Series, Barcelona, Spain, September 1998; 3499, 224-236. 3. Riegler, G.; Stolz, R.; Mauser, W. Geometric and radiometric corrections of ERS SAR data for biomass estimation of meadows in rugged terrain. In Proceedings of SPIE, EUROPTO Series, Barcelona, Spain, September 1998; 3499, 224-236. 4. Shimada, M.; Isoguchi, O.; Tadono, T.; Isono, K. PALSAR radiometric and geometric calibration. IEEE Trans. Geosci. Remote Sens. 2009, 47, 3915-3931. 4. Shimada, M.; Isoguchi, O.; Tadono, T.; Isono, K. PALSAR radiometric and geometric calibration. IEEE Trans. Geosci. Remote Sens. 2009, 47, 3915-3931. 5. Loew, A.; Mauser, W. Generation of geometrically and radiometrically terrain corrected SAR 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= ܣݔ൅ܦ൅݁ ݁~൫0, ߑ`൯ ݓ݄݁ݎ݁ߑ` = ߪ௢ ଶܲ`ାܽ݊݀ܲ`ܦ= 0 (A.1) ݕ= ܣݔ൅ܦ൅݁ ݁~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`ݕ൅ݔ்ܣ்ܲ`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`ݕ൅ݔ்ܣ்ܲ`ܣݔ= ݉݅݊|௫ (A.4) E ti (A 3) b i lifi d t െܣݔ)்ܲ`ሺݕെܣݔ) = ݕ்ܲ`ݕെݕ்ܲ`ܣݔെݔ்ܣ்ܲ`ݕ൅ݔ்ܣ்ܲ`ܣݔ= ݉݅݊|௫ (A.4) ߶ሺݔ) = ሺݕെܣݔ)்ܲ`ሺݕെܣݔ) = ݕ்ܲ`ݕെݕ்ܲ`ܣݔെݔ்ܣ்ܲ`ݕ൅ݔ்ܣ்ܲ`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`ݕ൅ݔ்ܣ்ܲ`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`൫ܫ௡െܣܰିଵܣ்ܲ`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`ା൅ሺܣݔ൅ܦ)ሺܣݔ൅ܦ)்= ߪ௢ ଶܲ`ା൅ܣݔݔ்ܣ்൅ܣݔܦ்൅ܦݔ்ܣ்൅ܦܦ் (A.16) S b i i E i (A 16) i E i (A 14) i ld ݕݕ்) = ߪ௢ ଶܲ`ା൅ሺܣݔ൅ܦ)ሺܣݔ൅ܦ)்= ߪ௢ ଶܲ`ା൅ܣݔݔ்ܣ்൅ܣݔܦ்൅ܦݔ்ܣ்൅ܦܦ் Substituting Equation (A.16) in Equation (A.14) yields: Substituting Equation (A.16) in Equation (A.14) yields: ܧ൫்݁̃ܲ`݁̃൯= ݐݎܲ`൫ܫെܣܰିଵܣ்ܲ`൯ൣߪ௢ ଶܲ`ା൅ܣݔݔ்ܣ்൅ܣݔܦ்൅ܦݔ்ܣ்൅ܦܦ்൧ (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`ା൅ܣݔݔ்ܣ்൅ܣݔܦ்൅ܦݔ்ܣ்൅ܦܦ்൧ (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`ା൅ܣݔݔ்ܣ்൅ܣݔܦ்൅ܦݔ்ܣ்൅ܦܦ்൧ (A.17) Gi th t P`ܦ 0 E ti (A 17) b i lifi d t ܧ൫்݁̃ܲ`݁̃൯= ݐݎܲ`൫ܫെܣܰିଵܣ்ܲ`൯ൣߪ௢ ଶܲ`ା൅ܣݔݔ்ܣ்൅ܣݔܦ்൅ܦݔ்ܣ்൅ܦܦ்൧ (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/).
https://openalex.org/W3168701222
http://eprints.gla.ac.uk/241359/1/241359.pdf
English
null
Role of Common Genetic Variants for Drug-Resistance to Specific Anti-Seizure Medications
Frontiers in pharmacology
2,021
cc-by
5,599
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, Samuel F. Berkovic, Josemir W. Sander, Graeme J. Sills, Hreinn Stefansson, Pasquale Striano, Federico Zara, and Sanjay M. Sisodiya REFERENCES Androsova, G., Krause, R., Borghei, M., Wassenaar, M., Auce, P., Avbersek, A., et al. (2017). Comparative Effectiveness of Antiepileptic Drugs in Patients with Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis. Epilepsia 58 (10), 1734–1741. doi:10.1111/epi.13871 Chen, Z., Brodie, M. J., Liew, D., and Kwan, P. (2018). Treatment Outcomes in Patients with Newly Diagnosed Epilepsy Treated with Established and New Antiepileptic Drugs. JAMA Neurol. 75 (3), 279–286. doi:10.1001/jamaneurol. 2017.3949 Chung, W.-H., Hung, S.-I., Hong, H.-S., Hsih, M.-S., Yang, L.-C., Ho, H.-C., et al. (2004). A Marker for Stevens-Johnson Syndrome. Nature 428 (6982), 486. doi:10.1038/428486a Beyenburg, S., Stavem, K., and Schmidt, D. (2010). Placebo-corrected Efficacy of Modern Antiepileptic Drugs for Refractory Epilepsy: Systematic Review and Meta-Analysis. Epilepsia 51 (1), 7–26. doi:10.1111/j.1528-1167.2009.02299.x Brodie, M. J., Barry, S. J. E., Bamagous, G. A., Norrie, J. D., and Kwan, P. (2012). Patterns of Treatment Response in Newly Diagnosed Epilepsy. Neurology 78 (20), 1548–1554. doi:10.1212/wnl.0b013e3182563b19 Cross-Disorder Group of the Psychiatric Genomics Consortium (2013). Identification of Risk Loci with Shared Effects on Five Major Psychiatric Disorders: a Genome-wide Analysis. Lancet 381 (9875), 1371–1379. doi:10. 1016/S0140-6736(12)62129-1 Bulik-Sullivan, B. K., Loh, P-R., Loh, P.-R., Finucane, H. K., Ripke, S., Yang, J., et al. (2015). LD Score Regression Distinguishes Confounding from Polygenicity in Genome-wide Association Studies. Nat. Genet. 47 (3), 291–295. doi:10.1038/ng.3211 Daly, A. K. (2017). Pharmacogenetics: a General Review on Progress to Date. Br. Med. Bull., 124, 1–15. doi:10.1093/bmb/ldx035 June 2021 | Volume 12 | Article 688386 Frontiers in Pharmacology | www.frontiersin.org 6 Common Variants in Drug-Resistant Epilepsy Wolking et al. Hypersensitivity Reactions in Europeans. N. Engl. J. Med. 364 (12), 1134–1143. doi:10.1056/nejmoa1013297 Hypersensitivity Reactions in Europeans. N. Engl. J. Med. 364 (12), 1134–1143. doi:10.1056/nejmoa1013297 Euesden, J., Lewis, C. M., and O’Reilly, P. F. (2015). PRSice: Polygenic Risk Score Software. Bioinformatics 31 (9), 1466–1468. doi:10.1093/ bioinformatics/btu848 McCormack, M., Gui, H., Ingason, A., Speed, D., Wright, G. E. B., Zhang, E. J., et al. (2018). Genetic Variation in CFH Predicts Phenytoin-Induced Maculopapular Exanthema in European-descent Patients. Neurology 90 (4), e332–e341. doi:10. 1212/wnl.0000000000004853 Fang, M., Xi, Z.-Q., Wu, Y., and Wang, X.-F. (2011). A New Hypothesis of Drug Refractory Epilepsy: Neural Network Hypothesis. Med. Hypotheses 76 (6), 871–876. doi:10.1016/j.mehy.2011.02.039 Menashe, I., Rosenberg, P. S., and Chen, B. E. (2008). PGA: Power Calculator for Case-Control Genetic Association Analyses. BMC Genet. 9, 36. doi:10.1186/ 1471-2156-9-36 Glauser, T. A., Cnaan, A., Shinnar, S., Hirtz, D. G., Dlugos, D., Masur, D., et al. (2013). REFERENCES Ethosuximide, Valproic Acid, and Lamotrigine in Childhood Absence Epilepsy: Initial Monotherapy Outcomes at 12 Months. Epilepsia 54 (1), 141–155. doi:10.1111/epi.12028 Moreau, C., Rébillard, R.-M., Wolking, S., Michaud, J., Tremblay, F., Girard, A., et al. (2020). Polygenic Risk Scores of Several Subtypes of Epilepsies in a Founder Population. Neurol. Genet. 6 (3), e416. doi:10.1212/nxg. 0000000000000416 Heavin, S. B., McCormack, M., Wolking, S., Slattery, L., Walley, N., Avbersek, A., et al. (2019). Genomic and Clinical Predictors of Lacosamide Response in Refractory Epilepsies. Available:at: https://onlinelibrary.wiley.com/doi/abs/10. 1002/epi4.12360. Rogawski, M. A. (2013). The Intrinsic Severity Hypothesis of Pharmacoresistance to Antiepileptic Drugs. Epilepsia 54, 33–40. doi:10.1111/epi.12182 International League Against Epilepsy Consortium on Complex Epilepsies (2018). Genome-wide Mega-Analysis Identifies 16 Loci and Highlights Diverse Biological Mechanisms in the Common Epilepsies. Nat. Commun. 9 (1), 5269. doi:10.1038/s41467-018-07524-z Scheffer, I. E., Berkovic, S., Capovilla, G., Connolly, M. B., French, J., Guilhoto, L., et al. (2017). ILAE Classification of the Epilepsies: Position Paper of the ILAE Commission for Classification and Terminology. Epilepsia 58 (4), 512–521. doi:10.1111/epi.13709 Kobow, K., El-Osta, A., and Blümcke, I. (2013). The Methylation Hypothesis of Pharmacoresistance in Epilepsy. Epilepsia 54 (Suppl. 2), 41–47. doi:10.1111/epi. 12183 Silvennoinen, K., Lange, N., Zagaglia, S., Balestrini, S., Androsova, G., Wassenaar, M., et al. (2019). Comparative Effectiveness of Antiepileptic Drugs in Juvenile Myoclonic Epilepsy. Available:at: https://onlinelibrary.wiley.com/doi/abs/10. 1002/epi4.12349. Kwan, P., Arzimanoglou, A., Berg, A. T., Brodie, M. J., Allen Hauser, W., Mathern, G., et al. (2010). Definition of Drug Resistant Epilepsy: Consensus Proposal by the Ad Hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia 51 (6), 1069–1077. doi:10.1111/j.1528- 1167.2009.02397.x Vilhjálmsson, B. J., Yang, J., Finucane, H. K., Gusev, A., Lindström, S., Ripke, S., et al. (2015). Modeling Linkage Disequilibrium Increases Accuracy of Polygenic Risk Scores. Am. J. Hum. Genet. 97 (4), 576–592. doi:10.1016/j.ajhg.2015.09.001 Leu, C., Stevelink, R., Smith, A. W., Goleva, S. B., Kanai, M., Ferguson, L., et al. (2019). Polygenic burden in Focal and Generalized Epilepsies. Available:at: https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awz292/ 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 of Pharmacogenetics on the Treatment of Epilepsy. Epilepsia 50 (1), 1–23. doi:10.1111/j.1528-1167.2008.01716.x Wolking, S., Moreau, C., Nies, A. T., Schaeffeler, E., McCormack, M., Auce, P., et al. (2020b). June 2021 | Volume 12 | Article 688386 Frontiers in Pharmacology | www.frontiersin.org REFERENCES Testing Association of Rare Genetic Variants with Resistance to Three 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 in Epilepsy: Clinical Impact, Potential Mechanisms, and New Innovative Treatment Options. Pharmacol. Rev. 72 (3), 606–638. doi:10.1124/pr.120. 019539 Yang, J., Lee, S. H., Goddard, M. E., and Visscher, P. M. (2011). GCTA: a Tool for 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 Carbamazepine, Gabapentin, Lamotrigine, Oxcarbazepine, or Topiramate for Treatment of Partial Epilepsy: an Unblinded Randomised Controlled Trial. The Lancet 369 (9566), 1000–1015. doi:10.1016/s0140-6736(07) 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 Epilepsy: an Unblinded Randomised Controlled Trial. The Lancet 369 (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
https://openalex.org/W2992055595
https://europepmc.org/articles/pmc7064452?pdf=render
English
null
Being Friends with or Rejected by Classmates: Aggression Toward Same- and Cross-Ethnic Peers
Journal of youth and adolescence
2,019
cc-by
11,671
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 Exp. Exp. Exp. References mechanisms. Annual Review of Sociology, 36, 91–115. https:// doi.org/10.1146/annurev.soc.34.040507.134743. Beiser, M., Puente-Duran, S., & Hou, F. (2015). Cultural distance and emotional problems among immigrant and refugee youth in Canada: Findings from the New Canadian Child and Youth Study (NCCYS). International Journal of Intercultural Relations, 49, 33–45. https://doi.org/10.1016/j.ijintrel.2015.06.005. Schiefer, D., Möllering, A., & Daniel, E. (2012). Cultural value fit of immigrant and minority adolescents: the role of acculturation orientations. International Journal of Intercultural Relations, 36, 486–497. https://doi.org/10.1016/j.ijintrel.2012.02.001. Sentse, M., Dijkstra, J. K., Salmivalli, C., & Cillessen, A. H. N. (2013). The dynamics of friendships and victimization in ado- lescence: a longitudinal social network perspective. Aggressive Behavior, 39, 229–238. https://doi.org/10.1002/ab.21469. Berry, J. W. (2003). A psychology of immigration. Journal of Social Issues, 57, 615–631. https://doi.org/10.1111/0022-4537.00231. Bikmen, N. (2011). Asymmetrical effects of contact between minority groups: Asian and Black students in a small college. Cultural Diversity and Ethnic Minority Psychology, 17, 186–194. https:// doi.org/10.1037/a0023230. Sijtsema, J. J., Veenstra, R., Lindenberg, S., & Salmivalli, C. (2009). Empirical test of bullies’ status goals: assessing direct goals, aggression, and prestige. Aggressive Behavior, 35, 57–67. https:// doi.org/10.1002/ab.20282. Boda, Z., & Néray, B. (2015). Inter-ethnic friendship and negative ties in secondary school. Social Networks, 43, 57–72. https://doi.org/ 10.1016/j.socnet.2015.03.004. Snijders, T. A. B., Van de Bunt, G. G., & Steglich, C. E. G. (2010). Introduction to stochastic actor-based models for network dynamics. Social Networks, 32, 44–60. https://doi.org/10.1016/j. socnet.2009.02.004. Cameron, A. C., & Trivedi, P. K. (2009). Microeconometrics using Stata. College Staton, TX: Stata Press. Stark, T. H., & Flache, A. (2012). The double edge of common interest: Ethnic segregation as an unintended byproduct of opi- nion homophily. Sociology of Education, 85, 179–199. https:// doi.org/10.1177/0038040711427314. Cameron, A. C., & Trivedi, P. K. (2013). Regression analysis of count data (2nd edn). New York, NY: Cambridge University Press. https://doi.org/10.1017/CBO9781139013567 Dollmann, J., Jacob, K., & Kalter, F. (2014). Examining the diversity of youth in Europe. A classification of generations and ethnic origins using CILS4EU data. www.mzes.uni-mannheim.de StataCorp. (2017). Stata statistical software: release 15. College Station, TX: StataCorp LP. Statistics Netherlands (2018). StatLine - Bevolking; kerncijfers. Retrieved August 9, 2019, from https://opendata.cbs.nl/statline/ #/CBS/nl/dataset/37296ned/table?dl=1989F Ganzeboom, H. B. G., De Graaf, P. M., Treiman, D. J., & De Leeuw, J. (1992). A standard international socio-economic index of occupational status. Social Science Research, 21(1), 1–56. https:// doi.org/10.1016/0049-089X(92)90017-B. Strohmeier, D., Spiel, C., & Gradinger, P. (2008). Social relationships in multicultural schools: Bullying and victimization. Appendix 1 Variance classroom na na na na na na Log likelihood −1889.76 −1876.39 −1875.73 −600.32 −590.63 −588.85 The number of Dutch or Surinamese 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 = 704.50, p < 0.001; test of multilevel model: Χ2 = 0.78, p = 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 = 0.25) *It indicates an interaction effect ber of Dutch or Surinamese 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 = 704.50, p < 0.001; test of multilevel model: Χ2 = 0.78, p = ng 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 = Journal of Youth and Adolescence (2020) 49:678–692 691 References European Journal of Developmental Psychology, 5, 262–285. https://doi. org/10.1080/17405620701556664. Hamm, J. V. (2000). Do birds of a feather flock together? The variable bases for African American, Asian American, and European American adolescents’ selection of similar friends. Develop- mental Psychology, 36, 209–219. https://doi.org/10.1037/0012- 1649.36.2.209. Syed, M., Juang, L. P., & Svensson, Y. (2018). Toward a new understanding of ethnic-racial settings for ethnic-racial identity development. Journal of Research on Adolescence, 28(2), 262–276. https://doi.org/10.1111/jora.12387. Ibarra, H. (1992). Homophily and differential returns: sex differences in network structure and access in an advertising firm. Adminis- trative Science Quarterly, 37, 422–447. https://doi.org/10.2307/ 2393451. Tajfel, H. (1982). Social psychology of intergroup relations. Annual Review of Psychology, 33, 1–39. https://doi.org/10.1146/annurev. ps.33.020182.000245. Kalter, F., Heath, A. F., Hewstone, M., Jonsson, J. O., Kalmijn, M., Kogan, I., & Van Tubergen, F. (2016). Children of immigrants longitudinal survey in Four European countries (CILS4EU) – Reduced version. (ZA5656 Data file Version 1.2.0). Cologne: GESIS Data Archive. https://doi.org/10.4232/cils4eu.5656.1.2.0 Tajfel, H., & Turner, J. (1979). An integrative theory of intergroup conflict. In W. G. Austin & S. Worchel (Eds.), The Social Psy- chology of Intergroup Relations (pp. 33–47). Monterey, CA: Brooks/Cole. https://doi.org/10.1016/S0065-2601(05)37005-5 Tolsma, J., Van Deurzen, I., Stark, T. H., & Veenstra, R. (2013). Who is bullying whom in ethnically diverse primary schools? Exploring links between bullying, ethnicity, and ethnic diversity in Dutch primary schools. Social Networks, 35, 51–61. https:// doi.org/10.1016/j.socnet.2012.12.002. Knifsend, C. A., Bell, A. N., & Juvonen, J. (2016). Identification with multiple groups in multiethnic middle schools: What predicts social ingroup overlap? Journal of Youth and Adolescence, 46(2), 317–327. https://doi.org/10.1007/s10964-016-0535-x. Leszczensky, L., & Pink, S. (2015). Ethnic segregation of friendship networks in school: Testing a rational-choice argument of dif- ferences in ethnic homophily between classroom- and grade-level networks. Social Networks, 42, 18–26. https://doi.org/10.1016/j. socnet.2015.02.002. g j Travaglino, G. A., Abrams, D., Randsley de Moura, G., Marques, J. M., & Pinto, I. R. (2014). How groups react to disloyalty in the context of intergroup competition: Evaluations of group deserters and defectors. Journal of Experimental Social Psychology, 54, 178–187. https://doi.org/10.1016/J.JESP.2014.05.006. Lindenberg, S. (2001). Social rationality versus rational egoism. In J. H. Turner (Ed.), Handbook of sociological theory (pp. 635–668). New York, NY: Plenum. https://doi.org/10.1007/0-387-36274-6_29 Van der Ploeg, R., Steglich, C., Salmivalli, C., & Veenstra, R. (2015). The intensity of victimization: associations with chil- dren’s psychosocial well-being and social standing in the classroom. PLoS ONE, 10, e0141490 https://doi.org/10.1371/ journal.pone.0141490. Long, J. S., & Freese, J. References (2006). Regression models for categorical variables using stata. 2nd edition College Station, TX: Stata Press. g g Lundborg, P. (2013). Refugees’ employment integration in Sweden: cultural distance and labor market performance. Review of International Economics, 21, 219–232. https://doi.org/10.1111/ roie.12032. j p Veenstra, R., Dijkstra, J. K., & Kreager, D. A. (2018). Pathways, networks, and norms: A sociological perspective on peer research. In W. M. Bukowski, B. Laursen & K. H. Rubin (Eds.), Handbook of peer interactions, relationships, and groups. 2nd ed. (pp. 45–63). New York, NY: The Guilford Press. Muthen, B. O., & Satorra, A. (1995). Complex sample data in struc- 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 Vitoroulis, I., & Vaillancourt, T. (2018). Ethnic group differences in bullying perpetration: a meta-analysis. Journal of Research on Adolescence, 28, 752–771. https://doi.org/10.1111/jora.12393. Andreas Flache is a full Professor at the University of Groningen. His 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.
https://openalex.org/W1508825410
https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/1471-2105-6-248
English
null
Better prediction of protein contact number using a support vector regression analysis of amino acid sequence
BMC bioinformatics
2,005
cc-by
6,729
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 material). be found in the additional file 1 (supplementary material). 9. Kabsch W, Sander C: Dictionary of protein secondary struc- ture: pattern recognition of hydrogen-bonded and geometri- cal features. Biopolymers 1983, 22:2577-2637. 10. p y 10. Thompson MJ, Goldstein RA: Predicting solvent accessibility: higher accuracy using Bayesian statistics and optimized res- idue substitution classes. Proteins 1996, 25:38-47. The proteins are randomly divided into three groups with each group having 315 chains. Each group is in turn used for training with the remaining two groups used for test- ing. Therefore, each group is tested twice by the two func- tions derived from the other groups, and as a result we have six groups of examination results. 11. Rost B, Sander C: Conservation and prediction of solvent accessibility in protein families. Proteins 1994, 20:216-226. 12. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lip- man DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402. 13. Jones DT: Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 1999, 292:195-202. Pearson's correlation coefficients and root mean square errors are calculated with respects to all residues and individual proteins. In addition, the absolute errors are calculated for the residues with different contact numbers. In order to compare with previous classification methods, we use different thresholds to classify contact numbers as "contacted" or "non-contacted" and compute the overall accuracy. The accuracy is defined as the ratio between the number of correctly predicted residues and the total number. 14. Vapnik V: The Nature of Statistical Learning Theory. Springer, New York, ; 2000. 15. Smola A, Scholkopf B: A tutorial on support vector regression. NeuroCOLT Technical Report Series, NC-TR-1998-030 1998 [http:// www.neurocolt.com]. ] 16. Joachims T: Making large-Scale SVM Learning Practical. In Advances in Kernel Methods - Support Vector Learning Edited by: Schölkopf B, Burges C and Smola A. , MIT Press; 1999. 17. Noguchi T, Akiyama Y: PDB-REPRDB: a database of represent- ative protein chains from the Protein Data Bank (PDB) in 2003. Nucleic Acids Res 2003, 31:492-493. References 1. Karchin R, Cline M, Karplus K: Evaluation of local structure alphabets based on residue burial. Proteins 2004, 55:508-518. 1. Karchin R, Cline M, Karplus K: Evaluation of local structure alphabets based on residue burial. Proteins 2004, 55:508-518. p 2. Hamelryck T: An amino acid has two sides: A new 2D measure provides a different view of solvent exposure. Proteins 2005, 59:38-48. 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 Page 9 of 9 (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 3. Kinjo AR, Horimoto K, Nishikawa K: Predicting absolute contact numbers of native protein structure from amino acid sequence. Proteins 2005, 58:158-165. 3. Kinjo AR, Horimoto K, Nishikawa K: Predicting absolute contact numbers of native protein structure from amino acid sequence. Proteins 2005, 58:158-165. q , 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 (page number not for citation purposes) Page 8 of 9 (page number not for citation purposes) (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/6/248
https://openalex.org/W2004976876
https://revistas.uniandes.edu.co/index.php/antipoda/article/download/1918/216
Spanish; Castilian
null
Trabajadores indígenas en el Chaco argentino: algunos sentidos estigmatizadores
Antipoda/Antípoda
2,013
cc-by
16,397
* 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. ** D t A t l í U i id d d B Ai A 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 extrañamiento. Buenos Aires, Ediciones Cervantes. 6. Bórmida, Marcelo. 1969. Mito y cultura. Bases para una ciencia de la conciencia mítica y una etnología 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 Argentina. Buenos Aires, Asociación de Comunidades Aborígenes Lhaka Honhat - IWGIA Grupo Internacional de Trabajo sobre Asuntos Indígenas. 9. Briones, Claudia y Walter Delrio. 2002. Patria sí, colonias también. Estrategias diferenciadas de radicación de indígenas en Pampa y Patagonia (1885-1900). En Fronteras, ciudades y Estados, comps. Ana Teruel, Mónica Lacarrieu y Omar Jerez, pp. 45-78. Córdoba, Alción. 9. Briones, Claudia y Walter Delrio. 2002. Patria sí, colonias también. Estrategias diferenciadas de radicación de indígenas en Pampa y Patagonia (1885-1900). En Fronteras, ciudades y Estados, comps. Ana Teruel, 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 Trabajo y Previsión. 12. Cordeu, Edgardo y Alejandra Siffredi. 1971. De la algarroba al algodón. Movimiento mesiánico de los guaycurú. Buenos Aires, Juárez Editor. 13. Decreto N° 1363. Creación de colonias aborígenes. Formosa, 08/08/1963. 14. Dirección de Información Parlamentaria. 1991. Conclusión 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. . 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- 1930. Buenos Aires, Centro Editor de América Latina. 28. Lagos, Marcelo. 2000. La cuestión indígena en el Estado y la sociedad nacional. Gran Chaco 1870- 1920. San Salvador de Jujuy, Universidad Nacional de Jujuy. 29. Ley N° 113. Régimen de Colonización y Tierras Fiscales. Formosa, 1963. 30. Marx, Karl. 2001. El capital. Crítica de la economía política. México, Fondo de Cultura Económica. 31. Marx, Karl. 1973. Miseria de la filosofía. Respuesta a la “Filosofía de la miseria” del Señor Proudhon. Buenos Aires, Cartago. 32. Marx, Karl y Friedrich Engels. 1973. La ideología alemana. Buenos Aires, Ediciones Pueblos Unidos. 33. Mashnshnek, Celia. 1975. Aportes para una comprensión de la economía de los Mataco. Scripta Ethnologica 3 (1), pp. 7-39. 34. Menéndez, Eduardo. 2002. El malestar actual de la antropología o de la casi imposibilidad de pensar lo ideológico. Revista de Antropología Social 11, pp. 39-87. 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 20. Instituto Provincial de Colonización y Tierras Fiscales. 2012. Balance general del estado legal de las tierras de la provincia de Formosa. Año 1960-2012. Consultado el 4 de marzo de 2013 en http:// www.formosa.gob.ar/. 20. Instituto Provincial de Colonización y Tierras Fiscales. 2012. Balance general del estado legal de las tierras de la provincia de Formosa. Año 1960-2012. Consultado el 4 de marzo de 2013 en http:// www.formosa.gob.ar/. 21. Iñigo Carrera, Valeria. 2012a. Movilización indígena en el Chaco argentino. Acción y conciencia políticas entre los qom del este de Formosa. Indiana 29, pp. 273-301. 21. Iñigo Carrera, Valeria. 2012a. Movilización indígena en el Chaco argentino. Acción y conciencia políticas entre los qom del este de Formosa. Indiana 29, pp. 273-301. 22. Iñigo Carrera, Valeria. 2012b. Movilización indígena en el noreste formoseño. Notas a propósito de un caso. Relaciones de la Sociedad Argentina de Antropología 37 (2), pp. 457-462. 22. Iñigo Carrera, Valeria. 2012b. Movilización indígena en el noreste formoseño. Notas a propósito de un caso. Relaciones de la Sociedad Argentina de Antropología 37 (2), pp. 457-462. 23. Iñigo Carrera, Valeria. 2009. De trabajadores asalariados y productores independientes de mercancías a población obrera sobrante: el desplazamiento de los tobas del este de Formosa de la producción algodonera. En Fronteras del desarrollo. Impacto social y económico en la cuenca del río Pilcomayo, coords. R e f e r e n c i a s Tratamiento de la cuestión indígena. Buenos Aires, Congreso de la Nación. 15. Goffman, Erving. 2003. Estigma: la identidad deteriorada. Buenos Aires, Amorrortu. 16. Golbert, Laura. 2004. ¿Derecho a la inclusión o paz social? Plan Jefas y Jefes de Hogar Desocupados. Santiago de Chile, Comisión Económica para América Latina y el Caribe. 17. Gordillo, Gastón. 2006. En el Gran Chaco. Antropologías e historias. Buenos Aires, Prometeo. 18. Gordillo, Gastón. 2004. Landscapes of Devils. Tensions of Place and Memory in the Argentinean Chaco. Durham, Duke University Press. 18. Gordillo, Gastón. 2004. Landscapes of Devils. Tensions of Place and Memory in the Argentinean Chaco. Durham, Duke University Press. 19. Grassi, Estela. 2003. Políticas y problemas sociales en la sociedad neoliberal. La otra década infame (I). Buenos Aires, Espacio. Antipod. Rev. Antropol. Arqueol. No. 17, Bogotá, julio-diciembre 2013, 296 pp. ISSN 1900-5407, pp. 229-251 2 5 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 20. Instituto Provincial de Colonización y Tierras Fiscales. 2012. Balance general del estado legal de las tierras de la provincia de Formosa. Año 1960-2012. Consultado el 4 de marzo de 2013 en http:// www.formosa.gob.ar/. 21. Iñigo Carrera, Valeria. 2012a. Movilización indígena en el Chaco argentino. Acción y conciencia políticas entre los qom del este de Formosa. Indiana 29, pp. 273-301. 22. Iñigo Carrera, Valeria. 2012b. Movilización indígena en el noreste formoseño. Notas a propósito de un caso. Relaciones de la Sociedad Argentina de Antropología 37 (2), pp. 457-462. 23. Iñigo Carrera, Valeria. 2009. De trabajadores asalariados y productores independientes de mercancías a población obrera sobrante: el desplazamiento de los tobas del este de Formosa de la producción algodonera. En Fronteras del desarrollo. Impacto social y económico en la cuenca del río Pilcomayo, coords. Héctor Hugo Trinchero y Elena Belli, pp. 173-210. Buenos Aires, Biblos. 24. Iñigo Carrera, Valeria. 2008. Sujetos productivos, sujetos políticos, sujetos indígenas: las formas de su objetivación mercantil entre los tobas del este de Formosa. Tesis de Doctorado, Facultad de Filosofía y Letras, Universidad de Buenos Aires, Buenos Aires. 25. Iñigo Carrera, Juan. 2008. El capital: razón histórica, sujeto revolucionario y conciencia. Buenos Aires: Imago Mundi. 26. Iñigo Carrera, Juan. 2004. Trabajo infantil y capital. Buenos Aires, Ediciones Cooperativas. 27. Iñigo Carrera, Nicolás. 1984. Indígenas y fronteras. Campañas militares y clase obrera. R e f e r e n c i a s Héctor Hugo Trinchero y Elena Belli, pp. 173-210. Buenos Aires, Biblos. 24. Iñigo Carrera, Valeria. 2008. Sujetos productivos, sujetos políticos, sujetos indígenas: las formas de su objetivación mercantil entre los tobas del este de Formosa. Tesis de Doctorado, Facultad de Filosofía y Letras, Universidad de Buenos Aires, Buenos Aires. 25. Iñigo Carrera, Juan. 2008. El capital: razón histórica, sujeto revolucionario y conciencia. Buenos Aires: Imago Mundi. 26. Iñigo Carrera, Juan. 2004. Trabajo infantil y capital. Buenos Aires, Ediciones Cooperativas. 27. Iñigo Carrera, Nicolás. 1984. Indígenas y fronteras. Campañas militares y clase obrera. Chaco, 1870- 1930. Buenos Aires, Centro Editor de América Latina. 28. Lagos, Marcelo. 2000. La cuestión indígena en el Estado y la sociedad nacional. Gran Chaco 1870- 1920. San Salvador de Jujuy, Universidad Nacional de Jujuy. 29. Ley N° 113. Régimen de Colonización y Tierras Fiscales. Formosa, 1963. 30. Marx, Karl. 2001. El capital. Crítica de la economía política. México, Fondo de Cultura Económica. 31. Marx, Karl. 1973. Miseria de la filosofía. Respuesta a la “Filosofía de la miseria” del Señor Proudhon. Buenos Aires, Cartago. 32. Marx, Karl y Friedrich Engels. 1973. La ideología alemana. Buenos Aires, Ediciones Pueblos Unidos. 33. Mashnshnek, Celia. 1975. Aportes para una comprensión de la economía de los Mataco. Scripta Ethnologica 3 (1), pp. 7-39. 34. Menéndez, Eduardo. 2002. El malestar actual de la antropología o de la casi imposibilidad de pensar lo ideológico. Revista de Antropología Social 11, pp. 39-87. 35. Miller, Elmer. 1979. Los tobas argentinos. Armonía y disonancia en una sociedad. México, Siglo XXI Editores. 36. Movimiento Campesino de Formosa (Mocafor). 2005. Parte de prensa. General Belgrano, 20/07/2005. 37. Sapkus, Sergio. 2002. Campesinado y protesta rural en el nordeste argentino. El Movimiento Campesino de Formosa (1995-2000). Tesis de Maestría, Programa de Postgrado en Antropología Social, Facultad de Humanidades y Ciencias Sociales, Universidad Nacional de Misiones, Misiones. 38. Trinchero, Héctor Hugo. 2000. Los dominios del demonio. Civilización y barbarie en las fronteras de la Nación. El Chaco Central. Buenos Aires, Eudeba. 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 39. R e f e r e n c i a s Trinchero, Héctor Hugo. 1994. Compromiso y distanciamiento: configuraciones de la crítica etnográfica contemporánea. Runa 21, pp. 317-333. 40. Trinchero, Héctor Hugo y Juan Martín Leguizamón. 1995. Fronteras de la modernización. Reproducción del capital y de la fuerza de trabajo en el umbral al Chaco argentino. En Producción doméstica y capital. Estudios desde la antropología económica, ed. Héctor Hugo Trinchero, pp. 15-44. Buenos Aires, Biblos. 40. Trinchero, Héctor Hugo y Juan Martín Leguizamón. 1995. Fronteras de la modernización. Reproducción del capital y de la fuerza de trabajo en el umbral al Chaco argentino. En Producción doméstica y capital. Estudios desde la antropología económica, ed. Héctor Hugo Trinchero, pp. 15-44. Buenos Aires, Biblos. 41. Vázquez, Héctor. 2002. Procesos identitarios, “minorías” étnicas y etnicidad: los mapuches de la República Argentina. Amnis, Revue de Civilisation Contemporaine Europes/Amériques 2. Disponible en: http://amnis.revues.org/167 [Consultado el 16 de abril de 2011]. 41. Vázquez, Héctor. 2002. Procesos identitarios, “minorías” étnicas y etnicidad: los mapuches de la República Argentina. Amnis, Revue de Civilisation Contemporaine Europes/Amériques 2. Disponible en: http://amnis.revues.org/167 [Consultado el 16 de abril de 2011]. 42. Vivaldi, Ana. 2010. El monte en la ciudad: (des)localizando identidades en un barrio toba. En Movilizaciones indígenas e identidades en disputa en la Argentina, eds. Gastón Gordillo y Silvia Hirsch, pp. 101-121. Buenos Aires, La Crujía. 42. Vivaldi, Ana. 2010. El monte en la ciudad: (des)localizando identidades en un barrio toba. En Movilizaciones indígenas e identidades en disputa en la Argentina, eds. Gastón Gordillo y Silvia 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
https://openalex.org/W4304187573
https://trialsjournal.biomedcentral.com/counter/pdf/10.1186/s13063-022-06743-6
English
null
Managing clustering effects and learning effects in the design and analysis of randomised surgical trials: a review of existing guidance
Trials
2,022
cc-by
6,774
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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​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.​equat​or-​netwo​rk.​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 Not applicable. 1. Evans D. Hierarchy of evidence: a framework for ranking evidence evalu- ating healthcare interventions. J Clin Nurs. 2003;12(1):77–84. 2. Cook JA. The challenges faced in the design, conduct and analysis of surgical randomised controlled trials. Trials. 2009;10:9. https://​doi.​org/​10.​ 1186/​1745-​6215-​10-9. Abbreviations GEE G li d Blencowe NS, Mills N, Cook JA, Donovan JL, Rogers CA, Whiting P, et al. Standardizing and monitoring the delivery of surgical interventions in randomized clinical trials. Br J Surg. 2016;103(10):1377–84. 10. Conroy EJ, Rosala-Hallas A, Blazeby JM, Burnside G, Cook JA, Gamble C. Randomized trials involving surgery did not routinely report considera- tions of learning and clustering effects. J Clin Epidemiol. 2019a;107:27–35. 28. Blencowe NS, Mills N, Cook JA, Donovan JL, Rogers CA, Whiting P, et al. Standardizing and monitoring the delivery of surgical interventions in randomized clinical trials. Br J Surg. 2016;103(10):1377–84. 29. Hyman WA. Medical device and diagnostic industry online, FDA flirts with the learning curve. 2019. Available from: https://​www.​mddio​nline.​com/​ regul​atory-​quali​ty/​fda-​flirts-​learn​ing-​curve. 11. Conroy EJ, Rosala-Hallas A, Blazeby JM, Burnside G, Cook JA, Gamble C. Funders improved the management of learning and clustering effects through design and analysis of randomized trials involving surgery. J Clin Epidemiol. 2019b;113:28–35. y yl 30. Ritchey ME. Chapter 25 - Special considerations of interventions: Medi- cal devices. In: Girman CJ, Ritchey ME, editors. Pragmatic Randomized Clinical Trials. Academic Press; 2021. p. 387–95. https://​doi.​org/​10.​1016/​ B978-0-​12-​817663-​4.​00032-5. 12. Conroy EJ, Blazeby JM, Burnside G, Cook JA, Gamble C. Managing clustering effects and learning effects in the design and analysis of multicentre randomised trials: a survey to establish current practice. Trials. 2020;21(1):433. 31. Shaw W, Semb G. The Scandcleft randomised trials of primary surgery for unilateral cleft lip and palate: 11. What next? J Plast Surg. Hand Surg. 2017;51(1):88–93. 31. Shaw W, Semb G. The Scandcleft randomised trials of primary surgery for unilateral cleft lip and palate: 11. What next? J Plast Surg. Hand Surg. 2017;51(1):88–93. 13. QSR International Pty Ltd. NVivo (Version 12). 2018. https://​www.​qsrin​ terna​tional.​com/​nvivo-​quali​tative-​data-​analy​sis-​softw​are/​home. 14. Boutron I, Moher D, Altman DG, Schulz KF, Ravaud P, Grp C. Extend- ing the CONSORT statement to randomized trials of nonpharma- cologic treatment: explanation and elaboration. Ann Intern Med. 2008;148(4):295–309. 32. Barkun JS, Aronson JK, Feldman LS, Maddern GJ, Strasberg SM, Bal- liol C, et al. Evaluation and stages of surgical innovations. Lancet. 2009;374(9695):1089–96. 32. Barkun JS, Aronson JK, Feldman LS, Maddern GJ, Strasberg SM, Bal- liol C, et al. Evaluation and stages of surgical innovations. Lancet. 2009;374(9695):1089–96. 15. National Institute for Health Research. Planning a Randomised Controlled Trial (RTC) – points to consider 2016. Available from: https://​www.​ct-​toolk​ it.​ac.​uk/​route​map/​trial-​plann​ing-​and-​design/​downl​oads/​plann​ing-a-​ rando​mised-​contr​olled-​trial.​pdf/. Accessed 30 Nov 2021. Abbreviations GEE G li d 1. Evans D. Hierarchy of evidence: a framework for ranking evidence evalu- ating healthcare interventions. J Clin Nurs. 2003;12(1):77–84. 2. Cook JA. The challenges faced in the design, conduct and analysis of surgical randomised controlled trials. Trials. 2009;10:9. https://​doi.​org/​10.​ 1186/​1745-​6215-​10-9. Conroy et al. Trials (2022) 23:869 Conroy et al. Trials (2022) 23:869 Page 8 of 8 3. Craig P, Dieppe P, Macintyre S, Michie S, Nazareth I, Petticrew M. Developing and evaluating complex interventions2019. Available from: https://​mrc.​ukri.​org/​docum​ents/​pdf/​compl​ex-​inter​venti​ons-​guida​nce/; Accessed 30 Nov 2021. ema.​europa.​eu/​en/​docum​ents/​scien​tific-​guide​line/​ich-e-​8-​gener​al-​consi​ derat​ions-​clini​cal-​trials-​step-5_​en.​pdf. Accessed 3 Aug 2022. ema.​europa.​eu/​en/​docum​ents/​scien​tific-​guide​line/​ich-e-​8-​gener​al-​consi​ derat​ions-​clini​cal-​trials-​step-5_​en.​pdf. Accessed 3 Aug 2022. 25. 25. Group IEW. ICH harmonised tripartite guideline: structure and content of clinical study reports E3 ICH harmonisation for better health web site: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; 1995. Available from: https://​datab​ase.​ich.​org/​sites/​defau​lt/​files/​E3_​Guide​line.​pdf. Accessed 3 Aug 2022. 4. Ergina PL, Cook JA, Blazeby JM, Boutron I, Clavien PA, Reeves BC, et al. Challenges in evaluating surgical innovation. Lancet. 2009;374:1097–104. 5. McCulloch P, Altman DG, Campbell B, Flum DR, Glasziou P, Marshall J, et al. No surgical innovation without evaluation: the IDEAL recommendations. Lancet. 2009;374:1105–12. 26. Bilbro NA, Hirst A, Paez A, Vasey B, Pufulete M, Sedrakyan A, et al. The IDEAL reporting guidelines: a Delphi consensus statement stage specific recommendations for reporting the evaluation of surgical innovation. Ann Surg. 2021;273(1):82–5. 6. Cook JA, Bruckner T, MacLennan GS, Seller CM. Clustering in surgical tri- als–database of intracluster correlations. Trials. 2012;13:2. 7. Cook JA, Ramsay CR, Fayers P. Statistical evaluation of learning curve effects in surgical trials. Clin Trials. 2004;1:421–7. 7. Cook JA, Ramsay CR, Fayers P. Statistical evaluation of learning curve effects in surgical trials. Clin Trials. 2004;1:421–7. 27. Committee for Medicinal Products for Human Use (CHMP). Guideline on adjustment for baseline covariates in clinical trials European Medicines Agency Science Medicines Health web site: European Medicines Agency; 2015. Available from: https://​www.​ema.​europa.​eu/​en/​docum​ents/​scien​ tific-​guide​line/​guide​line-​adjus​tment-​basel​ine-​covar​iates-​clini​cal-​trials_​ en.​pdf. Accessed 30 Nov 2021. f 8. Lee KJ, Thompson SG. Clustering by health professional in individually randomised trials. BMJ. 2005;330:142. f 8. Lee KJ, Thompson SG. Clustering by health professional in individually randomised trials. BMJ. 2005;330:142. 9. ICH Harmonised Tripartite Guideline. Statistical principles for clinical trials. International Conference on Harmonisation E9 Expert Working Group. Stat Med. 1999;18(15):1905-42. 9. ICH Harmonised Tripartite Guideline. Statistical principles for clinical trials. International Conference on Harmonisation E9 Expert Working Group. Stat Med. 1999;18(15):1905-42. 28. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 16. Zwarenstein M, Treweek S, Gagnier JJ, Altman DG, Tunis S, Haynes B, et al. Improving the reporting of pragmatic trials: an extension of the CONSORT statement. Brit Med J. 2008;337:a2390. 17. Campbell MK, Piaggio G, Elbourne DR, Altman DG, Group C. Consort 2010 statement: extension to cluster randomised trials. BMJ. 2012;345:e5661. 17. Campbell MK, Piaggio G, Elbourne DR, Altman DG, Group C. Consort 2010 statement: extension to cluster randomised trials. BMJ. 2012;345:e5661. 18. Jackson DL. Reporting results of latent growth modeling and multilevel modeling analyses: some recommendations for rehabilitation psychol- ogy. Rehabil Psychol. 2010;55(3):272–85. 18. Jackson DL. Reporting results of latent growth modeling and multilevel modeling analyses: some recommendations for rehabilitation psychol- ogy. Rehabil Psychol. 2010;55(3):272–85. 19. Group IEW. ICH harmonised tripartite guideline: guideline for good clinical practice E6(R2) ICH Harmonisation for better health web site: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; 2016 Available from: https://​datab​ase.​ich.​org/​sites/​defau​lt/​files/​E6_​R2_​Adden​dum.​pdf. Accessed 3 Aug 2022. 19. Group IEW. ICH harmonised tripartite guideline: guideline for good clinical practice E6(R2) ICH Harmonisation for better health web site: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; 2016 Available from: https://​datab​ase.​ich.​org/​sites/​defau​lt/​files/​E6_​R2_​Adden​dum.​pdf. Accessed 3 Aug 2022. 20. Medicines & Healthcare products Regulatory Agency. Clinical investiga- tions of medical devices – statistical considerations 2021. Available from: https://​assets.​publi​shing.​servi​ce.​gov.​uk/​gover​nment/​uploa​ds/​system/​ uploa​ds/​attac​hment_​data/​file/​989415/​Stati​stical_​consi​derat​ions_​clini​ cal_​inves​tigat​ions_-_​May_​2021.​pdf. Accessed 30 Nov 2021. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Publisher’s Note Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 21. Boutron I, Altman DG, Moher D, Schulz KF, Ravaud P, Group CN. CONSORT statement for randomized trials of nonpharmacologic treatments: a 2017 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 (RECOvER) checklist: a joint statement by the ERAS(I) and ERI(R) USA societies. World J Surg. 2019;43(1):1–8. 23. Vanhie A, Meuleman C, Tomassetti C, Timmerman D, D’Hoore A, Wolthuis A, et al. Consensus on recording deep endometriosis surgery: the CORDES statement. Hum Reprod. 2016;31(6):1219–23. 23. Vanhie A, Meuleman C, Tomassetti C, Timmerman D, D’Hoore A, Wolthuis A, et al. Consensus on recording deep endometriosis surgery: the CORDES statement. Hum Reprod. 2016;31(6):1219–23. 24. ICH Expert Working Group. ICH harmonised tripartite guidelines: general considerations for clinical trials E8. 1998. Available from: https://​www.​ 24. ICH Expert Working Group. ICH harmonised tripartite guidelines: general considerations for clinical trials E8. 1998. Available from: https://​www.​
https://openalex.org/W2694046640
https://pure.uva.nl/ws/files/29064528/10.1007_s11214_017_0369_1.pdf
English
null
Earth as a Tool for Astrobiology—A European Perspective
Space science reviews
2,017
cc-by
25,893
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 It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Download date:24 Oct 2024 Download date:24 Oct 2024 Download date:24 Oct 2024 Space Sci Rev (2017) 209:43–81 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 This article is distributed under the terms of the Creative Commons Attribution 4.0 Inter- national 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. References W. Abbey, E. Salas, R. Bhartia, L.W. Beegle, The Mojave vadose zone: a subsurface biosphere analogue for Mars. Astrobiology 13, 637–646 (2005) L.J. Allamandola, S.A. Sandford, G.J. Valero, Photochemical and thermal evolution of interstel- lar/precometary ice analogs. Icarus 76, 225–252 (1988) p y g C.C. Allen et al., Lunar and Planetary Science Conference XXXI Abstracts (1998a), p. 1287 C.C. Allen et al., Lunar and Planetary Science Conference XXIX Abstracts (1998b), p. 1690 X. Amashukeli et al., J. Geophys. Res. 112, G04S16 (2007) R. Amils et al., Extreme environments as Mars terrestrial analogs: the Rio Tinto case. Planet. Space Sci. 55, 370–381 (2007) M. Ansdell, P. Ehrenfreund, C. McKay, Stepping stones toward international space exploration. Acta Astro- naut. 68, 2098–2113 (2011) S.K. Atreya, A. Wong, N. Renno, W. Farrell, G. Delory, D. Sentman, S. Cummer, J. Marshall, S. Rafkin, D. Catling, Oxidant Enhancement in Martian Dust Devils and Storms: implications for Life and Habitabil- ity. Astrobiology 6, 439–450 (2006) y gy K.C. Benison, B.B. Bowen, Icarus 183, 225–229 (2006) M.P. Bernstein, J.P. Dworkin, S.A. Sandford, G.W. Cooper, L.J. Allamandola, Racemic amino acids from the ultraviolet photolysis of interstellar ice analogues. Nature 416, 401–403 (2002) D.A. Beysens, J.J.W.A. van Loon (eds.), Generations and Applications of Extraterrestrial Environments on Earth (River Publishers, Aalborg, 2015) J.M. Bigham, D.K. Nordstrom, in Reviews in Mineralogy and Geochemistry, vol. 40 (2000), pp. 351–403 A. Borst et al., Lunar and Planetary Science Conference XLI Abstracts (2010), p. 2744 N. Bost, F. Westall, C. Ramboz, F. Foucher, D. Pullan, A. Meunier, S. Petit, I. Fleicher, G. Klingelhöfer, J. Vago, Missions to Mars: characterization of Mars analogue rocks for the International Space Analogue Rockstore (ISAR). Planet. Space Sci. 82, 113–127 (2013) Rockstore (ISAR). Planet. Space Sci. 82, 113 p P.J. Boston, M.V. Ivanov, C.P. McKay, On the possibility of chemosynthetic ecosystems in subsurface habitats on Mars. Icarus 95, 300–308 (1992) A. Bouquet, O. Mousis, J.H. Waite, S. Picaud, Possible evidence for a methane source in Enceladus’ ocean. Geophys. Res. Lett. 42, 1334–1339 (2015) 72 Z. Martins et al. B.B. Bowen et al., Earth Planet. Sci. Lett. 268, 52–63 (2008) B.B. Bowen et al., Earth Planet. Sci. Lett. 268, 52–63 (2008) G. Briani, N. Fray, H. Cottin, Y. Benilan, M.-C. Gazeau, S. Perrier, HMT production and sublimation during thermal process of cometary organic analogs. Implications for its detection with the ROSETTA instru- ments. Icarus 226, 541–551 (2013) M.E. Brown, K.P. References Hand, Astron. J. 145, 1–7 (2013) A. Buch et al., Planet. Space Sci. 54, 1592–1599 (2006) M.L. Cable et al., Titan tholins: simulating titan organic chemistry in the Cassini-Huygens Era. Chem. Rev. 112, 1882–1909 (2012) N. Carrasco, H. Cottin, M. Cloix, Y. Bénilan, P. Coll, M.-C. Gazeau, R. Raulin, K. Saiagh, C. Szopa, The AMINO experiment: methane photolysis under solar VUV irradiation. Int. J. Astrobiol. 14, 79–87 (2014) N. Carrasco, A. Giuliani, J-J. Correia, G. Cernogora, VUV photochemistry simulation of planetary upper atmosphere using synchrotron radiation. J. Synchrotron Radiat. 20, 587–590 (2013a) N. Carrasco, J. Westlake, P. Pernot, H. Waite Jr., Nitrogen in Titan’s atmospheric aerosol factory, in The Early Evolution of the Atmospheres of Terrestrial Planets (Springer, New York, 2013b) S.C. Cary, I.R. McDonald, J.E. Barrett, D.A. Cowan, On the rocks: the microbiology of Antarctic Dry Valley soils. Nat. Rev. Microbiol. 8, 129–138 (2010) D.C. Catling, M.W. Claire, K.J. Zahnle, R.C. Quinn, B.C. Clark, M.H. Hecht, S. Kounaves, Atmospheric origins of perchlorate on Mars and in the Atacama. J. Geophys. Res. 115, E00E11 (2010) H S Chan et al Planet Space Sci 68 42 47 (2012) H.-S. Chan et al., Planet. Space Sci. 68, 42–47 (2012) C.S. Cockell, Geomicrobiology beyond Earth: microbe–mineral interactions in space exploration and settle- ment. Trends Microbiol. 18, 308–314 (2010) C.S. Cockell, S. Payler, S. Paling, D. McLuckie, Boulby International Subsurface Astrobiology Laboratory. Astron. Geophys. 54, 2.25–2.27 (2013) L. Colangeli, J.R. Brucato, A. Bar-Nun, R.L. Hudson, M.H. Moore, Laboratory experiments on cometary materials, in Comets II, ed. by M.C. Festou, H.U. Keller, H.A. Weaver (University of Arizona Press, Tucson, 2004), pp. 695–717 P. Coll, D. Coscia, N. Smith, M.C. Gazeau, S.I. Ramirez, G. Cernogora, G. Israel, F. Raulin, Experimental laboratory simulation of Titan’s atmosphere: aerosols and gas phase. Planet. Space Sci. 47, 1331–1340 (1999) H. Cottin, M.C. Gazeau, F. Raulin, Cometary organic chemistry: a review from observations, numerical and experimental simulations. Planet. Space Sci. 47, 1141–1162 (1999) H. Cottin, J.M. Kotler, K. Bartik, H.J. Cleaves, C.S. Cockell, J-P.P. de Vera, P. Ehrenfreund, S. Leuko, I.L. ten Kate, Z. Martins, R. Pascal, R. Quinn, P. Rettberg, F. Westall, Astrobiology and the Possibility of Life on Earth and Elsewhere.... Space Sci. Rev. (2015). doi:10.1007/s11214-015-0196-1 ten Kate, Z. Martins, R. Pascal, R. Quinn, P. Rettberg, F. Westall, Astrobiology and the Possibility of Life on Earth and Elsewhere.... Space Sci. Rev. (2015). doi:10.1007/s11214-015-0196-1 p Cottin, J.M. Kotler, C.S. References Möhlmann, T. Spohn, Adaptation of an Antarctic lichen to Martian niche conditions can occur within 34 days. Planet. Space Sci. 98, 182–190 (2014b) J.-P. de Vera, G. Horneck, P. Rettberg, S. Ott, The potential of the lichen symbiosis to cope with the extreme conditions of outer space II: germination capacity of lichen ascospores in response to simulated space conditions. Adv. Space Res. 33, 1236–1243 (2004) p J.P. de Vera, P. Rettberg, S. Ott, Life at the limits: capacities of isolated and cultured lichen symbionts to resist extreme environmental stresses. Orig. Life Evol. Biosph. 38, 457–468 (2008) S.O.L. Direito, P. Ehrenfreund, A. Marees, M. Staats, B. Foing, W. Röling, A wide variety of putative ex- tremophiles and large beta-diversity at the Mars Desert Research Station (Utah). Int. J. Astrobiol. 10, 191–208 (2011) R. dos Santos, M. Patel, J. Cuadros, Z. Martins, Influence of mineralogy on the preservation of amino acids under simulated Mars conditions. Icarus 277, 342–353 (2016) M.B. Duke et al., PISCES: Hawaii facility for simulation and training, in Proc., 38th Lunar and Planetary Science Conf., Lunar and Planetary Institute (LPI), Houston 2007) Ehrenfreund et al., Astrobiology and habitability studies in preparation for future Mars missions: trends from investigating minerals, organics and biota. Int. J. Astrobiol. 10, 239–254 (2011) S.A. Ewing et al., Geochim. Cosmochim. Acta 70, 5293–5322 (2006) W. Farrell, G. Delory, S.K. Atreya, Martian dust storms as a possible sink of atmospheric methane. Geophys. Res. Lett. 33, L21203 (2006) D.C. Fernandez-Remolar et al., Earth Planet. Sci. Lett. 240, 149–167 (2005) D.C. Fernandez-Remolar et al., Earth Planet. Sci. Lett. 240, 149–167 (2005) D.C. Fernández-Remolar, O. Prieto-Ballesteros, N. Rodriguez, F. Gómez, R. Amils, J. Gómez-Elvira, C.R. Stoker, Underground habitats in the Rio Tinto Basin: a model for subsurface life habitats on Mars. Astrobiology 8, 1023–1047 (2008) Stoker, Underground habitats in the Rio Tinto Basin: a model for subsurface life habitats on Mars. Astrobiology 8, 1023–1047 (2008) gy ( ) B. Fleury, N. Carrasco, T. Gautier, A. Mahjoub, J. He, C. Szopa, E. Hadamcik, A. Buch, G. Cernogora, Influence of CO on Titan atmospheric reactivity. Icarus 238, 221–229 (2014) B.H. Foing, C. Stoker, J. Zavaleta, P. Ehrenfreund, C. Thiel, P. Sarrazin, D. Blake, J. Page, V. Pletser, J. Hen- drikse, S. Direito, J.M. Kotler, Z. Martins, G. Orzechowska, C. Gross, L. Wendt, J. Clarke, A.M. Borst, S.T.M. Peters, M.B. Wilhelm, G.R. References Pace, Microbial diversity of cryptoendolithic communi- ties from the McMurdo Dry Valleys, Antarctica. Appl. Environ. Microbiol. 69, 3858–3867 (2003) R. de la Torre, G. Horneck, L.G. Sancho, A. Pintado, K. Scherer, R. Facius, U. Deutschmann, M. Reina, P. Baglioni, R. Demets, Studies of LICHENS from high mountain regions in outer space: the BIOPAN experiment, in Proceedings of the III European Workshop on Exo-Astrobiology, vol. 545 (2004), pp. 193–194 R. de la Torre, L.G. Sancho, G. Horneck, C. Ascaso, A. de los Ríos, K. Olsson-Francis, C.S. Cockell, P. Rettberg, T. Berger, J.P.P. de Vera, S. Ott, J.M. Frías, J. Wierzchos, M. Reina, A. Pintado, R. Demets, Survival of lichens and bacteria exposed to outer space conditions—results of the Lithopanspermia experiments. Icarus 208, 735–748 (2010) P. de Marcellus, C. Meinert, M. Nuevo, J.J. Filippi, G. Danger, D. Deboffle, L. Nahon, L.L.S. d’Hendecourt, U.J. Meierhenrich, Non-racemic amino acid production by ultraviolet irradiation of achiral interstellar ice analogs with circularly polarized light. Astrophys. J. Lett. 727, L27 (2011) P. de Marcellus, C. Meinert, I. Myrgorodska, L. Nahon, T. Buhse, L.L.S. d’Hendecourt, U.J. Meierhenrich, Aldehydes and sugars from evolved precometary ice analogs: importance of ices in astrochemical and prebiotic evolution. Proc. Natl. Acad. Sci. USA 112, 965–970 (2015) L. d’Hendecourt, M. Jourdain de Muizon, E. Dartois, M. Breitfellner, P. Ehrenfreund, J. Benit, F. Boulanger, J.L. Puget, H.J. Habing, ISO-SWS observations of solid state features towards RAFGL 7009S. Astron. Astrophys. 315L, 365–369 (1996) p y , ( ) D. Deamer, The origin of life, in The Princeton Guide to Evolution (Princeton University Press, Princeton, 2014), pp. 121–126 J.W. Deming, Psychrophiles and polar regions. Curr. Opin. Microbiol. 3, 301–309 (2002) S. Derenne, C. Coelho, C. Anquetil, C. Szopa, A.S. Rahman, P.F. McMillan, F. Corà, C.J. Pickard, E. Quirico, C. Bonhomme, New insights into the structure and chemistry of Titan’s tholins via 13C and 15N solid state nuclear magnetic resonance spectroscopy. Icarus 221, 844–853 (2012) g p py J.P. de Vera, D. Möhlmann, F. Butina, A. Lorek, R. Wernecke, S. Ott, Survival potential and photosynthetic activity of lichens under Mars-like conditions: a laboratory study. Astrobiology 10, 215–227 (2010) J.-P. de Vera, S. Dulai, A. Kereszturi, A. Koncz, A. Lorek, D. Mohlmann, M. Marschall, T. Pocs, Results on the survival of cryptobiotic cyanobacteria samples after exposure to Mars-like environmental conditions. Int. J. Astrobiol. 13, 35–44 (2014a) J.-P. de Vera, D. Schulze-Makuch, A. Khan, A. Lorek, A. Koncz, D. References Cockell, R. Demets, P. Ehrenfreund, A. Elsaesser, L. d’Hendecourt, J. van Loon, H. Cottin, J.M. Kotler, C.S. Cockell, R. Demets, P. Ehrenfreund, A. Elsaesser, L. d’Hendecourt, J. van Loon, Z. Martins, S. Onofri, R. Quinn, E. Rabbow, P. Rettberg, A.J. Ricco, K. Slenzka, R. de la Torre, J.- P. de Vera, F. Westall, N. Carrasco, A. Fresneau, Y. Kawaguchi, Y. Kebukawa, D. Nguyen, O. Poch, K. Saiagh, F. Stalport, A. Yamagishi, H. Yano, B.A. Klamm, Space as a tool for astrobiology: re- view and recommendations for experimentations in Earth orbit and beyond. Space Sci. Rev. (2017). doi:10.1007/s11214-017-0365-5 C.R. Cousins, I.A. Crawford, Volcano-Ice Interaction as a Microbial Habitat on Earth and Mars. Astrobiology 11, 695–710 (2011) I. Couturier-Tamburelli, M.S. Gudipati, A. Lignell, R. Jacovi, N. Piétri, Spectroscopic studies of non-volatile residue formed by photochemistry of solid C4N2: a model of condensed aerosol formation on Titan. Icarus 234, 81–90 (2014) D. Cremers, L. Radziemski, History and fundamentals of LIBS, in Laser Induced Breakdown Spectroscopy: Fundamentals and Applications (Cambridge University Press, Cambridge, 2006), pp. 9–16. Chapter 1 G E C hi T N Tit J J W PR Ch i t THEMIS b ibl k li ht M D. Cremers, L. Radziemski, History and fundamentals of LIBS, in Laser Induced Breakdown Spectroscopy: Fundamentals and Applications (Cambridge University Press, Cambridge, 2006), pp. 9–16. Chapter 1 G.E. Cushing, T.N. Titus, J.J. Wynne, P.R. Christensen, THEMIS observes possible cave skylights on Mars. Geophys Res Lett 34 L17201 (2007) G.E. Cushing, T.N. Titus, J.J. Wynne, P.R. Christensen, THEMIS observes possible cave skylights on Mars. Geophys. Res. Lett. 34, L17201 (2007) G. Danger, F.R. Orthous-Daunay, P.D. Marcellus, P. Modica, V. Vuitton, F. Duvernay, L. Flandinet, L.L.S. d’Hendecourt, R. Thissen, T. Chiavassa, Characterization of laboratory analogs of interstellar/cometary organic residues using very high resolution mass spectrometry. Geochim. Cosmochim. Acta 118, 184– 201 (2013) L.R. Dartnell, L. Desorgher, J.M. Ward, A.J. Coates, Modelling the surface and subsurface Martian radiation environment: implications for astrobiology. Geophys. Res. Lett. 34, L02207 (2007) L. Dartnell, M.R. Patel, Degradation of microbial fluorescence biosignatures by solar ultraviolet radiation on Mars. Int. J. Astrobiol. 13, 112–123 (2013) R. de la Torre Noetzel, Estudio de los efectos de la radiación UV sobre ecosistemas epilíticos. Tesis Doctoral, Universidad Complutense de Madrid (2002) 73 Earth as a Tool for Astrobiology—A European Perspective J.R. de la Torre, B.M. Goebel, E.I. Friedmann, N.R. References Pollack, Threshold windspeeds for sand on Mars—wind-tun simulations. Geophys. Res. Lett. 7, 121–124 (1980) R. Greeley, R. Leach, B. White, J. Iversen, J. Pollack, Threshold windspeeds for sand on Mars—wind-tunnel simulations. Geophys. Res. Lett. 7, 121–124 (1980) R. Greeley, G. Wilson, R. Coquilla, B. White, R. Haberle, Wind-blown dust on Mars: laboratory simulations R. Greeley, G. Wilson, R. Coquilla, B. White, R. Haberle, Wind-blown dust on Mars: laboratory simulati of flux as a function of surface roughness. Planet. Space Sci. 48, 1349–1355 (2000) R. Greeley, Saltation impact as a means for raising dust on Mars. Planet. Space Sci. 50, 151–155 (2002) R. Greeley, M.R. Balme, J.D. Iversen, M. Metzger, R. Mickelson, J. Phoreman, B. White, Martian dust devils: laboratory simulations of particle threshold. J. Geophys. Res. 108, 1–12 (2003) J.M. Greenberg, What are comets made of? A model based on interstellar dust, in Comets, e ing (University of Arizona Press, Tucson, 1982), pp. 131–163 J.P. Grotzinger et al., Earth Planet. Sci. Lett. 240, 11–72 (2005) H.P. Gunnlaugsson et al., Hyperfine Interact. 144/145, 365–370 (2002) E. Hadamcik, J.-B. Renard, A. Mahjoub, T. Gautier, N. Carrasco, G. Cernogora, C. Szopa, Optical properties of analogs of Titan’s aerosols produced by dusty plasma. Earth Planets Space 65, 1175–1184 (2013) E. Hadamcik, J-B. Renard, G. Alcouffe, G. Cernogora, A-C. Levasseur-Regourd, C. Szopa, Light scattering measurements on Titan’s aerosols analogues produced by a dusty plasma. Planet. Space Sci. 57, 1631– 1641 (2009) Hansen, European Space Agency, ESA Communications SP-1299 (2007) A. Hansen et al., Int. J. Astrobiol. 4, 135–144 (2005) C. He, M.A. Smith, A comprehensive NMR structural study of Titan aerosol analogs: implications for Titan’s atmospheric chemistry. Icarus 243, 31–38 (2014) C. He, M.A. Smith, A comprehensive NMR structural st atmospheric chemistry. Icarus 243, 31–38 (2014) p y J. He, A. Buch, N. Carrasco, C. Szopa, Thermal degradation of organics for pyrolysis in space: Titan’s atmo- spheric aerosol case study. Icarus 248, 205–212 (2014) J. He, A. Buch, N. Carrasco, C. Szopa, Thermal degradation of organics for pyrolysis in space: Titan’s atm spheric aerosol case study. Icarus 248, 205–212 (2014) J. He, A. Buch, N. Carrasco, C. Szopa, Thermal degradation o spheric aerosol case study. Icarus 248, 205–212 (2014) J.W. Head, J.F. Mustard, M.A. Kreslavsky, R.E. Milliken, D.R. Marchant, Recent ice ages on Mars. Nature 426, 797–802 (2003) P.E. Hintze, C.R. Buhler, A.C. Schuerger, L.M. Calle, C.I. References Davies (ILEWG EuroGeoMars 2009 Team), Field astrobiology re- search in Moon–Mars analogue environments: instruments and methods. Int. J. Astrobiol. 10, 141–160 (2011) 74 Z. Martins et al. B.H. Foing, C. Orgel, C. Stoker, P. Ehrenfreund et al., Gale crater analogue geology studies at multiple scales, in LPI, vol. 45 (2014), p. 2675 p B.H. Foing, C. Stoker, L. Rodrigues, Å. Svendsen, I. Rammos et al., Astrobiology, geology and habitability field studies supporting Mars research, in LPI, vol. 44 (2013), p. 3057 A.G. Fountain, T.H. Nylen, A. Monaghan, H.J. Basagic, D. Bromwich, Snow in the McMurdo Dry Valleys, Antarctica. Int. J. Climatol. 30, 633–642 (2009) G. Freier, The electric field of a large dust devil. J. Geophys. Res. 65, 3504 (1960) E.I. Friedmann, Ocampo, Endolithic blue-algae in the dry valleys: primary producers in the Antarctic desert ecosystem. Science 193, 1247–1249 (1976) y , ( ) E.I. Friedmann, Endolithic microorganisms in the Antarctic cold desert. Science 215, 1045–1053 (1982) E.I. Friedmann (ed.), Antarctic Microbiology (Wiley, New York, 1993) dmann (ed.), Antarctic Microbiology (Wiley, New Y J.R. Garry et al., Meteorit. Planet. Sci. 41, 391–405 (2006) T. Gautier, N. Carrasco, A. Mahjoub, S. Vinatier, A. Giuliani, C. Szopa, C.M. Anderson, J.-J. Correia, P. Dumas, G. Cernogora, Mid- and far-infrared absorption spectroscopy of Titan’s aerosols analogues. Icarus 221, 320–327 (2012) T. Gautier, N. Carrasco, I. Schmitz-Afonso, D. Touboul, C. Szopa, A. Buch, P. Pernot, Nitrogen incorporation in Titan’s tholins inferred by high resolution orbitrap mass spectrometry and gas chromatography–mass spectrometry. Earth Planet. Sci. Lett. 404, 33–42 (2014) p y ( ) P.A. Gerakines, R.L. Hudson, Glycine’s radiolytic destruction in ices: first in situ laboratory measurements for Mars. Astrobiology 13, 647–655 (2013) P.A. Gerakines, R.L. Hudson, Glycine’s radiolyti for Mars. Astrobiology 13, 647–655 (2013) D. Gilichinsky et al., Astrobiology 3, 331–341 (2003) D.A. Gilichinsky et al., Astrobiology 7, 275–311 (2007) F. Gomez, E. Mateo-Martí, O. Prieto-Ballesteros, J.A. Martín-Gago, R. Amils, Protection of chemolithoau- totrophic bacteria exposed to simulated mars environmental conditions. Icarus 209, 482–487 (2010) J. Goordial, A.F. Davila, D. Lacelle, W. Pollard, M. Marinova, C. Greer, J. DiRuggiero, C.P. McKay, L. Whyte, Nearing the cold-arid limits of microbial life in permafrost of an upper dry valley, Antarctica. ISME J. 10, 1613–1624 (2016) R. Greeley, B.R. White, J.B. Pollack, J.D. Iversen, R.N. Leach, Dust storms on Mars: considerations and simulations. NASA Technical Memorandum 78423: 1–32 (1977) R. Greeley, R. Leach, B. White, J. Iversen, J. References Calle, Alteration of five organic compounds by glow discharge plasma and UV light under simulated Mars conditions. Icarus 208, 749–757 (2010) J.W. Holt, A. Safaeinili, J.J. Plaut, J.W. Head, R.J. Phillips, R. Seu, S.D. Kempf, P. Choudhary, D.A. Young, N.E. Putzig, D. Biccari, Y. Gim, Radar sounding evidence for buried glaciers in the southern mid- latitudes of Mars. Science 322, 1235–1238 (2008) B. Hong et al., in Abstracts, American Society for Microbiology 106th General Meeting (2006), p. 388 G. Horneck et al., AstRoMap European Astrobiology Roadmap. Astrobiology 16, 201–243 (2016) Earth as a Tool for Astrobiology—A European Perspective 75 N.H. Horowitz, R.E. Cameron, J.S. Hubbard, Microbiology of the Dry Valleys of Antarctica. Science 193, 242–245 (1972) S.M. Hörst, M.A. Tolbert, In situ measurements of the size and density of Titan aerosol analogs. Astrophys. J. Lett. 770, L10 (2013) ( ) S. Hörst et al., Formation of amino acids and nucleotide bases in a Titan atmosphere simulation experiment. Astrobiology 12, 809–817 (2012) G. Horvath, J.D. Skalny, N.J. Mason, M. Klas, M. Zahoran, R. Vladoiu, M. Manole, Corona discharge exper- iments in admixtures of N2 and CH4: a laboratory simulation of Titan’s atmosphere. Plasma Sources Sci. Technol. 18, 034016 (2009) H.-W. Hsu et al., Ongoing hydrothermal activities within Enceladus. Nature 519, 207–210 (2015) H. Imanaka, B.N. Khare, J.E. Elsila, E.L.O. Bakes, C.P. McKay, D.P. Cruikshank, S. Sugita, T. Matsui, R.N. Zare, Laboratory experiments of Titan next term tholin formed in cold plasma at various pressures: implications for nitrogen-containing polycyclic aromatic compounds in previous termTitan next term haze. Icarus 168, 344–366 (2004) H. Imanaka, P. Dale Cruikshank, B.N. Khare, C.P. McKay, Optical constants of Titan tholins at mid-infrared wavelengths (2.5–25 µm) and the possible chemical nature of Titan’s haze particles. Icarus 218, 247–261 (2012) ( ) H. Imanaka, M.A. Smith, Formation of nitrogenated organic aerosols in the Titan upper atmosphere. Proc. Natl. Acad. Sci. 107, 12423–12428 (2010) J. Jänchen, J. Meeßen, T.H. Herzog, M. Feist, R. de la Torre, J.-P.P. de Vera, Humidity interaction of lichens under astrobiological aspects: the impact of UVC exposure on their water retention properties. Int. J. Astrobiol. 14, 445–456 (2015) J. Jänchen, A. Bauermeister, N. Feyh, J.-P. de Vera, P. Rettberg, H.-C. Flemming, U. Szewzyk, Water retention of selected microorganisms and Martian soil simulants under close to Martian environmental conditions. Planet. Space Sci. 98, 163–168 (2014) p ( ) B. Jakosky et al., Astrobiology 3, 343–350 (2003) L.L. References Jensen, J. Merrison, A.A. Hansen, K.A. Mikkelsen, T. Kristoffersen, P. Nørnberg, B.A. Lomstein, K. Fin- ster, A facility for long-term Mars simulation experiments: the Mars Environmental Simulation Cham- ber (MESCH). Astrobiology 8, 537–548 (2008) ( ) gy , ( ) A.P. Johnson, L.M. Pratt, Metal-catalyzed degradation and racemization of amino acids in iron sulfate brines under simulated Martian surface conditions. Icarus 207, 124–132 (2010) K. Junge, H. Eicken, B.D. Swanson, J.W. Deming, Bacterial incorporation of leucine into protein down to −20◦C with evidence for potential activity in sub-eutectic saline ice formations. Cryobiology 52, 417– 429 (2006) ( ) D. Kapitulˇcinová et al., The interlayer regions of sheet silicates as a favorable habitat for endolithic microor- ganisms. Geomicrobiol. J. 32, 530–537 (2015) D.M. Karl et al., Science 286, 2144–2147 (1999) K. Kashefi, D.R. Lovley, Science 301, 934 (2003) F. Keppler, I. Vigano, A. McLeod, U. Ott, M. Früchtl, T. Röckmann, Ultraviolet-radiation-induced methane emissions from meteorites and the Martian atmosphere. Nature 486, 93–96 (2012) S.J. Kim, A. Jung, C.K. Sim, R. Courtin, A. Bellucci, B. Sicardy, I.O. Song, Y.C. Minh, Retrieval and tentative identification of the 3 µm spectral feature in Titan’s haze. Planet. Space Sci. 59, 699–704 (2011) G. Kminek, J.L. Bada, The effect of ionizing radiation on the preservation of amino acids on Mars. Ea Planet. Sci. Lett. 245, 1–5 (2006) K. Kobayashi, T. Kasamatsu, T. Kaneko, J. Koike, T. Oshima, T. Saito, T. Yamamoto, H. Yanagawa, Forma- tion of amino acid precursors in cometary ice environments by cosmic radiation. Adv. Space Res. 16, 21–26 (1995) ( ) Kotler, R.C. Quinn, Z. Martins, B.H. Foing, P. Ehrenfreund, Analysis of mineral matrices of planetary so analogs from the Utah Desert. Int. J. Astrobiol. 10, 221–230 (2011) Kotler, R.C. Quinn, Z. Martins, B.H. Foing, P. Ehrenfreund, Analysis of mi analogs from the Utah Desert. Int. J. Astrobiol. 10, 221–230 (2011) T.A. Krala, T.S. Altheide, A.E. Lueders, A.C. Schuerger, Low pre methanogens: implications for life on Mars. Planet. Space Sci. 59, 2 T.A. Krala, T.S. Altheide, A.E. Lueders, A.C. Schuerger, Low pressure and desiccation effects on methanogens: implications for life on Mars. Planet. Space Sci. 59, 264–270 (2011) g p p M. Kuga, N. Carrasco, B. Marty, Y. Marrocchi, S. Bernard, T. Rigaudier, B. Fleury, L. Tissandier, Nitrogen isotopic fractionation during abiotic synthesis of organic solid particles. Earth Planet. Sci. Lett. 393, 2–13 (2014) O.L. References Lange, CO2-Gaswechsel von Moosen nach Wasserdampfaufnahme aus dem Luftraum. Planta 89, 90–94 (1969) O L L E D S h l W K h E i ll k l i h U h Fl h d N O.L. Lange, E.-D. Schulze, W. Koch, Experimentell-ökologische Untersuchungen an Flechten der Negev- Wüste. III. CO2-Gaswechsel und Wasserhaushalt von Krusten- und Blattflechten am natürlichen Stan- dort während der sommerlichen Trockenperiode. Flora 12, 525–538 (1970) O.L. Lange, T.G.A. Green, H. Reichenberger, A. Meyer, Photosynthetic depression at high thallus water contents in lichens: concurrent use of gas exchange and fluorescence techniques with a cyanobacterial and a green algal Peltigera species. Bot. Acta, Ber. Dtsch. Bot. Gesellschaft 109, 43–50 (1996) Z. Martins et al. 76 P. Lee, Haughton-Mars Project 1997–2007: a decade of Mars analog science and exploration research at Haughton Crater, Devon Island, High Arctic, in Proc., 2nd Int. Workshop on Exploring Mars and its Earth Analogs International Research School of Planetary Sciences (IRSPS), Pescara, Italy (2007) P. Lee, G.R. Osinski, The Haughton-Mars Project: overview of science investigations at the Haughton impact structure and surrounding terrains, and relevance to planetary studies. Meteorit. Planet. Sci. 40, 1755– 1758 (2005) E. Lester et al., Soil Biol. Biochem. 39, 704–708 (2007) R. Léveillé, Validation of astrobiology technologies and instrument operations in terrestrial analogue envi- ronments. C. R., Palévol 8, 637–648 (2009) D.S.S. Lim, A.L. Brady (Pavilion Lake Research Project (PLRP) Team), A historical overview of the Pavilion Lake Research Project—analog science and exploration in an underwater environment, in Special Paper Geological Society of America, vol. 483 (2011), pp. 85–116 A. Lorek, A. Koncz, Simulation and measurement of extraterrestrial conditions for experiments on habitabil- ity with respect to Mars, in Habitability of other Planets and Satellites, Cellular Origin, Life in Extreme Habitats and Astrobiology, vol. 18, ed. by J.P. de Vera, J. Seckbach (2013), pp. 145–162 P.R. Mahaffy et al., The sample analysis at Mars investigation and instrument suite. Space Sci. Rev. 170, 401–478 (2012) P.R. Mahaffy et al., The sample analysis at Mars investigation and instrument suite. Space Sci. Rev. 170, 401–478 (2012) A M hj b N C P R D h T G ti C S G C I fl f th t A. Mahjoub, N. Carrasco, P.-R. Dahoo, T. Gautier, C. Szopa, G. Cernogora, Influence of methane concen tion on the optical indices of Titan’s aerosols analogues. Icarus 221, 670–677 (2012) D. Marchant, G.H. Denton, C.C. References Swisher III., N. Potter Jr., Late Cenozoic Antarctic paleoclimate recon- structed from volcanic ashes in the Dry Valleys region of southern Victoria Land. Bull. Geol. Soc. Am. 108, 181–194 (1996) D.R. Marchant, J.W. Head, Antarctic dry valleys: microclimate zonation, variable geomorphic processes, a implications for assessing climate change on Mars. Icarus 192, 187–222 (2007) J.J. Marlow et al., Astron. Geophys. 49, 2.2–2.5 (2008) J.J. Marlow et al., Int. J. Astrobiol. 10, 31–44 (2011 D. Martin, C.S. Cockell, PELS (Planetary Environmental Liquid Simulator): a new type of simulation facility to study extraterrestrial aqueous environments. Astrobiology 15, 111–118 (2015) y q gy W. Martin et al., Hydrothermal vents and the origin of life. Nat. Rev. Microbiol. 6, 805–814 (2008) Z. Martins, M.A. Sephton, B.H. Foing, P. Ehrenfreund, Extraction of amino acids from soils close to th Desert Research Station (MDRS), Utah. Int. J. Astrobiol. 10, 231–238 (2011) Z. Martins, P. Modica, B. Zanda, L.L.L.S. D’Hendecourt, The amino acid and hydrocarbon contents of the Paris meteorite: insights into the most primitive CM chondrite. Meteorit. Planet. Sci. J. 50, 926–943 (2015) B. Maté, I. Tanarro, R. Escribano, M.A. Moreno, V.J. Herrero, Stability of extraterrestrial glycine under energetic particle radiation estimated from 2 keV electron bombardment experiments. Astrophys. J. 806, 151 (2015) E. Mateo-Martí, O. Prieto-Ballesteros, J.M. Sobrado, J. Gómez-Elvira, J.A. Martín-Gago, A chamber for studying planetary environments and its applications to astrobiology. Meas. Sci. Technol. 17, 2274– 2280 (2006) ( ) E. Mateo-Martí, C.M. Pradier, J.A. Martín-Gago, UV-Photostability of adenine on gold and silicon surfaces. Astrobiology 9, 573–579 (2009) E. Mateo-Martí, C.M. Pradier, UV irradiation study of a tripeptide isolated in an argon matrix: a tautomerism process evidenced by infrared and X-ray photoemission spectroscopies. Spectrochim. Acta, Part A, Mol. Biomol. Spectrosc. 109, 247–252 (2013) A. Maturilli, J. Helbert, M. D’Amore, Dehydration of phyllosilicates under low temperatures: an application to Mars, in 41st Lunar and Planetary Science Conference, abstract 1533 (2010), p. 1299 104 T.M. McCollom, J. Geophys. Res. 104, 30729–30742 (1999) p y ( ) G.D. McDonald, B.N. Khare, W.R. Thompson, C. Sagan, CH4/NH3/H2O spark tholin: chemical analysis and interaction with Jovian aqueous clouds. Icarus 94, 354–367 (1991) G.D. McDonald, W.R. Thompson, M. Heinrich, B.N. Khare, C. Sagan, Chemical investigation of Titan and Triton tholins. Icarus 108, 137–145 (1994) A.S. McEwen et al., Seasonal flows on warm Martian slopes. Science 333, 740–743 (2011) J. Meeßen, P. Wuthenow, P. Schille, E. Rabbow, J.-P.P. References de Vera, S. Ott, Resistance of the Lichen Buellia frigida to simulated space conditions during the preflight tests for BIOMEX—viability assay and morphological stability. Astrobiology 15, 601–615 (2015) U.J. Meierhenrich, L. Nahon, C. Alcaraz, J.H. Bredehoft, S.V. Hoffmann, B. Barbier, A. Brack, Asymmetric vacuum UV photolysis of the amino acid leucine in the solid state. Angew. Chem., Int. Ed. Engl. 44, 5630–5634 (2005) C. Meinert, J.-J. Filippi, P.D. Marcellus, L.L.S. d’Hendecourt, U.J. Meierhenrich, N-(2-Aminoethyl)glycine and amino acids from interstellar ice analogues. ChemPlusChem 77, 186–191 (2012) Earth as a Tool for Astrobiology—A European Perspective 77 C. Meinert, I. Myrgorodska, P. de Marcellus, T. Buhse, L. Nahon, S.V. Hoffmann, L.L.S. d’Hendecourt, U.J. Meierhenrich, Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs. Science 352, 208–213 (2016) J.P. Merrison, P. Bertelsen, C. Frandsen, P. Gunnlaugsson, J.M. Knudsen, S. Lunt, M.B. Madsen, L.A. Mossin, J. Nielsen, P. Nørnberg, K.R. Rasmussen, E. Uggerhoj, Simulation of the Martian dust aerosol at low wind speeds. J. Geophys. Res., Planets 107, 5133 (2002) p p y J. Merrison, J. Jensen, K. Kinch, R. Mugford, P. Nørnberg, The electrical properties of Mars analogue dust. Planet. Space Sci. 52, 279–290 (2004) J.P. Merrison, H. Gunnlaugsson, P. Nørnberg, A. Jensen, K. Rasmussen, Determination of the wind induced detachment threshold for granular material on Mars using wind tunnel simulations. Icarus 191(2), 568– 580 (2007) J.P. Merrison, H. Bechtold, H. Gunnlaugsson, A. Jensen, K. Kinch, P. Nørnberg, K. Rasmussen, An environ- mental simulation wind tunnel for studying aeolian transport on Mars. Planet. Space Sci. 56, 426–437 (2008) ( ) J.P. Merrison, H.P. Gunnlaugsson, S. Knak Jensen, P. Nørnberg, Mineral alteration induced by sand transport: a source for the reddish color of Martian dust. Icarus 205(2), 716–718 (2010) J.P. Merrison, H.P. Gunnlaugsson, M.R. Hogg, M. Jensen, J.M. Lykke, M. Bo Madsen, M.B. Nielsen, P. Nørnberg, T.A. Ottosen, R.T. Pedersen, S. Pedersen, A.V. Sørensen, Factors affecting the electrification of wind-driven dust studied with laboratory simulations. Planet. Space Sci. 60, 328–335 (2012) D M i l Ad S R 39 337 344 (2007) D. Meunier et al., Adv. Space Res. 39, 337–344 (2007) P. Modica, C. Meinert, P.D. Marcellus, L. Nahon, U.J. Meierhenrich, L.L.S. d’Hendecour, Enantiomeric excesses induced in amino acids by ultraviolet circularly polarized light irradiation of extraterrestrial ice analogs: a possible source of asymmetry for prebiotic chemistry. Astrophys. J. 788, 79 (2014) M.H. Moore, R.L. References Hudson, Production of complex molecules in astrophysical ices, in Astrochemistry: Re- cent Successes and Current Challenges. Proceedings IAU Sym., vol. 231 (Cambridge University Press, Cambridge, 2005), pp. 247–260 g ) pp M.R. Mormile et al., Instruments, methods, and missions for astrobiology, in Proceedings of the SPIE Annual Meeting, vol. 33 (2007), p. 6694 M.R. Mormile et al., Astrobiology 9, 919–930 (2009) M. Morisson, C. Szopa, N. Carrasco, A. Buch, T. Gautier, Titan’s organic aerosols: molecular composi- tion and structure of laboratory analogues inferred from systematic pyrolysis gas chromatography mass spectrometry analysis. Icarus 277, 442–454 (2016) K. Motamedi, Validating the performance of a Raman laser spectrometer (RLS) instrument under Martian conditions. PhD thesis, VU University, Amsterdam, The Netherlands (2013) M. Muñoz-Caro, U.J. Meierhenrich, W.A. Schutte, B. Barbier, A. Arcones Segovia, H. Rosenbauer, W.H.- G.M. Muñoz-Caro, U.J. Meierhenrich, W.A. Schutte, B. Barbier, A. Arcones Segovia, H. Rosenbauer, W.H.- P. Thiemann, A. Brack, J.M. Greenberg, Amino acids from ultraviolet irradiation of interstellar ice analogues. Nature 416, 403–406 (2002) g P. Thiemann, A. Brack, J.M. Greenberg, Amino acids from ultraviolet irradiation of interstellar ice analogues. Nature 416, 403–406 (2002) g G.M. Muñoz Caro, W.A. Schutte, UV-photoprocessing of interstellar ice analogs: new infrared spectroscopic results. Astron. Astrophys. 412, 121–132 (2003) G.M. Muñoz-Caro, E. Mateo-Martí, J. Martínez-Frías, Near-UV transmittance of basalt dust as an analog the Martian regolith: implications for sensor calibration and astrobiology. Sensors 6, 688–696 (2006 G.M. Muñoz-Caro, E. Dartois, P. Boduch, H. Rothard, A. Domaracka, A. Jiménez-Escobar, Comparison UV and high-energy ion irradiation of methanol: ammonia ice. Astron. Astrophys. 566, 93–99 (2014 G.M. Muñoz-Caro, E. Dartois, P. Boduch, H. Rothard, A. Domaracka, A. Jiménez-Escobar, Comparison of UV and high-energy ion irradiation of methanol: ammonia ice. Astron. Astrophys. 566, 93–99 (2014) R. Navarro-González et al., Mars-like soils in the Atacama desert, Chile, and the dry limit of microbial life. Science 302, 1018–1021 (2003) g gy p y R. Navarro-González et al., Mars-like soils in the Atacama desert, Chile, and the dry limit of microbial life. Science 302, 1018–1021 (2003) , ( ) L.D.V. Neakrase, R. Greeley, J.D. Iversen, M.R. Balme, E.E. Eddlemon, Dust flux within dust devils: prelim- inary laboratory simulations. Geophys. Res. Lett. 33, L19S09 (2006) L.D.V. Neakrase, R. Greeley, Dust devil sediment flux on and Mars: laboratory simulations. Icarus 206, 306– 318 (2010) D. Nna-Mvondo, J.L. de la Fuente, M. Ruiz-Bermejo, B. Khare, C.P. References Holzer, The photolytic degradation and oxidation of organic compounds under simulated Martian conditions. J. Mol. Evol. 14, 153–160 (1979) G. Orzechowska, R.D. Kidd, B.H. Foing, I. Kanik, C. Stoker, P. Ehrenfreund, Analysis of Mars analog soil samples using solid phase microextraction, organic solvent extraction and gas chromatography/mass spectrometry. Int. J. Astrobiol. 10, 209–220 (2011) p y M.E. Palumbo, G. Ferini, G.A. Baratta, Infrared and Raman spectroscopy of refractory residues left over after ion irradiation of nitrogen bearing icy mixtures. Adv. Space Res. 33, 49–56 (2004) S. Pannewitz, M. Schlensog, T.G.A. Green, L.G. Sancho, B. Schroeter, Are lichens active under snow in continental Antarctica. Oecologia 135, 30–38 (2003) M.R. Patel, K. Miljkovic, T.J. Ringrose, M.R. Leese, The hypervelocity impact facility and environmental simulation at the Open University, in European Planetary Science Congress, vol. 5, EPSC (2010), p. 655 S J P l t l Pl t i d l ti i th d b f lt f th MINAR P M.R. Patel, K. Miljkovic, T.J. Ringrose, M.R. Leese, The hypervelocity impact facility and environmental simulation at the Open University, in European Planetary Science Congress, vol. 5, EPSC (2010), p. 655 S.J. Payler et al., Planetary science and exploration in the deep subsurface: results from the MINAR Program, Boulby Mine, UK. Int. J. Astrobiol. 16, 114–129 (2017) simulation at the Open University, in European Planetary Science Congress, vol. 5, EPSC (2010), p. 655 S.J. Payler et al., Planetary science and exploration in the deep subsurface: results from the MINAR Program, Boulby Mine, UK. Int. J. Astrobiol. 16, 114–129 (2017) S.J. Payler et al., Planetary science and exploration in the deep subsurf Boulby Mine, UK. Int. J. Astrobiol. 16, 114–129 (2017) Z. Peeters et al., Int. J. Astrobiol. 8, 301–315 (2009) H. Perko et al., J. Aerosp. Eng. 19, 169–176 (2006) p g ( ) P. Pernot, N. Carrasco, R. Thissen, I. Schmitz-Afonso, Tholinomics-chemical analysis of nitrogen-rich poly- mers. Anal. Chem. 82, 1371–1380 (2010) O. Poch, A. Noblet, F. Stalport, J.J. Correia, N. Grand, C. Szopa, P. Coll, Chemical evolution of organic molecules under Mars-like UV radiation conditions simulated in the laboratory with the “Mars Organic Molecule Irradiation and Evolution” (MOMIE) setup. Planet. Space Sci. 85, 188–197 (2013) O. Poch, S. Kaci, F. Stalport, C. Szopa, P. Coll, Laboratory insights into the chemical and kinetic evolution of several organic molecules under simulated Mars surface UV radiation conditions. Icarus 242, 50–63 (2014) O. References McKay, Thermal characterization of Titan’s tholins by simultaneous TG–MS, DTA, DSC analysis. Planet. Space Sci. 85, 279–288 (2013) P Nørnberg H P Gunnlaugsson J P Merrison A L Vendelboe Planet Space Sci 57 628–631 (2008) P. Nørnberg, H.P. Gunnlaugsson, J.P. Merrison, A.L. Vendelboe, Planet. Space Sci. 57, 628–631 (2008) P. Nørnberg, U. Schwertmann, H. Stanjek, T. Anderson, H.P. Gunnlaugsson, Clay Miner. 39, 85–98 (200 P. Nørnberg, U. Schwertmann, H. Stanjek, T. Anderson, H.P. Gunnlaugsson, Clay Miner. 39, 85–98 (2004) M. Nuevo, G. Auger, D. Blanot, L. D’Hendecourt, A detailed study of the amino acids produced from the vacuum UV irradiation of interstellar ice analogs. Orig. Life Evol. Biosph. 38, 37–56 (2008) M. Nuevo, G. Auger, D. Blanot, L. D’Hendecourt, A detailed study of the amino acids produced from the vacuum UV irradiation of interstellar ice analogs. Orig. Life Evol. Biosph. 38, 37–56 (2008) g g p M. Nuevo, S.N. Milam, S.A. Sandford, J.E. Elsila, J.P. Dworkin, Formation of uracil from the ultraviolet photo-irradiation of pyrimidine in pure H2O ices. Astrobiology 9, 683–695 (2009) M. Nuevo, C. Materese, S.A. Sandford, Photochemistry of pyrimidine in realistic astrophysical ices and the production of nucleobases. Astrophys. J. 793, 125–132 (2014) K. Olsson-Francis, C. Cockell, Experimental methods for studying microbial survival in extraterrestrial envi- ronments. J. Microbiol. Methods 80, 1–13 (2010) Z. Martins et al. 78 S. Onofri, L. Selbmann, L. Zucconi, S. Pagano, Antarctic microfungi as models for exobiology. Planet. Space Sci. 52, 229–237 (2004) S. Onofri, D. Barreca, L. Selbmann, D. Isola, E. Rabbow, G. Horneck, J.-P.P. de Vera, J. Hatton, L. Zucconi, Resistance of Antarctic black fungi and cryptoendolithic communities to simulated space and Mars conditions. Stud. Mycol. 61, 99–109 (2008) S. Onofri, R. de la Torre, J.-P. de Vera, S. Ott, L. Zucconi, L. Selbmann, G. Scalzi, K. Venkateswaran, E. Rabbow, F.J. Sánchez Inigo, G. Horneck, Survival of rock-colonizing organisms after 1.5 years in outer space. Astrobiology 12, 508–516 (2012) p gy S. Onofri, J.-P. de Vera, L. Zucconi, L. Selbmann, G. Scalzi, K.J. Venkateswaran, E. Rabbow, R. de la Torre, G. Horneck, Survival of Antarctic cryptoendolithic fungi in simulated Martian conditions on board the international space station. Astrobiology 15, 1052–1059 (2015) F. Orange, A. Chabin, A. Gorlas, S. Lucas-Staat, C. Geslin, M. Le Romancer, D. Prangishvili, P. Forterre, F. Westall, Experimental fossilisation of viruses from extremophilic Archaea. Biogeosciences 8, 1465– 1475 (2011) J. Oro, G. References Nimmo, Transient water vapor at Europa’s south pole. Science 343, 171–174 (2014) M.J. Russell, A.J. Hall, The emergence of life from iron monosulphide bubbles at a submarine hydrotherm redox and pH front. J. Geol. Soc. Lond. 154, 377–402 (1997) C. Sagan, B.N. Khare, Tholins: organic chemistry of interstellar grains and gas. Nature 277, 102 (1979) C. Sagan, B.N. Khare, W.R. Thompson, G.D. McDonald, M.R. Wing, J.L. Bada, T. Vo-Dinh, E.T. Arakawa, Polycyclic aromatic hydrocarbons in the atmospheres of Titan and Jupiter. Astrophys. J. 414, 399–405 (1993) F.J. Sánchez Iñigo, E. Mateo-Marti, J. Raggio, J. Meeßen, J. Martínez-Frías, L.G. Sancho, S. Ott, R. de la Torre, The resistance of the lichen Circinaria gyrosa (nom. provis.) towards simulated Mars conditions— a model test for the survival capacity of an eukaryotic extremophile. Planet. Space Sci. 72, 102–110 (2012) L.G. Sancho, B. Schroeter, R. Del-Prado, Ecophysiology and morphology of the globular erratic lichen As- picilia fruticulosa (Evers.) Flag. from Central Spain. Bibl. Lichenol. 75, 137–147 (2000) L.G. Sancho, R. de la Torre, G. Horneck, C. Ascaso, A. de Los Rios, A. Pintado, J. Wierzchos, M. Schus Lichens survive in space: results from the 2005 LICHENS experiment. Astrobiology 7, 443–454 (200 p p gy ( ) J. Schirmack, M. Böhm, C. Brauer, H.-G. Löhmannsröben, J.-P. de Vera, D. Möhlmann, D. Wagner, Laser spectroscopic real time measurements of methanogenic activity under simulated Martian subsurface analogue conditions. Planet. Space Sci. 98, 198–204 (2014) F. Schowengerdt, R. Fox, M. Duke, N. Marzwell, B. McKnight, PISCES: developing technologies for sus- tained human presence on the Moon and Mars, in Proc., 3rd AIAA Space Conf. and Exposition, Ameri- can Institute of Aeronautics and Astronautics (AIAA), Reston, VA (2007), pp. 3029–3038 B. Schroeter, T.G.A. Green, L. Kappen, R.D. Seppelt, Carbon dioxide exchange at subzero temperatures. Field measurements on “Umbilicaria aprina” in Antarctica. Cryptogam. Bot. 4, 233–241 (1994) A.C. Schuerger, R.L. Mancinelli, R.G. Kern, L.J. Rothschild, C.P. McKay, Survival of endospores of Bacil- lus subtilis on spacecraft surfaces under simulated Martian environments: implications for the forward contamination of Mars. Icarus 165, 253–276 (2003) A.C. Schuerger, P. Fajardo-Cavazos, C.A. Clausen, J.E. Moores, P.H. Smith, W.L. Nicholson, Slow degra- dation of ATP in simulated Martian environments suggests long residence times for the biosignature molecule on spacecraft surfaces on Mars. Icarus 194, 86–100 (2008) A.C. Schuerger, R. Ulrich, B.J. Berry, W.L. Nicholson, Growth of Serratia liquefaciens under 7 mbar, 0°C, and CO2-enriched anoxic atmospheres. References Poch, M. Jaber, F. Stalport, S. Nowak, T. Georgelin, J.F. Lambert, C. Szopa, P. Coll, Effect of nontronite smectite clay on the chemical evolution of several organic molecules under simulated Martian surface ultraviolet radiation conditions. Astrobiology 15, 221–237 (2015) W. Pollard, T. Haltigin, L. Whyte, T. Niederberger, D. Andersen, C. Omelon, J. Nadeau, M. Ecclestone, M. Lebeuf, Overview of analogue science activities at the McGill Arctic Research Station, Axel Heiberg Island, Canadian High Arctic. Planet. Space Sci. 57, 646–659 (2009) g p M.W. Powner, B. Gerland, J.D. Sutherland, Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions. Nature 459, 239–242 (2009) p L.J. Preston, L.R. Dartnell, Planetary habitability: lessons learned from terrestrial analogues. Int. J. Astrobiol. 13, 81–98 (2014) L.J. Preston, S. Barber, M. Grady, CAFE—Concepts for Activities in the Field for Exploration—Executive summary report. ESA Contract #4000104716/11/NL/AF. http://esamultimedia.esa.int/docs/gsp/ C4000104716ExS.pdf (last visited on Nov. 15, 2013) L.J. Preston, S. Barber, M. Grady, CAFE—Concepts for Activities in the Field for Exploration— TN2: the Catalogue of Planetary Analogues. http://esamultimedia.esa.int/docs/gsp/The_Catalogue_of_ Planetary_Analogues.pdf (last visited on Nov. 15, 2013) P.B. Price, T. Sowers, Temperature dependence of metabolic rates for microbial growth, maintenance, and survival. Proc. Natl. Acad. Sci. 101, 4631–4636 (2004) R.G. Prinn, B. Fegley, The atmosphere of Venus, Earth, and Mars: a critical comparison. Annu. Rev. Earth Planet. Sci. 15, 171–212 (1987) J.C. Priscu et al., Science 286, 2141–2144 (1999) R. Quinn et al., Planet. Space Sci. 53, 1376–1388 (2005) F. Raulin, C. Brasse, O. Poch, P. Coll, Prebiotic-like chemistry on Titan. Chem. Soc. Rev. 41, 5380–5393 (2012) 79 Earth as a Tool for Astrobiology—A European Perspective E. Rabbow, P. Rettberg, S. Barczyk, M. Bohmeier, A. Parpart, C. Panitz, G. Horneck, R. von Heise-Rotenburg, T. Hoppenbrouwers, R. Willnecker, P. Baglioni, R. Demets, J. Dettmann, G. Reitz, Astrobiology 12, 374–386 (2012) E. Rabbow, A. Parpart, G. Reitz, The planetary and space simulation facilities at DLR cologne. Microgravity Sci. Technol. 28, 215–229 (2016) P. Rettberg, E. Rabbow, C. Panitz, G. Horneck, Biological space experiments for the simulation of Mart conditions: UV radiation and Martian soil analogues. Adv. Space Res. 33, 1294–1301 (2004) E.M. Rivkina et al., Appl. Environ. Microbiol. 66, 3230–3233 (2000) A. Ross, J. Kosmo, B. Janoiko, Historical synopses of Desert RATS 1997–2010 and a preview of desert RATS 2011. Acta Astronaut. 90, 182–202 (2013) L. Roth, J. Saur, K.D. Retherford, D.F. Strobel, P.D. Feldman, M.A. McGrath, F. A. Skelley et al., J. Geophys. Res. 112, G04S11 (2007) References Astrobiology 13, 115–131 (2013) D.W. Schwartzman, C.H. Lineweaver, The hyperthermophilic origin of life revisited. Biochem. Soc. Trans. 32, 168–171 (2004) E. Sciamma-O’Brien, N. Carrasco, C. Szopa, A. Buch, G. Cernogora, Titan’s atmosphere: an optimal gas mixture for aerosol production? Icarus 209, 704–714 (2010) J.A. Sebree, M.G. Trainer, M.J. Loeffler, C.M. Anderson, Titan aerosol analog absorption features produced from aromatics in the far infrared. Icarus 236, 146–152 (2014) I.L. Schlacht, S. Voute, S. Irwin, M. Mikolajczak, B. Foing, A. Westenberg, C. Stoker, M. Masali, M. Rötting (Crew 91 & Mission Support), Moon-Mars Analogue Mission at the MDRS. EuroMoonMars-1 Mission, in IAC GLUC Global Lunar Conference 2010, Beijing (2010) B. Schroeter, C. Scheidegger, Water relations in lichens at subzero temperatures: structural changes and carbon dioxide exchange in the lichen “Umbilicaria aprina” from continental Antarctica. New Phytol. 131, 273–285 (1995) D. Schulze-Makuch et al., Astrobiology 11, 241–258 (2011) I.A. Shkrob, S.D. Chemerisov, T.W. Marin, Photocatalytic decomposition of carboxylated molecules on light- exposed Martian regolith and its relation to methane production on Mars. Astrobiology 10, 425–436 (2010) Y.M. Shtarkman et al., PLoS ONE 8, e67221 (2013) A. Skelley et al., J. Geophys. Res. 112, G04S11 (2007) 80 Z. Martins et al. J.J. Smith, L.A. Tow, W. Stafford, C. Cary, D.A. Cowan, Bacterial diversity in three different Antarctic cold desert mineral soils. Microb. Ecol. 51, 413–421 (2006) mith, in American Astronomical Society, AAS Meeting P. Smith, in American Astronomical Society, AAS Meeting (2009), p. 213 P. Smith et al., Science 325, 58–61 (2009) A. Somogyi, C-H. Oh, M.A. Smith, J.I. Lunine, Organic environments on Saturn’s Moon, Titan: simulating chemical reactions and analyzing products by FT-ICR and ion-trap mass spectrometry. Am. Soc. Mass Spectrom. 16, 850–859 (2005) F. Stalport, P. Coll, C. Szopa, F. Raulin, Search for organic molecules at the Mars surface: the “Martian Organic Material Irradiation and Evolution” (MOMIE) project. Adv. Space Res. 42, 2014–2018 (2008) F. Stalport, P. Coll, C. Szopa, F. Raulin, Search for organic molecules at the Mars surface: the “Martian Organic Material Irradiation and Evolution” (MOMIE) project. Adv. Space Res. 42, 2014–2018 (2008) F. Stalport, P. Coll, C. Szopa, F. Raulin, Investigating the photostability of carboxylic acids exposed to Mars surface radiation conditions. Astrobiology 9, 543–549 (2009) g p j p F. Stalport, P. Coll, C. Szopa, F. Raulin, Investigating the photostability of carboxylic acids exposed to Mars surface radiation conditions. Astrobiology 9, 543–549 (2009) F. Stalport, Y.Y. Guan, P. References Coll, C. Szopa, F. Macari, F. Raulin, D. Chaput, H. Cottin, UVolution, a photo- chemistry experiment in low Earth orbit: investigation of the photostability of carboxylic acids exposed to Mars surface UV radiation conditions. Astrobiology 10, 449–461 (2010) A. Steele, H.E.F. Amundsen (AMASE 07 Team), Arctic Mars Analog Svalbard Expedition 2007, in Proc., 38th Lunar and Planetary Science Conf., Lunar and Planetary Institute (LPI), Houston (2007) C.R. Stoker, M.A. Bullock, Organic degradation under simulated Martian conditions. J. Geophys. Res. 102, 10881–10888 (1997) B. Sutter et al., Lunar and Planetary Science Conference XXXVI Abstracts (2005), p. 2182 Szopa, G. Cernogora, L. Boufendi, J-J. Correia, P. Coll, PAMPRE: a dusty plasma experiment for Titan’s tholins production and study. Planet. Space Sci. 54, 394–404 (2006) I.L. ten Kate, M. Reuver, PALLAS: Planetary Analogues Laboratory for Light, Atmosphere, and Surface Simulations. Neth. J. Geosci. 95, 183–189 (2015) I.L. ten Kate, J.R.C. Garry, Z. Peeters, R. Quinn, B. Foing, P. Ehrenfreund, Amino acid photostability on the Martian surface. Meteorit. Planet. Sci. 40, 1185–1193 (2005) I.L. ten Kate, J.R.C. Garry, Z. Peeters, B. Foing, P. Ehrenfreund, The effects of Martian near surface condi- tions on the photochemistry of amino acids. Planet. Space Sci. 54, 296–302 (2006) I.L. ten Kate, R. Armstrong, B. Bernhardt, M. Blumers, J. Craft, D. Boucher, E. Caillibot, J. Captain, G.M.T. D’Eleuterio, J.D. Farmer, D.P. Glavin, T. Graff, J.C. Hamilton, G. Klingelhöfer, R.V. Morris, J.I. Nuñez, J.W. Quinn, G.B. Sanders, R.G. Sellar, L. Sigurdson, R. Taylor, K. Zacny, Mauna Kea, Hawaii, as an analogue site for future planetary resource exploration: results from the 2010 ILSO-ISRU field-testing campaign. J. Aerosp. Eng. 26, 183–196 (2013) Thiel, P. Ehrenfreund, B. Foing, V. Pletser, O. Ullrich, PCR-based analysis of microbial communities dur- ing the EuroGeoMars campaign at Mars Desert Research Station, Utah. Int. J. Astrobiol. 10, 177–190 (2011) R. Thirsk, D. Williams, M. Anvari, NEEMO 7 undersea mission. Acta Astronaut. 60, 512–517 (2007) B. Todd, M. Reagan, The NEEMO Project: A Report on How NASA Utilizes the “Aquarius” Undersea Habitat as an Analog for Long-Duration Space Flight. Engineering, Construction, and Operations in Challenging Environments, pp. 751–758 (2004). doi:10.1061/40722(153)103 M.G. Trainer, J.L. Jimenez, Y.L. Yung, O.B. Toon, M.A. Tolbert, Nitrogen incorporation in CH(4)-N(2) photochemical aerosol produced by far ultraviolet irradiation. Astrobiology 12, 315–326 (2012) M.G. Trainer, A.A. Pavlov, D.B. Curtis, C.P. McKay, D. Worsnop, A.E. Delia, D.W. Toohey, O.B. Toon, M.A. References Tolbert, Haze aerosols in the atmosphere of early Earth: manna from Heaven. Astrobiology 4, 409–419 (2004) M.G. Trainer, A.A. Pavlov, H. Langley DeWitt, J.L. Jimenez, C.P. McKay, O.B. Toon, M.A. Tolbert, Organic haze on Titan and the early Earth. Proc. Natl. Acad. Sci. 103, 18035–18042 (2006) B.N. Tran, M. Force, R.G. Briggs, J.P. Ferris, P. Persans, J.J. Chera, Titan’s atmospheric chemistry: photolysis of gas mixtures containing hydrogen cyanide and carbon monoxide at 185 and 254 nm. Icarus 193, 224– 232 (2008) T.A. Vishnivetskaya et al., Astrobiology 6, 400–414 (2006) E. Vorobyova et al., FEMS Microbiol. Rev. 20, 277–290 (1997) V. Vuitton, J.Y. Bonnet, M. Frisari, R. Thissen, E. Quirico, O. Dutuit, B. Schmitt, L. Le Roy, N. Fray, H. Cottin, E. Sciamma-O’Brien, N. Carrasco, C. Szopa, Very high resolution mass spectrometry of HCN polymers and tholins. Faraday Discuss. 147, 495–508 (2010) D. Wagner, A. Lipski, A. Embacher, A. Gattinger, Methane fluxes in permafrost habitats of the Lena Delta: effects of microbial community structure and organic matter quality. Environ. Microbiol. 7, 1582–1592 (2005) D. Wagner, A. Gattinger, A. Embacher, E.-M. Pfeiffer, M. Schloter, A. Lipinski, Methanogenic activity and biomass in Holocene permafrost deposits of the Lena Delta, Siberian Arctic and its implication for the global methane budget. Glob. Change Biol. 13, 1089–1099 (2007) Earth as a Tool for Astrobiology—A European Perspective 81 H. Walz, Photosynthesis yield analyzer mini-PAM, in Handbook of Operation 2nd edn. (Heinz-Walz GmbH, Effeltrich, 1999) F. Westall, The Early Earth, in Frontiers of Astrobiology, ed. by C. Impey, J. Lunine, J. Funes (Cambridge University Press, Cambridge, 2012), pp. 89–114 F. Westall, F. Foucher, B. Cavalazzi, S.T. de Vries, W. Nijman, V. Pearson, J. Watson, A. Verchovsky, I. Wright, J.N. Rouzaud, D. Marchesini, S. Anne, Early life on Earth and Mars: a case study from ∼ 3.5 Ga-old rocks from the Pilbara, Australia. Planet. Space Sci. 59, 1093–1106 (2011) F. Westall, F. Foucher, N. Bost, M. Bertrand, D. Loizeau, J.L. Vago, G. Kminek, F. Gaboyer, K.A. Campbell, J-B. Bréhéret, P. Gautret, C.S. Cockell, Biosignatures on Mars: what, where and how? Implications for the search for Martian life. Astrobiology 15, 998–1029 (2015a) gy F. Westall, K.A. Campbell, J.G. Bréhéret, F. Foucher, P. Gautret, A. Hubert, S. Sorieul, N. Grassineau, D.M. Guido, Archean (3.33 Ga) microbe-sediment systems were diverse and flourished in a hydrothermal context. Geology 43, 615–618 (2015b) gy E. Williams, C.P. McKay, O.B. Toon, J.W. References Head, Do ice caves exist on Mars? Icarus 209, 358–368 (2010) K.E. Williams, C.P. McKay, O.B. Toon, J.W. Head, Do ice caves exist on Mars? Icarus 209, 358–368 (2010) D.D. Wynn-Williams, H.G.M. Edwards, Antarctic ecosystems as models for extraterrestrial surface habitats. Planet. Space Sci. 48, 1065–1075 (2000) D.D. Wynn-Williams, H.G.M. Edwards, Antarctic ecosystems as models for extraterrestrial surface habitats. Planet. Space Sci. 48, 1065–1075 (2000) R.A. Yingst, P. Russell, I.L. ten Kate, S. Noble, T. Graff, L.D. Graham, D. Eppler, Designing remote opera- tions strategies to optimize science mission goals: lessons learned from the Moon Mars Analog Mission 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 hydrocarbons. Icarus 219, 161–167 (2012) M.P. Zorzano, E. Mateo-Martí, O. Prieto-Ballesteros, S. Osuna, N. Renno, The stability of liquid saline water on present day Mars. Geophys. Res. Lett. 36, L20201 (2009)
https://openalex.org/W2962945014
https://cadmus.eui.eu/bitstream/1814/63664/1/RSCAS_2019_52.pdf
English
null
'We Can Also Do Stupid': The EU's Response to America First Protectionism
Social Science Research Network
2,019
public-domain
14,375
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 Badia Fiesolana I – 50014 San Domenico di Fiesole (FI) Italy www.eui.eu/RSCAS/Publications/ 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
https://openalex.org/W2562786370
https://europepmc.org/articles/pmc5167254?pdf=render
English
null
A Mathematical Model of the Olfactory Bulb for the Selective Adaptation Mechanism in the Rodent Olfactory System
PloS one
2,016
cc-by
8,859
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; . . . ; P1805ðogފ 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; . . . ; c1805ðnqފ 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; . . . ; x1805Š 2 R18051 and Y ¼ ½y1; . . . ; yk; . . . ; y1805Š 2 R18051 are the internal states of the mitral and granular cells, respectively, I ¼ ½I1; . . . ; Ik; . . . ; I1805Š 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; . . . ; gyðy1805ފ 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;1805ފ 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;1805ފ 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;1805ފ 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;1805ފ 2 R18051805 is that from mitral units to granular units, and L0 ¼ ½L0ð1;1Þ; . . . ; L0ðj;kÞ; . . . ; L0ð1805;1805ފ 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;ogŠ 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;1Š 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;2Š 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 December 19, 2016 References 1. Atanosova B, Thomas-Danquin T, Chabanet C, Langlois D, Nicklaus S, Etievant P. Perceptual interac- tions in odour mixtures: odour quality in binary mixtures of woody and fruity wine odorants. Chem Senses. 2005; 30: 209–217. doi: 10.1093/chemse/bji016 PMID: 15741601 2. Haddad R, Khan R, Takahashi YK, Mori K, Harel D, Sobel N. A metric for odorant comparison. Nat Methods. 2008; 5(5): 425–429. doi: 10.1038/nmeth.1197 PMID: 18376403 3. Khan RM, Luk C-H, Flinker A, Aggarwal A, Lapid H, Haddad R, et al. Predicting odor pleasantness from odorant structure: pleasantness as a reflection of the physical world. J Neurosci. 2007; 27(37): 10015– 10023. doi: 10.1523/JNEUROSCI.1158-07.2007 PMID: 17855616 4. Soh Z, Tsuji T, Takiguchi N, Ohtake H. An artificial neural network approach for glomerular activity pat- tern prediction using the graph kernel method and the Gaussian mixture functions. Chem Senses. 2011; 36(5): 413–24. doi: 10.1093/chemse/bjq147 PMID: 21343242 5. Soh Z, Saito M, Kurita Y, Takiguchi N, Ohtake H, Tsuji T. A comparison between the human sense of smell and neural activity in the olfactory bulb of rats. Chem Senses. 2014; 39(2): 91–105. doi: 10.1093/ chemse/bjt057 PMID: 24252998 6. Snitz K, Yablonka A, Weiss T, Frumin I, Khan RM, Sobel N. Predicting odor perceptual similarity from odor structure. PLoS Comput Biol. 2013; 9(9): e1003184. doi: 10.1371/journal.pcbi.1003184 PMID: 24068899 7. Haddad R, Medhanie A, Roth Y, Harel D, Sobel N. Predicting odor pleasantness with an electronic nose. PLoS Comput Biol. 2010; 6(4): e1000740. doi: 10.1371/journal.pcbi.1000740 PMID: 20418961 8. Mori K, Nagao H, Yoshihara Y. The olfactory bulb: coding and processing of odor molecule information. Science. 1999; 286(22): 711–715. 9. Sakano H. Neural map formation in the mouse olfactory system. Neuron. 2010; 67(4):530–542. doi: 10. 1016/j.neuron.2010.07.003 PMID: 20797531 10. Belluscio L, Katz C. Symmetry, stereotypy, and topography of odorant representations in mouse olfac- tory bulbs. J Neurosci. 2001; 21(6): 2113–2122. PMID: 11245695 11. Uchida N, Takahashi Y, Tanifuji M, Mori K. Odor maps in the mammalian olfactory bulb: domain organi- zation and odorant structural features. Nat Neurosci. 2000; 3: 1035–1043. doi: 10.1038/79857 PMID: 11017177 12. Johnson BA, Woo CC, Leon M. Spatial coding of odorant features in the glomerular layer of the rat olfac- tory bulb. J Comp Neurol. 1998; 393: 457–471. PMID: 9550151 13. Fletcher ML, Masurkar AV, Xing J, Imamura F, Xiong W, Nagayama S, et al. Optical imaging of postsyn- aptic odor representation in the glomerular layer of the mouse olfactory bulb. Project administration: TT YK. Resources: NT. Resources: NT. Software: SN. PLOS ONE | DOI:10.1371/journal.pone.0165230 December 19, 2016 14 / 16 Olfactory Model for Selective Adaptation Mechanism Supervision: TT YK. Validation: ZS. Visualization: ZS. Writing – original draft: ZS. Writing – review & editing: YK. Writing – original draft: ZS. Writing – review & editing: YK. Writing – review & editing: YK. PLOS ONE | DOI:10.1371/journal.pone.0165230 December 19, 2016 References J Neurophysiol. 2009; 102: 817–830. doi: 10.1152/jn.00020.2009 PMID: 19474178 14. Youngentob SL, Johnson BA, Leon M, Sheehe PR, Kent PF. Predicting odorant quality perceptions from multidimensional scaling of olfactory bulb glomerular activity patterns. Behav Neurosci. 2006; 120 (6): 1337–1345. doi: 10.1037/0735-7044.120.6.1337 PMID: 17201479 15. Grossman KJ, Mallik AK, Ross J, Kay LM, Issa NP. Glomerular activation patterns and the perception of odor mixtures. Eur J Neurosci. 2008; 27(10): 2676–2685. doi: 10.1111/j.1460-9568.2008.06213.x PMID: 18445053 16. Takiguchi N. Performance of mice in discrimination of liquor odors: behavioral evidence for olfactory attention. Chem Senses Advance Access. 2008; 33(3): 283–290. 17. Goyert HF, Frank ME, Gent JF, Hettinger TP. Characteristic component odors emerge from mixtures after selective adaptation. Brain Res Bull. 2007; 72(1):1–9. doi: 10.1016/j.brainresbull.2006.12.010 PMID: 17303501 18. Giraudet P, Berthommier F, Chaput M. Mitral cell temporal response patterns evoked by odor mixtures in the rat olfactory bulb. Neurophysiol. 2002; 88: 829–838. PLOS ONE | DOI:10.1371/journal.pone.0165230 December 19, 2016 15 / 16 Olfactory Model for Selective Adaptation Mechanism 19. Li Z, Hopfield JJ. Modeling the olfactory bulb and its neural oscillatory processings. Biol Cybern. 1989; 61: 379–392. PMID: 2551392 20. Erdi P, Grobler T, Barna G, Kaski K. Dynamics of the olfactory bulb: bifurcations, learning, and memory. Biol Cybern. 1993; 69: 57–66. PMID: 8334190 21. Maresh A, Rodriguez GD, Whitman MC, Greer CA. Principles of glomerular organization in the human olfactory bulb—implications for odor processing. PLoS One. 2008; 3(7): e2640. doi: 10.1371/journal. pone.0002640 PMID: 18612420 22. Yamaguchi M, Yamazaki K, Beauchamp GK, Bard J, Thomas L, Boyse EA. Distinctive urinary odors governed by the major histocompatibility locus of the mouse. Proc Natl Acad Sci USA. 1981; 78(9): 5817–5820. PMID: 6946517 23. Yamazaki K, Beauchamp GK, Singer A, Bard J, Boyse EA. Odortypes: Their origin and composition. Proc Natl Acad Sci USA. 1999; 96: 1522–1525. PMID: 9990056 24. Hebb DO. The Organization of Behavior: A Neuropsychological Theory. New York: Wiley & Sons; 1949. 25. Linster C, Menon A V, Singh CY, Wilson DA. Odor-specific habituation arises from interaction of afferent synaptic adaptation and intrinsic synaptic potentiation in olfactory cortex. Learn Mem. 2009; 16(7): 452–459. doi: 10.1101/lm.1403509 PMID: 19553383 26. Naritsuka H, Sakai K, Hashikawa T, Mori K, Yamaguch M. Perisomatic-targeting granule cells in the mouse olfactory bulb. J Comp Neuron. 2009; 515(4): 409–426. 27. Johnson BA, Woo CC, Leon M. Spatial coding of odorant features in the glomerular layer of the rat olfac- tory bulb. PLOS ONE | DOI:10.1371/journal.pone.0165230 December 19, 2016 References J Comp Neurol. 1998; 393(4): 457–451. PMID: 9550151 28. Johnson BA et al. Functional mapping of the rat olfactory bulb using diverse odorants reveals modular responses to functional groups and hydrocarbon structural features. J Comp Neurol. 2002; 449: 180– 194. doi: 10.1002/cne.10284 PMID: 12115688 29. Johnson BA, Farahbod H, Leon M. Interactions between odorant functional group and hydrocarbon structure influence activity in glomerular response modules in the rat olfactory bulb. J Comp Neurol. 2005; 483: 205–216. doi: 10.1002/cne.20409 PMID: 15678471 30. Johnson BA, Leon M. Chemotopic odorant coding in a mammalian olfactory system. J Comp Neurol. 2007; 503: 1–34. doi: 10.1002/cne.21396 PMID: 17480025 31. Glomerular response archive. [accessed 7 March 2016]. [Internet]. http://gara.bio.uci.edu/index.jsp 32. Miyamichi K, Shlomai-Fuchs Y, Shu M, Weissbourd BC, Luo L, Mizrahi A. Dissecting local circuits: par- valbumin interneurons underlie broad feedback control of olfactory bulb output. Neuron. 2013; 80(5): 1232–1245. doi: 10.1016/j.neuron.2013.08.027 PMID: 24239125 33. Haberly LB, Price JL. Association and commissural fiber systems of the olfactory cortex of the rat II. Systems originating in the olfactory peduncle. J Comp Neurol. 1978; 181(4): 781–807. doi: 10.1002/ cne.901810407 PMID: 690285 34. Boyd AM, Sturgill JF, Poo C, Isaacson JS. Cortical feedback control of olfactory bulb circuits. Neuron. 2012; 76(6): 1161–1174. doi: 10.1016/j.neuron.2012.10.020 PMID: 23259951 35. Cleland TA, Linster C. On-Center/Inhibitory-Surround Decorrelation via Intraglomerular Inhibition in the Olfactory Bulb Glomerular Layer. Front. Integr. Neurosci. 2012; 6(5) 1–10. 36. Linster C, Menon AV, Singh CY, Wilson DA. Odor-specific habituation arises from interaction of afferent synaptic adaptation and intrinsic synaptic potentiation in olfactory cortex. Learn. Mem. 2009; 16(7): 452–459 doi: 10.1101/lm.1403509 PMID: 19553383 16 / 16
https://openalex.org/W196078183
https://europepmc.org/articles/pmc5210221?pdf=render
English
null
Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics
Journal of nanobiotechnology
2,017
cc-by
7,801
© 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 Immunol Infect. 2010;43:347–9. 8. Afshari A, Schrenzel J, Ieven M, Harbarth S. Bench-to-bedside review: rapid molecular diagnostics for bloodstream infection—a new frontier? Crit Care. 2012;16:222. 8. Afshari A, Schrenzel J, Ieven M, Harbarth S. Bench-to-bedside review: rapid molecular diagnostics for bloodstream infection—a new frontier? Crit Care. 2012;16:222. 9. Lin C-C, Wang J-H, Wu H-W, Lee G-B. Microfluidic immunoassays. J Assoc Lab Autom. 2010;15:253–74. 9. Lin C-C, Wang J-H, Wu H-W, Lee G-B. Microfluidic immunoassays. J Assoc Lab Autom. 2010;15:253–74. 10. Mayeux PR. Pathobiology of lipopolysaccharide. J Toxicol Environ Health. 1997;51:415–35. Additional file 3: Figure S3. 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. 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 systems. Anal Bioanal Chem. 2010;397:991–1007. 11. Ng AHC, Uddayasankar U, Wheeler AR. Immunoassays in micro systems. Anal Bioanal Chem. 2010;397:991–1007. 12. Nguyen HB, Corbett SW, Steele R, Banta J, Clark RT, Hayes SR, et al. Imple- mentation of a bundle of quality indicators for the early management of severe sepsis and septic shock is associated with decreased mortality. Crit Care Med. 2007;35:1105–12. 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 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 insignificant magnitude of forces on bacteria. 13. Yung CW, Fiering J, Mueller AJ, Ingber DE. Micromagnetic–microfluidic blood cleansing device. Lab Chip. 2009;9:1171. 14. Demirci U, Wang S, Inci F, Chaunzwa TL, Ramanujam A, Vasudevan A, et al. Portable microfluidic chip for detection of Escherichia coli in produce and blood. Int J Nanomedicine. 2012;7:2591. Additional file 5: Figure S5. Elasto-inertial microfluidics based bacteria separation from PBS (a) and whole blood sample (b) for a range of flow rates. The bacteria recovery from the side outlet is relatively constant (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 separation from PBS (a) and whole blood sample (b) for a range of flow rates. The bacteria recovery from the side outlet is relatively constant (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 multicenter study. Intensive Care Med. 2007;33:606–18. y 2. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med. 2006;34:1589–96. 3. Kumar A, Ellis P, Arabi Y, Roberts D, Light B, Parrillo JE, et al. Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock. Chest. 2009;136:1237–48. p 4. Mancini N, Carletti S, Ghidoli N, Cichero P, Burioni R, Clementi M. The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin Microbiol Rev. 2010;23:235–51. p 5. Lebovitz EE, Burbelo PD. Commercial multiplex technologies for the microbiological diagnosis of sepsis. Mol Diagn Ther. 2013;17:221–31. 6. Mancini N, Carletti S, Ghidoli N, Cichero P, Ossi CM, Ieri R, et al. Molecular p 5. Lebovitz EE, Burbelo PD. Commercial multiplex technologies for the microbiological diagnosis of sepsis. Mol Diagn Ther. 2013;17:221–31. g g p g 6. Mancini N, Carletti S, Ghidoli N, Cichero P, Ossi CM, Ieri R, et al. Molecular diagnosis of polymicrobial sepsis. J Clin Microbiol. 2009;47:1274–5. 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. Additional file 2: Figure S2. Particle defocussing due to shear thinning effect as flow rate is increased. References 2004;26:133–46. 23. Hou HW, Bhattacharyya RP, Hung DT, Han J. Direct detection and drug- resistance profiling of bacteremias using inertial microfluidics. Lab Chip. 2015;15:2297–307. 37. Wiesinger-Mayr H, Jordana-Lluch E, Martró E, Schoenthaler S, Noeham- mer C. Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits. J Microbiol Methods. 2011;85:206–13. 24. Caserta S, D’Avino G, Greco F, Guido S, Maffettone PL. Migration of a sphere in a viscoelastic fluid under planar shear flow: experiments and numerical predictions. Soft Matter. 2011;7:1100–6. 38. Al-Soud WA, Radstrom P. Purification and characterization of PCR-inhibi- tory components in blood cells. J Clin Microbiol. 2001;39:485–93. 25. D’Avino G, Romeo G, Villone MM, Greco F, Netti PA, Maffettone PL. Single line particle focusing induced by viscoelasticity of the suspending liquid: theory, experiments and simulations to design a micropipe flow-focuser. Lab Chip. 2012;12:1638. 39. Nam J, Namgung B, Lim CT, Bae J-E, Leo HL, Cho KS, et al. Microfluidic device for sheathless particle focusing and separation using a viscoelastic fluid. J Chromatogr A. 2015;1406:244–50. l 40. Won Seo K, Ran Ha Y, Joon Lee S. Vertical focusing and cell ordering in a microchannel via viscoelasticity: applications for cell monitoring using a digital holographic microscopy. Appl Phys Lett. 2014;104:213702. 26. Ho BP, Leal LG. Migration of rigid spheres in a two-dimensional unidirec- tional shear flow of a second-order fluid. J Fluid Mech. 1976;76:783. ll 27. Leshansky AM, Bransky A, Korin N, Dinnar U. Tunable nonlinear viscoelas- tic “focusing” in a microfluidic device. Phys Rev Lett. 2007;98:234501. 41. Lim EJ, Ober TJ, Edd JF, Desai SP, Neal D, Bong KW, et al. Inertio-elastic focusing of bioparticles in microchannels at high throughput. Nat Com- mun. 2014;5:4120. l 28. Romeo G, D’Avino G, Greco F, Netti PA, Maffettone PL. Viscoelastic flow-focusing in microchannels: scaling properties of the particle radial distributions. Lab Chip. 2013;13:2802–7. 42. Yang S, Lee SS, Ahn SW, Kang K, Shim W, Lee G, et al. Deformability-selec- tive particle entrainment and separation in a rectangular microchannel using medium viscoelasticity. Soft Matter. 2012;8:5011. p 29. Villone MM, D’Avino G, Hulsen MA, Greco F, Maffettone PL. Particle motion in square channel flow of a viscoelastic liquid: migration vs. secondary flows. J Non Newton Fluid Mech. 2013;195:1–8. 43. Cha S, Shin T, Lee SS, Shim W, Lee G, Lee SJ, et al. Cell stretching measure- ment utilizing viscoelastic particle focusing. Anal Chem. 2012;84:10471–7. l 30. References 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%). 15. Lee J-J, Jeong KJ, Hashimoto M, Kwon AH, Rwei A, Shankarappa SA, et al. Synthetic ligand-coated magnetic nanoparticles for microfluidic bacterial separation from blood. Nano Lett. 2014;14:1–5. 16. Leevy WM, Johnson JR, Lakshmi C, Morris J, Marquez M, Smith BD. Selective recognition of bacterial membranes by zinc(ii)-coordination complexes. Chem Commun. 2006;1595–7. doi:10.1039/b517519d. 16. Leevy WM, Johnson JR, Lakshmi C, Morris J, Marquez M, Smith BD. Selective recognition of bacterial membranes by zinc(ii)-coordination complexes. Chem Commun. 2006;1595–7. doi:10.1039/b517519d. Faridi et al. J Nanobiotechnol (2017) 15:3 Page 9 of 9 17. Leevy WM, Lambert TN, Johnson JR, Morris J, Smith BD. Quantum dot probes for bacteria distinguish Escherichia coli mutants and permit in vivo imaging. Chem Commun. 2008;2331–3. doi:10.1039/B803590C. 31. Ahn SW, Lee SS, Lee SJ, Kim JM. Microfluidic particle separator utilizing sheathless elasto-inertial focusing. Chem Eng Sci. 2015;126:237–43. 32. Yang S, Kim JY, Lee SJ, Lee SS, Kim JM. Sheathless elasto-inertial particle focusing and continuous separation in a straight rectangular microchan- nel. Lab Chip. 2011;11:266–73. 18. White AG, Fu N, Leevy WM, Lee J-J, Blasco MA, Smith BD. Optical imaging of bacterial infection in living mice using deep-red fluorescent squaraine rotaxane probes. Bioconjug Chem. 2010;21:1297–304. 33. Nam J, Lim H, Kim D, Jung H, Shin S. Continuous separation of microparti- cles in a microfluidic channel via the elasto-inertial effect of non-Newto- nian fluid. Lab Chip. 2012;12:1347. 19. Di Carlo D. Inertial microfluidics. Lab Chip. 2009;9:3038. 20. Wu Z, Willing B, Bjerketorp J, Jansson JK, Hjort K. Soft inertial microfluidics for high throughput separation of bacteria from human blood cells. Lab Chip. 2009;9:1193. 34. Liu C, Xue C, Chen X, Shan L, Tian Y, Hu G. Size-based separation of parti- cles and cells utilizing viscoelastic effects in straight microchannels. Anal Chem. 2015;87:6041–8. p 21. Mach AJ, Di Carlo D. Continuous scalable blood filtration device using inertial microfluidics. Biotechnol Bioeng. 2010;107:302–11. 35. Li G, McKinley GH, Ardekani AM. Dynamics of particle migration in chan- nel flow of viscoelastic fluids. J Fluid Mech. 2015;785:486–505. 22. Hou HW, Warkiani ME, Khoo BL, Li ZR, Soo RA, Tan DS-W, et al. Isolation and retrieval of circulating tumor cells using centrifugal forces. Sci Rep. 2013;3:1259. ll 36. Rådström P, Knutsson R, Wolffs P, Lövenklev M, Löfström C. Pre-PCR pro- cessing. Mol Biotechnol. References Young Kim J, Won Ahn S, Sik Lee S, Min Kim J. Lateral migration and focusing of colloidal particles and DNA molecules under viscoelastic flow. Lab Chip. 2012;12:2807. 44. Hansson J, Karlsson JM, Haraldsson T, Brismar H, van der Wijngaart W, Russom A. Inertial microfluidics in parallel channels for high-throughput applications. Lab Chip. 2012;12:4644. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W2063084942
https://dialnet.unirioja.es/servlet/citart?info=link&codigo=3326355&orden=273152
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 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 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 fields, Duke Math. J. 98 (1999), 541–564. MR1695801 (2000j:11074) [RVZ] 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] D. Rohrlich, The nonvanishing of certain Hecke L-functions at the center of the critical strip. Duke Math. J. 47 (1980), 223–232. MR563377 (81k:12017) [R2] D. Rohrlich, On the L-functions of canonical Hecke characters of imaginary quadratic fields. Duke Math. J. 47 (1980), 547–557. MR587165 (81m:12020) [R3] D. Rohrlich, Galois conjugacy of unramified twists of Hecke characters. Duke Math. J. 47 (1980), 695–703. MR587174 (82a:12009) [Sh] G. Shimura, On periods of modular forms. Math. Ann. 229 (1977), 211–221. MR0463119 (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
https://openalex.org/W2152332660
https://europepmc.org/articles/pmc3035531?pdf=render
English
null
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
https://openalex.org/W1534982716
https://dergipark.org.tr/tr/download/article-file/191135
Turkish
null
KOMPAKT VE RİNG İPLİKLERDEN ELDE EDİLMİŞ ÖRME KUMAŞLARIN PATLAMA MUKAVEMETİ VE BONCUKLANMA PERFORMANSI ÜZERİNE BİR ARAŞTIRMA
DergiPark (Istanbul University)
2,005
cc-by
2,345
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